Role of the transcription factor Tog1 in Candida glabrata

virulence and oxidative stress resistance

Susana Cristina Vagueiro Santos

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisor: Prof. Dr. Miguel Nobre Parreira Cacho Teixeira

Examination Committee

Chairperson: Prof. Dr. Ana Cristina Anjinho Madeira Viegas

Supervisor: Prof. Dr. Miguel Nobre Parreira Cacho Teixeira

Member of the Committee: Dr. Margarida Isabel Rosa Bento Palma

October 2018

ii

Acknowledgements

Firstly, I would like to express my sincere gratitude to my advisor, Professor Miguel Teixeira, for the

opportunity given by accepting me in his team and in this project. His guidance and patience

helped me in all the time of research and writing of this thesis. I am grateful for his insightful comments

and knowledge shared through this year.

I would like to thank Professor Isabel Sá Correia for giving me the chance to join the Biological Sciences

Research Group to develop my master thesis work.

My sincere thanks also go to everyone who was always kind to give me a hand during the experimental

work. Specially to Mafalda Cavalheiro, whose methodical and organized work was a true example to

follow, and my laboratory mentor Pedro Pais, for always being available to exchange ideas with me and

for all the time spent teaching me doing bioinformatic analysis fundamental to develop this work. I also

would like to thank Andreia Pimenta, for her essential help through the virulence assays and for always

amusing me with her humorous character.

Lastly, I would like to thank all my friends who made this academic experience a lot more enjoyable:

Alexandra, Cristiana, Lampreia, João, Jorge, Mariana, Pedro, Raquel and Rita. Also, my partner

Ricardo, who always makes me laugh even in the hardest days, and my family for the continuous

encouragement in pursuing my educational path.

This work was supported by FEDER and “Fundação para a Ciência e a Tecnologia” (FCT) (Contracts

PTDC/BBB-BIO/4004/2014 and PTDC/BII-BIO/28216/2017) and Programa Operacional Regional de

Lisboa 2020, contract LISBOA-01-0145-FEDER-022231. Funding was received by iBB – Institute for

Bioengineering and Biosciences from FCT (UID/BIO/04565/2013) and from Programa Operacional

Regional de Lisboa 2020 (Project N. 007317).

iii

Abstract

The opportunistic human fungal pathogen Candida glabrata is the second cause of Candida infections

worldwide, being a major clinical challenge with severe economic and health impact. Therefore, it is

urgent to understand the mechanisms underlying virulence in this pathogenic yeast.

In this work, the transcription factor Tog1 was studied as a new determinant of C. glabrata virulence

using Galleria mellonella as an infection model. TOG1 expression was found to increase C. glabrata

ability to proliferate inside G. mellonella hemocytes. Given the predicted physiological role of Tog1,

based on that of its close homolog in Saccharomyces cerevisiae, the effect of TOG1 deletion in the

ability to grow in non-fermentable carbon sources and under oxidative stress was evaluated. Although

Tog1 did not appeared to have a role in utilizing alternative carbon sources, TOG1 was identified as a

determinant of resistance to hydrogen peroxide.

Analysis of C. glabrata transcriptome enlightened the role of Tog1 in respiratory and energy metabolism

during oxidative stress with hydrogen peroxide. The mitochondrial function was vastly affected by the

deletion of TOG1. Tog1 seems to activate genes belonging to biological groups of carnitine transport,

assembly and function of the oxidative phosphorylation pathway, and glyoxylate and TCA cycle.

Dysfunctional mitochondrial function may lead to iron accumulation exacerbating the generation of

reactive oxygen species (ROS) by the Fenton reaction. Additionally, Tog1 was found to activate two

genes linked directly to oxidative stress: ALD6 and RCK2, an aldehyde dehydrogenase, whose activity

leads to NADPH regeneration, and protein kinase implicated in translational regulation, respectively.

The results gathered through this work lead us to suggest that Tog1 determines virulence in C. glabrata

through adaptation to oxidative stress found inside macrophage-like cells.

Keywords: C. glabrata, virulence, CgTog1, oxidative stress, energy metabolism.

iv

Resumo

Candida glabrata é um fungo patogénico oportunista humano, sendo a segunda maior causa de

infecções de candidíase no mundo, tornando-se assim num desafio clínico que possui um grande

impacto na economia e saúde. Portanto, é cada vez mais urgente compreender quais os mecanismos

que definem a virulência desta levedura patogénica.

Neste trabalho, o factor de transcrição Tog1 foi estudado como um novo determinante da virulência de

C. glabrata recorrendo ao modelo de infecção de Galleria mellonella. Observou-se que a expressão do

TOG1 aumentou a capacidade de proliferação de C. glabrata dentro dos hemócitos de G. mellonella.

Sendo já conhecida a função do homólogo de Tog1 em Saccharomyces cerevisiae, o efeito da delecção

do gene TOG1 na capacidade de crescer em fontes de carbono não fermentáveis e sob o feito de stress

oxidativo foi avaliado. Apesar de Tog1 não aparentar possuir um papel na utilização de fontes de

carbono alternativas, TOG1 foi identificado como um determinante da resistência ao peróxido de

hidrogénio.

A análise do transcriptoma de C. glabrata forneceu evidências da função de Tog1 no metabolismo

respiratório e energético durante o stress oxidativo com peróxido de hidrogénio. O funcionamento

mitocondrial foi largamente afectado pela delecção do TOG1. Tog1 parece activar genes pertencentes

a grupos biológicos de transporte de carnitina, montagem e funcionamento da via de fosforilação

oxidativa e do ciclo do TCA e glioxilato. Funcionamento anormal da mitocôndria pode levar à

acumulação de ferro, exacerbando a formação de espécies reactivas de oxigénio pela reacção de

Fenton. Adicionalmente, Tog1 parece activar dois genes envolvidos na resposta ao stress oxidativo:

ALD6 e RCK2, um aldeído desidrogenase, cuja actividade leva à regeneração de NADPH, e uma

proteína cinase com um potencial papel na regulação da translação.

Os resultados obtidos através deste trabalho levam-nos a sugerir que o Tog1 determina a virulência

em C. glabrata conferindo protecção contra o stress oxidativo inerente ao interior de células fagocitárias

como macrófagos.

v

Table of contents

1. Introduction ..................................................................................................................................... 1

1.1. C. glabrata as an opportunistic pathogen ............................................................................... 1

1.2. Mechanisms of Immune System Evasion ............................................................................... 3

1.3. Mechanisms of Response and Resistance to Oxidative Stress .............................................. 5

1.3.1. Antioxidant defenses....................................................................................................... 5

1.3.1. Transcriptional Control of the OSR ................................................................................. 6

1.3.1. Cellular response to oxidative damage caused by hydrogen peroxide ........................... 8

1.4. Mechanisms of Adaptation to Growth in Different Host Niches ............................................ 10

1.4.1. Carbon Metabolic Adaptation to the Host Niche ........................................................... 10

1.4.2. Carbon Metabolism Regulatory Network ...................................................................... 11

1.5. Predicted Role of the Transcription Factor CgTog1 (ORF CAGL0F09229g) ........................ 14

1.5.1. Role of the transcription factor Tog1 in S. cerevisiae .................................................... 14

1.6. Motivation and work outline: The possible role of transcription factor CgTog1 in C. glabrata

virulence ........................................................................................................................................... 15

2. Materials and Methods ................................................................................................................. 16

2.1. Yeast strains and media ....................................................................................................... 16

2.2. Phenotypic analysis .............................................................................................................. 16

2.3. G. mellonella survival assays ............................................................................................... 17

2.4. C. glabrata phagocytosis assays in G. mellonella hemocytes .............................................. 17

2.5. CgTOG1 sub-cellular localization assessment ..................................................................... 17

2.6. RNA isolation ........................................................................................................................ 18

2.7. Quantitative RT-PCR analysis (qRT-PCR) ........................................................................... 18

2.8. RNA-Sequencing .................................................................................................................. 18

2.8.1. Library preparation ........................................................................................................ 18

2.8.2. Gene expression analysis ............................................................................................. 19

2.9. Statistical analysis ................................................................................................................ 19

3. Results ......................................................................................................................................... 20

3.1. Tog1 overexpression does not grant hypervirulence to C. glabrata ...................................... 20

vi

3.2. Tog1 is required for proliferation upon phagocytosis by G. mellonella hemocytes ............... 21

3.3. C. glabrata Tog1 is a determinant of oxidative stress resistance, but is not essential for growth

in non-fermentable carbon-sources .................................................................................................. 22

3.4. Tog1 is constitutively localized in the cell nucleus ................................................................ 25

3.5. Oxidative stress condition induces CTA1 expression ........................................................... 25

3.6. Transcriptomic analysis of the role of Tog1 in C. glabrata response to oxidative stress ....... 27

3.6.1. C. glabrata transcriptional adaptation to oxidative stress: role of Tog1 ......................... 28

3.6.2. The possible role of Tog1 in C. glabrata virulence ........................................................ 35

3.6.3. In silico prediction of Tog1 binding sites ....................................................................... 36

4. Discussion .................................................................................................................................... 38

5. References ................................................................................................................................... 43

6. Supplemental Material .................................................................................................................. 50

vii

List of Figures

Figure 1.1 - Pathways of the OSR in C. glabrata suggested by Brione-Martins-del-Campo et al. 2014:

C. glabrata responds with antioxidant defences against external sources of oxidative stress such as

menadione (MD) or hydrogen peroxide (H2O2). Data suggest that H2O2 activates the transcription factors

Yap1, Skn7 and Msn4. Yap1 response to H2O2 requires Ybp1. Yap1 and Skn7 activate transcription of

the catalase (CTA1), thioredoxin (TRX2), and thioredoxin peroxidase (TSA1). The catalase detoxifies

H2O2 in cytoplasm and peroxisomes. The superoxide dismutase (Sod1) and the glutathione (GSH)

system neutralize the superoxide (O2•−). Sod1 converts O2

− to H2O2. GSH participates in maintaining the

redox balance. The transcription of CTA1, SOD1 and SOD2 is induced by glucose starvation (↓Glc).

SOD1 and SOD2 do not respond to H2O2. Msn2 and Msn4 are involved in the activation of the general

stress response. The discontinuous arrows and dashed-line circles represent not-well established

pathways. ............................................................................................................................................... 7

Figure 1.2 – Cellular response to molecular damage by hydrogen peroxide. Catalases are antioxidant

enzymes capable of detoxify H2O2 into H2O and O2 to prevent ROS generation. H2O2 can be reduced

by iron (Fe2+) in the Fenton reaction to produce the highly reactive hydroxyl radical. The ROS generated

cause lipid peroxidation producing reactive products like lipid hydroperoxides (LOOH) and lipid radicals

(L•), which can form highly reactive aldehydes and react with other lipids and damage the membrane.

The GSH system protects susceptible aminoacids residues, like cysteine and methionine, from being

oxidised by forming thiol reversible bonds. If protein oxidation occurs, an irreversible bond is formed,

and proteins are target do be degraded. ROS can also induce genetic damage through the modification

of DNA. ................................................................................................................................................... 9

Figure 1.3 - Expression of key carbon TFs between S. cerevisiae (Scer), C. glabrata (Cgla) and C.

albicans (Calb). Shown is the expression of selected transcription factors that regulate glucose

metabolism genes (rows) in the Thompson et al. 2013 glucose depletion data set. Figure adapted from

Roy et al. 2015. For each species, the data are from five time points (columns) during growth on glucose:

Lag, Late Log, Diauxic Shift, Post Shift and Plateau (left to right). Genes are matched based on orthology

and clustered. Red: induced; green: repressed; black: no change; white: ortholog absent in species. 11

Figure 1.4 - Schematic overview of C. glabrata infection survival strategies. C. glabrata resides in

phagosomes of macrophages which are not fully matured and acidified. High stress resistance, efficient

nutrient acquisition and blocking of host cell activation contribute to intracellular survival of C. glabrata.

............................................................................................................................................................. 13

Figure 1.5 - CgTOG1 deletion decreases C. glabrata virulence in the G. mellonella infection model. The

survival of larvae injected with 5×107 cells /larvae of Kchr606 wild-type (full line), and ∆cgtog1 deletion

mutant (dashed line). (A) CgTOG1 expression increases C. glabrata proliferation in G. mellonella

viii

hemocytes. The concentration of viable of Kchr606 wild-type (dark grey) and ∆cgtog1 cells (light grey)

(B). These results were obtained by Pedro Pais. ................................................................................. 15

Figure 3.1 - TOG1 overexpression has no effect in C. glabrata virulence in the G. mellonella infection

model. The survival of larvae injected with 5×107 cells/ larvae of L5U1 wild-type strain harbouring the

pGREG576 cloning vector or the pGREG576-MTI-TOG1 expression plasmid, is displayed as Kaplan-

Meier survival curves. The results and are the average of at least three independent experiments. ... 20

Figure 3.2 - TOG1 expression increases C. glabrata proliferation in G. mellonella hemocytes. The

concentration of viable L5U1 wild-type harbouring the pGREG576 cloning vector or the pGREG576-

MTI-TOG1 expression plasmid cells assessed upon 1, 24 and 48 h of co-culture with G. mellonella

hemocytes. The displayed results are relative to the concentration of viable cells initially inoculated in

each well containing a mono-layer of hemocytes and are the average of at least three independent

experiments. ......................................................................................................................................... 21

Figure 3.3 - Tog1 is not essential for growth in non-fermentable carbon-sources. Kchr606 wild-type and

∆cgtog1 cells were spotted on YP plates, containing different carbon sources. The C. glabrata cells with

OD600nm = 0,05 were serially diluted on a reason of 1:5 two times before being spotted. The displayed

images are representative of at least three independent experiments. ................................................ 22

Figure 3.4 - Expression of TOG1 is not essential for C. glabrata cells to grow on YP-Oleate. Growth of

the wildtype Kchr606 or deletion mutant ∆cgtog1 was followed by measuring optic density at 600 nm

during 30 h. Growth curves of Kchr606 in YPD (⚫), ∆cgtog1 in YPD (▲), Kchr606 in YP-Oleate 0,1%

(⚫) and ∆cgtog1 in YP-Oleate 0,1% (▲). ............................................................................................. 22

Figure 3.5 - Tog1 is a determinant of oxidative stress resistance. Kchr606 wild-type and ∆cgtog1 cells

were spotted on MMG plates supplemented with 7.5 or 25 mM H2O2 and 1 mM diamide menadione.

The C. glabrata cells with OD600nm = 0.05 were serially diluted on a reason of 1:5 two times before being

spotted. The displayed images are representative of at least three independent experiments. ........... 23

Figure 3.6 - TOG1 expression affects C. glabrata ability to grow on MMG + 15 mM H2O2 medium. Growth

of the wildtype Kchr606 or deletion mutant ∆cgtog1 was followed by measuring optic density at 600 nm

during 35 h, with an initial OD = 0.1. Growth curves of Kchr606 in MMG (⚫), ∆cgtog1 in MMG (▲),

Kchr606 in MMG + 15 mM H2O2 (⚫) and ∆cgtog1 in MMG + 15 mM (▲). ............................................ 24

Figure 3.7 - S. cerevisiae BY4741 does not display phenotypic differences upon ScTOG1 deletion.

Kchr606 wild-type and ∆cgtog1 cells were spotted on MMG plates supplemented with 7.5 or 25 mM

H2O2 and 1 mM diamide menadione. The C. glabrata cells with OD600nm = 0.05 were serially diluted on

a reason of 1:5 two times before being spotted. The displayed images are representative of at least

three independent experiments. ........................................................................................................... 24

ix

Figure 3.8 - Tog1 is constitutively localized in the cell nucleus. Fluorescence of exponential-phase L5U1

C. glabrata cells harbouring the pGREG576-MTI-TOG1 plasmid, after 5-6 h of copper induction in MMG

(A), YPD (B), YPD + 20 mM H2O2 (C) or YP-Oleate 0,1% (D). ............................................................. 25

Figure 3.9 - Expression of CTA1 in C. glabrata wild-type and ∆tog1 during oxidative stress. Analysis of

gene expression was performed by qRT-PCR from two independent experiments; The comparative Ct

method was used for quantification. Values are expressed as relative expression for CTA1 against CTA1

expression in Kchr606 control, after normalization with the endogenous reference ACT1 gene. ......... 26

Figure 3.10 – Genes down-regulated in ∆cgtog1 strain in control conditions, distributed according to

their biological functions. ...................................................................................................................... 27

Figure 3.11 - Genes up-regulated in ∆cgtog1 strain in control conditions, distributed according to their

biological functions. .............................................................................................................................. 28

Figure 3.12 – Genes down-regulated in hydrogen peroxide stressed ∆cgtog1 cells, distributed according

to their biological functions. .................................................................................................................. 29

Figure 3.13 – Genes up-regulated in hydrogen peroxide stressed ∆cgtog1 cells, distributed according

to their biological functions. .................................................................................................................. 29

Figure 3.14 - KEGG Mapper representation of oxidative phosphorylation. Blue boxes: proteins encoded

by genes down-regulated in ∆cgtog1 vs wild-type strain in H2O2 conditions; Green boxes: other proteins

belonging to C. glabrata proteome; White boxes: proteins that do not exist in C. glabrata. ................ 31

Figure 3.15 - KEGG Mapper representation of the carbon metabolism. Blue arrows: reactions catalysed

by enzymes encoded by genes down-regulated in ∆cgtog1 vs wild-type strain in H2O2 conditions; Green

arrows: other reactions belonging to C. glabrata metabolism; Black arrows: reactions that do not exist

in C. glabrata. ....................................................................................................................................... 32

Figure 3.16 - Schematic representation of the fermentation enzymatic reactions, where the enzymes

encoded by S. cerevisiae ortholog PDC6 (pyruvate decarboxylase) and ADH6 (alcohol dehydrogenase)

were up-regulated, and ALD6 (aldehyde dehydrogenase) was down-regulated in ∆tog1 cells. ........... 33

Figure 3.17 - Venn diagram illustrating the intersection between genes up-regulated in murine and/or

human macrophages and genes found down-regulated in ∆cgtog1 vs wild-type strain in H2O2 or control

conditions. ............................................................................................................................................ 35

Figure 4.1 - Hypothetical model of action of Tog1 in C. glabrata cells during stress response to hydrogen

peroxide: Tog1 activates several genes encoding enzymes involved in carbon metabolism: PYC1 and

CAGL0H06633g (ScPCK1) from gluconeogenesis, CAGL0E01705g (ScMDH2) and CAGL0L09273g

(CaICL1) from the glyoxylate cycle, PYC1, LSC2 and CAGL0E03850g (ScSDH2) from TCA cycle. Sdh2

x

is the responsible for reducing ubiquinone to ubiquinol during electrons transference to the ECT.

Synthesis of several subunits of the OXPHOS process are also controlled by Tog1. Malfunction during

the assembly of this process can lead to a deficient iron homeostasis regulated by Aft2, creating a pool

of Fe2+ which exacerbates the Fenton reaction and generates more ROS caused damage. Fe2+ can be

transported into the cell by Lso1 and afterwards into the mitochondria by Mrs3. The expression of these

proteins is repressed in Tog1 response to H2O2 and are represented in grey. Several proteins (Agp2,

Yat1, Crc1 and Cat2) involved in carnitine transportation of acyl /acetyl groups are also activated by

Tog1. Acetyl molecules can enter directly on the TCA cycle to generate more reducing power in the form

of NADH or succinate. NCE103 encodes a carbonic anhydrase that catalyses CO2 hydration to

bicarbonate, an important metabolic substrate. Rck2 is a MAP kinase-activated protein kinase required

for ribosomes reprogramming during oxidative stress. The activation of ALD6 by Tog1 can help the

cellular NAPDH regeneration to act as a co-factor of antioxidant enzymes. Between the CgTog1

predicted binding sites, GAAGAHGA and CGATGAGM were the most similar to the predicted binding

sites of ScTog1. .................................................................................................................................... 41

xi

List of Tables

Table 1.1 - Results of RT-PCR analyses showing downregulation of S. cerevisiae genes when oleate is

the sole carbon source (Thepnok et al. 2014). ..................................................................................... 14

Table 3.1 - List of genes involved in iron homeostasis which were up-regulated in ∆cgtog1 strain during

oxidative stress caused by H2O2 treatment. *Fold-change relative to differentially gene expression of

∆cgtog1 deletion mutant against wild-type strain exposed to H2O2. ..................................................... 33

Table 3.2 - List of Tog1 activated genes, which are up-regulated in oxidative stress caused by H2O2

conditions. *Fold-change relative to differentially gene expression of wild-type against ∆cgtog1 deletion

mutant exposed to H2O2. ...................................................................................................................... 34

Table 3.3 - Predicted binding sites for C. glabrata Tog1 and the more specific GO terms significantly

associated with these motifs. ................................................................................................................ 37

xii

Abbreviations

CFU Colony Forming Unit

ChIP Chromatin ImmunoPrecipitation

EPA Epithelial adhesion

GFP Green Fluorescence Protein

GO Genome Ontology

GPI Glycosylphosphatidylinositol

GSH Glutathione

ISC Iron-sulfer Cluster

KEGG Kyoto Encyclopaedia of Genes and Genomes

MAP Mitogen-activated protein

MDM Monocyte-derived Macrophages

MMG Minimal Medium with Glucose

OD Optical Density

ORF Open reading frame

OSR Oxidative stress response

PAMP Pathogen-associated pattern

PBS Phosphate-buffer-solution

PCR Polymerase Chain Reaction

PEP Phosphoenolpyruvate

PI3K Phosphatidylinositol 3-kinase

PPR Pattern Recognition Receptor

qRT-PCR Quantitative Real Time Polymerase Chain Reaction

ROS Reactive Oxygen Species

STRE Stress Regulatory Element

TF Transcription factor

YNB Yeast Nitrogen Base

YPD Yeast Extract Peptone Dextrose

YPS Yapsins

1

1. Introduction

1.1. C. glabrata as an opportunistic pathogen

Pathogenic fungi are eukaryotic organisms that cause both endemic and severe infections,

predominantly in immunocompromised and debilitated hosts. In the USA, candidiasis is responsible for

approximately 75% of all systemic fungal infections and is associated with a mortality rate of 40%

(Wilson et al. 2002; Pappas et al. 2018). Candidiasis refers to all infections caused by fungi of the

Candida genus, which can be cutaneous, mucosal and deep-seated organ. Candida species are

commensal yeasts that can be found in the human skin, gut microbiota and oral cavity, and they are

detectable in up to 60% of healthy individuals. However, some of them are able to become pathogenic

in favourable conditions, including mostly: Candida albicans, Candida glabrata, Candida tropicalis,

Candida parapsilosis and Candida krusei (McCarty and Pappas 2016).

Although C. albicans is still the major causative agent of candidiasis, in the past two decades non-

albicans Candida spp. have risen in incidence. (Diekema et al. 2012). Within non-albicans Candida

species, C. glabrata has been associated with longer hospital stays and higher costs, when compared

to C. albicans (Moran et al. 2010), being currently the second major cause of candidiasis worldwide

(Castanheira et al. 2016). Being a secondary pathogen, C. glabrata is able to cause infections in

immunocompromised individuals, those suffering from neutropenia (e.g. cancer or transplant patients),

or patients treated with prolonged antibiotic therapy (Castón-Osorio et al. 2008). The increasing

resistance to commonly used azole antifungals observed for C. glabrata, when compared to C. albicans,

is likely to underlie the emerging infection rate (Diekema et al. 2012). Worryingly, echinocandin

resistance is also developing among Candida spp., particularly in C. glabrata isolates (Pappas et al.

2018).

Despite its genus name, C. glabrata is phylogenetically more closely related to the budding yeast

Saccharomyces cerevisiae and quite distinct from the other pathogenic Candida species. It does not

display many of the typical pathogenesis traits of C. albicans. For instances, while C. albicans is able to

undergo dimorphic growth, switching from growth as yeast cells to growth as hyphae, an important

structure for tissue invasion and immune system evasion, there are only reports of C. glabrata cells

forming pseudohyphae, and only under nitrogen starvation in vitro (Calcagno et al. 2003).

The few of C. glabrata virulence determinants include the ability to adhere to mucosal surfaces and the

expression of cell surface proteases. Adherence of C. glabrata to epithelial surfaces is mediated largely

by members of the EPA (Epithelial adhesion) family, which, like Hwp1 or the Als proteins, are GPI-

anchored cell wall proteins. One of the most study adhesins in C. glabrata is the Ca2+-dependent lectin

Epa1 that recognizes host N-acetyllactosamine-containing glycoconjugates (Cormack 1999). Epa1 also

mediates adhesion by C. glabrata to human macrophage-like cells and, when heterologously expressed

in S. cerevisiae induces inflammatory cytokine production, including IL-8 and TNF-α, in peripheral blood

mononuclear cells-derived macrophages (Kuhn and Vyas 2012). The C. glabrata genome encodes a

2

total of 11 GPI-linked cell surface-associated aspartyl proteases (yapsins) (CgYps1–11) (Kaur et al.

2007). The promoters of CgYPS genes harbour STRE (stress regulatory element) consensus

sequences, suggesting their transcriptional control by the Msn4 transcription factor (Roetzer et al. 2008;

Bairwa and Kaur 2011). The C. glabrata yapsin family has been implicated in survival upon

internalization by murine macrophages, maintenance of the cell wall integrity, adherence to mammalian

cells. By monitoring the processing of Epa1, it is known that yapsins also play a role in cell wall

remodelling by removal and proteolytic processing of GPI-anchored cell wall protein. Furthermore,

growth of a C. glabrata mutant lacking all 11 yapsins is impaired in vitro under stress conditions and

limits virulence in a murine model of systemic infection (Kaur et al. 2007).

Given its limited array of virulence mechanisms, the relatively high prevalence of C. glabrata as a

pathogenic agent has been proposed to be linked to its unusual ability to tolerate and adapt to stress.

This ability may be responsible for high proliferative and colonization capacity exhibited by C. glabrata

within the host. Worth mention is also how more recent reports have been untangling this pathogen’s

stress tolerance to high genomic plasticity. A prime example is the high variability of the already

mentioned EPA family of genes, with different C. glabrata strains presenting different number of genes

present per genome, resulting from gene deletions and duplications (Gabaldón and Fairhead 2018;

López-Fuentes et al. 2018).

In this introduction, current knowledge on the mechanisms of response and adaptation to stress

exhibited by C. glabrata, focusing on stresses relevant for the proliferation of this pathogen in the human

host, is reviewed. These stresses include nutrient starvation and harsh oxidative stress conditions found

inside phagolysosomes (Seider et al. 2011). Additionally, C. glabrata has to cope with other aspects of

the human immune system, including the release of antimicrobial peptides, and with the specific

environmental conditions found in different colonization niches. C. glabrata survival strategies, involving

immune system evasion, the activation of oxidative stress defence mechanisms and switching towards

utilization of alternative carbon sources are evaluated. Finally, the possible role of the uncharacterized

transcription factor CgTog1 in this context is discussed.

3

1.2. Mechanisms of Immune System Evasion

Phagocytic cells of the innate immune system, such as macrophages, dendritic cells, and neutrophils,

are the first line of host defence against fungal infections, acting to eliminate microbial pathogens from

the bloodstream and tissue (Mansour and Levitz 2002). The first contact between a phagocytic cell and

a microbe is mediated by host receptors. These include pattern recognition receptors (PRRs), which

detect conserved basic molecular components of microorganisms, the pathogen-associated patterns

(PAMPs) and opsonic receptors, which recognize opsonized microbes (Gordon 2002).

Once recognized, a microbe is quickly engulfed by the phagocyte and enters the phagocytic pathway.

The phagosome then matures via fusion events with endosomal vesicles, which in turn leads to the

exchange of membrane proteins and finally yields the phagolysosome, an extremely hostile environment

to microbes (Janeway et al. 2005). The organelle harbours destructive and highly toxic mechanisms for

the elimination of ingested microbes. Low pH, hydrolytic enzymes, potent reactive oxygen and nitrogen

species causing damage to DNA, proteins and lipids of the pathogen, are the major antifungal

ingredients of the phagolysosome (Haas 2007). Furthermore, the activation of subsequent innate and

adaptive immune reactions is promoted by antigen presentation and the production of

immunomodulatory proteins such as cytokines (Kasper et al. 2015).

One key aspect of C. glabrata virulence seems to include immune evasion strategies, since during

infection very little inflammation is observed in mouse models (Jacobsen et al. 2010). After

internalization by macrophages, C. glabrata has the capacity of multiply inside these host cells and

remain intracellularly for 2-3 days without provoking damage or induce apoptosis, until they finally lyse

and release the pathogenic cells (Kasper et al. 2015). Additionally, they are capable of inhibiting the

production of reactive oxygen species, reduce levels of proinflammatory cytokines, prevent phagosome

acidification by hindering the recruitment of cathepsin D, and block phagosome maturation at a late

endosomal stage (Seider et al. 2011).

After maturation to the early and late endosomal stage, the phagosome would normally interact with

lysosomes to acquire hydrolytic enzymes and an acid pH. C. glabrata cells prevent fusion with

lysosomes, as phagosomes do not acquire cathepsin D, a lysosomal marker, and are weakly acidify.

The mechanism underlying this phenotype is still unknow. However, observations with heat-killed

yeasts, which inhibit phagosomal acidification, and with yeasts killed with UV light to preserve their cell

surface, which in their turn were still capable of preventing acidification, suggest that a heat-sensitive,

UV light-indestructible surface factor may be responsible for this phenotype. (Seider et al. 2011).

Meanwhile, the authors of Rai et al. demonstrated the impact of phosphatidylinositol 3-kinase (PI3K) in

phagosome modification. Macrophages infected with C. glabrata mutants that lack the two-class III PI3K

subunit-encoding genes VPS15 and VPS34 showed a slightly increased number of acidified

phagosomes. Although the mechanism remains to be known, the authors suggest that PI3K-dependent

cellular trafficking events in C. glabrata may be involved in modification of phagosome maturation (Rai

et al. 2015).

4

Another strategy to escape the host immune system includes covering the cell wall components that

mediate the activation of an immune response. Interestingly, ultrastructure analysis of C. glabrata versus

C. albicans cell walls revealed ~50% more proteins, higher amounts of mannan and lower levels of total

glucan in C. glabrata cell walls (de Groot et al. 2008). The presence of species-specific antigenic

proteins indicates that Candida species promote distinct immune responses. Regarding fungal PAMPs,

it was observed that deletions in C. glabrata affecting the cell wall integrity or modification of the

immunostimulatory cell wall components, like β-1,3-glucan and chitin, caused a stronger inflammatory

response by macrophages (Kaur et al. 2007; Seider et al. 2014).

By screening a library of 433 C. glabrata mutants 23 genes were found to be involved in survival during

phagocytosis by monocyte-derived macrophages (MDMs). This group represented mutations of genes

involved in cell wall biosynthesis, calcium homeostasis, nutritional response, response to stress, protein

glycosylation, and iron homeostasis, which were defective for surviving interactions with MDMs (Seider

et al. 2014).

In addition, none of the mutants lacking a gene with a known function in reactive oxygen species (ROS)

detoxification (e.g., genes encoding catalase, superoxide dismutases, peroxidases or thioredoxin

proteins), which were included in this mutant collection, were found to have reduced survival following

coincubation with MDMs. This suggests that C. glabrata possesses robust and redundant antioxidant

systems and that ROS play a relatively minor role in the killing of this fungus. However, since some of

the 23 mutants with reduced survival in MDMs were more susceptible to H2O2 or menadione in vitro, it

is possible that ROS act in combination with other stresses or microbiocidal mechanisms inside the

phagosome and thus may at least partially contribute to killing of C. glabrata (Kaloriti et al. 2012; Seider

et al. 2014).

5

1.3. Mechanisms of Response and Resistance to Oxidative Stress

Upon interaction with the pathogen, phagocytes rapidly produce ROS through the NADPH oxidase

complex. This multicomponent enzyme carries a redox centre and mediates the transfer of electrons

from cytoplasmic NADPH onto extra cellular and intra phagosomal molecular oxygen, thereby

generating superoxide, hydroxyl anions and hydroxyl radicals (Brown et al. 2009). Deficiencies in the

NADPH oxidase result in increased susceptibility to fungal infection, highlighting the importance of

pathogens’ antioxidant systems in the survival against the oxidative stress generated by host

phagocytes during infection (Missall et al. 2004). Moreover, C. glabrata inhibits phagosome maturation

and suppresses the production of ROS generated by the oxidative burst of phagocytes, ultimately

enabling cell proliferation inside phagosomes (Seider et al. 2011).

1.3.1. Antioxidant defenses

A variety of small antioxidant molecules, such as glutathione (GSH) and thioredoxin, are synthesized to

keep the redox homeostasis (Carmel-Harel and Storz 2000). In addition, the elimination of ROS is also

carried out by several well-characterized enzymes, such as the superoxide dismutases, catalases,

peroxidases, and glutathione peroxidases. These are well-conserved antioxidation mechanisms

displayed by many pathogens to evade phagocyte defences (González-Párraga 2003; Thorpe et al.

2004), suggesting that the production of these enzymes is directly related to virulence (Hwang et al.

2002; Saijo et al. 2010).

Gutiérrez-Escobedo et al. demonstrated how GSH is essential for OSR in C. glabrata, by constructing

GSH biosynthetic pathway mutants, devoid of GSH1 or GSH2 (Gutiérrez-Escobedo et al. 2013). The

mutant strains were sensitive to oxidative stress provoked by hydrogen peroxide (H2O2), menadione and

other peroxide species. The authors also showed that the catalase-independent adaptation response to

H2O2 is not mediated by GSH or thioredoxin, unlike what happens in S. cerevisiae (Izawa et al. 1996).

Previously, Yadav et al. had already confirmed the essential role of GSH1 in C. glabrata and its

importance for virulence in C. albicans (Yadav et al. 2011). Unlike C. albicans, which has six SOD genes

encoding superoxide dismutases (Martchenko et al. 2004), C. glabrata only has two SOD genes and

the encoded CgSod1 is involved in its OSR (Cuéllar-Cruz et al. 2008). In C. glabrata the core response

to oxidative stress also includes thioredoxin peroxidases (CgTsa1, CgTsa2), thioredoxin reductases

(CgTrr1, CgTrr2), the thioredoxin cofactor CgTrx2, the glutathione peroxidase CgGpx2, and the catalase

CgCta1 (Roetzer et al. 2011).

The CgCTA1 gene encodes the single catalase in C. glabrata, which has been shown to localize in the

cytosol and to accumulate in peroxisomes during respiration and inside phagocytic cells (Roetzer et al.

2010). The expression of CTA1 is induced during oxidative stress conditions and carbon source

deprivation (Roetzer et al. 2011). Although this catalase was absolutely required for resistance to H2O2

in vitro, in a mouse model of systemic infection the strain lacking CgCTA1 showed no impact in virulence.

C. glabrata can tolerate exposure to high H2O2 levels, making it more resistant than S. cerevisiae and

6

C. albicans. Surprisingly, catalase activity decreases in Candida spp. after exposure to high

concentrations of H2O2. (Ramírez-Quijas et al. 2015). Although C. glabrata presents the same activity

levels of catalase as other Candida spp, it is capable to resist higher ROS concentrations. (Cuéllar-Cruz

et al. 2009; Ramírez-Quijas et al. 2015). In summary, all these evidences point for additional elements,

yet to be unveiled, that determine C. glabrata increased resistance to oxidative stress.

1.3.1. Transcriptional Control of the OSR

In S. cerevisiae, the induction of the oxidative stress regulon is largely under control of the conserved

transcription factors Yap1 and Skn7 (Lee et al. 1999). Similarly, a genome-wide expression analysis in

C. glabrata revealed that CgYap1 and CgSkn7 control the core response to oxidative stress (Roetzer et

al. 2011). Although ∆cgyap1 mutant cells display higher susceptibility to hydrogen peroxide, in C.

glabrata, when compared to S. cerevisiae, the expression of superoxide dismutases is not regulated by

CgYap1, being dependent on carbon source. While CgSod1 is cytoplasmic and is the major contributor

to total SOD activity, CgSod2 is a mitochondrial protein and is required for growth in non-fermentable

carbon sources (Briones-Martin-del-Campo et al. 2015). Nonetheless, the combined loss of CgYAP1

and CgSOD1, reduced the survival of the yeast during macrophage phagocytosis (Roetzer et al. 2011).

The role of CgSkn7 in peroxide stress protection includes the induction of CgTRX2, CgTRR1, CgTSA1

and CgCTA1. Furthermore, the deletion of CgSKN7 leads to virulence attenuation in C. glabrata (Saijo

et al. 2010).

The S. cerevisiae transcriptions factors Msn2 and Msn4 are the principal factors for the activation of the

general stress response during carbon source starvation, heat shock and severe osmotic and oxidative

stresses (Martínez-Pastor et al. 1996). Although in C. albicans the two homologs of these transcription

factors seem to have no effective role in stress response (Nicholls et al. 2004), in C. glabrata the CgMsn2

and CgMsn4 appeared to work independently of each other. While both were needed for stationary

phase cells oxidative stress resistance, only CgMsn4 was required for log-phase cells resistance, along

with CgSkn7 and CgYap1, and required at least the activation and induction of the catalase gene

(Cuéllar-Cruz et al. 2008).

C. glabrata adaptation to H2O2 is completely abolished in ∆cgskn7 and ∆cgyap1 mutants (Fassler and

West 2011). A schematic representation of the possible pathways of OSR in C. glabrata is demonstrated

in Figure 1.1. (Briones-Martin-Del-Campo et al. 2014).

7

Figure 1.1 - Pathways of the OSR in C. glabrata suggested by Brione-Martins-del-Campo et al. 2014: C. glabrata

responds with antioxidant defences against external sources of oxidative stress such as menadione (MD) or

hydrogen peroxide (H2O2). Data suggest that H2O2 activates the transcription factors Yap1, Skn7 and Msn4. Yap1

response to H2O2 requires Ybp1. Yap1 and Skn7 activate transcription of the catalase (CTA1), thioredoxin (TRX2),

and thioredoxin peroxidase (TSA1). The catalase detoxifies H2O2 in cytoplasm and peroxisomes. The superoxide

dismutase (Sod1) and the glutathione (GSH) system neutralize the superoxide (O2•−). Sod1 converts O2

− to H2O2.

GSH participates in maintaining the redox balance. The transcription of CTA1, SOD1 and SOD2 is induced by

glucose starvation (↓Glc). SOD1 and SOD2 do not respond to H2O2. Msn2 and Msn4 are involved in the activation

of the general stress response. The discontinuous arrows and dashed-line circles represent not-well established

pathways.

Vacuolar H+-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular

compartments but also plays an important role in OSR. The V-ATPase activity has been associated with

virulence in C. neoformans and C. albicans (Erickson et al. 2001; Poltermann et al. 2005). During the

adaptation of C. glabrata cells to oxidative stresses, the V-ATPase indirectly controls CgCTA1 and

CgSOD2 expression levels (Nishikawa et al. 2016).

More recently, a new virulence factor, Ada2, was shown to have a role in OSR. When using Drosophila

larvae as an oral infection model, the deletion strain of ∆cgada2 was not able to activate the Toll

pathway, an immune response that occurs in the presence of a fungal infection. This transcription factor

showed susceptibility to oxidative stress caused by hydrogen peroxide or menadione. The survival of

larvae with suppressed ROS production was higher when infected with Δcgada2 compared to wild-type

(Kounatidis et al. 2018).

8

Interestingly, in the same year Yu et al. reporter that the deletion mutant ∆cgada2 exhibited susceptibility

to three classes of antifungal drugs (i.e., azoles, echinocandins, and polyenes) as well as cell-wall

perturbing agents, and it was hyper-virulent in a mouse model of systemic infection (Yu et al. 2018).

These differences could be explained by the different infection model approaches. Also, genome-wide

mapping in C. albicans of CaAda2 have demonstrated that this coactivator is recruited to 200 promoters

upstream of genes involved in different stress-response functions, as oxidative stress and drug

response, and metabolic processes, as the glycolytic pathway (Sellam et al. 2009). This may suggest

that Ada2 has a very broad function and is likely to be also involved in the up-regulation of several genes

in C. glabrata.

1.3.1. Cellular response to oxidative damage caused by hydrogen peroxide

Hydrogen peroxide is widely used as a model for oxidative stress condition, mainly due to its convenient

use since its water soluble and presents relative stability. It can damage cells by promoting protein and

DNA oxidation, and lipid peroxidation (Figure 1.2). Antioxidant enzymes, including catalases and

peroxides, must detoxify hydrogen peroxide to avoid Fenton reaction, which generates highly reactive

hydroxyl radicals (Morano et al. 2012).

Cysteine and methionine are the most oxidation-susceptible amino acids. Hydrogen peroxide decreases

the GSH/GSSG ratio and increases the levels of protein mixed disulphide between cysteine residues

and low molecular weight thiols such as glutathione (Grant et al. 1998). This process is called protein

S-thiolation and prevents the irreversible oxidation of cysteine residues. A major target of this process

is the glyceraldehyde-3-phosphate dehydrogenase (GPDH), encoded by TDH3, but also includes other

glycolytic enzymes. Blocking glycolysis during oxidative stress could provide a powerful means of

controlling cellular metabolism, since it could be translated in an increased flux of glucose equivalents

through the pentose phosphate pathway leading to the generation of NADPH, the reducing power of

antioxidant enzymes (Shenton and Grant 2003).

The initial phase of protein synthesis is a major regulatory step and represents a checkpoint for

eukaryotic gene expression. Indeed, initiation of translation is inhibited in Candida spp. in response to

H2O2 (Kaur et al. 2007; Sundaram and Grant 2014).

Lipid peroxidation is probably dependent on the fraction of lipids present in cell membranes. For

instances, increase of polyunsaturated fatty acids in membrane phospholipids leads to accumulation of

lipid hydroperoxides (LOOH), which decrease GSH levels and when fragmentated leads to highly

reactive aldehyde. These products, with a higher half-life than ROS, can cause extensive damage to

the cell. Imaging techniques have been used to show Candida cells after exposure to oxidative stress

undergoing morphological changes in its cell wall and decrease in size (Ramírez-Quijas et al. 2015).

DNA is also a target for oxidative damage after exposure to H2O2. As histones are closely associated

with DNA in the nucleus, and are known to bind copper ions that can catalyse hydroperoxide

decomposition to radicals, these reactions may contribute to DNA–protein cross-links in damaged cells

9

(Davies 2016). Among DNA oxidative damages, base modification has high impact due to its lethal or

mutagenic effect and oxidised bases have to be replaced by an enzymatic system (Moradas-Ferreira

and Costa 2000).

Figure 1.2 – Cellular response to molecular damage by hydrogen peroxide. Catalases are antioxidant enzymes

capable of detoxify H2O2 into H2O and O2 to prevent ROS generation. H2O2 can be reduced by iron (Fe2+) in the

Fenton reaction to produce the highly reactive hydroxyl radical. The ROS generated cause lipid peroxidation

producing reactive products like lipid hydroperoxides (LOOH) and lipid radicals (L•), which can form highly reactive

aldehydes and react with other lipids and damage the membrane. The GSH system protects susceptible aminoacids

residues, like cysteine and methionine, from being oxidised by forming thiol reversible bonds. If protein oxidation

occurs, an irreversible bond is formed, and proteins are target do be degraded. ROS can also induce genetic

damage through the modification of DNA.

10

1.4. Mechanisms of Adaptation to Growth in Different Host Niches

Environmental adaptation is essential for pathogenic organisms, like Candida spp, so they can cope

with the host defence mechanisms and survive in the different microenvironments of the host. Metabolic

adaptation, specially to the available carbon sources at specific niches during commensalism phase and

disease progression is tough to contribute to the pathogenicity of C. albicans and C. glabrata.

Various human anatomical sites are found to have different level of nutrients. Some niches supply low

concentrations of glucose: vaginal secretion and blood are found to contain 0.05-0.1% and

approximately 0.1% glucose, respectively (Ehrström et al. 2006). While others provide transient

exposure to fermentable sugars (e.g. oral cavity and gastrointestinal tract) or eventually complete lack

of glucose (e.g. skin and nails) (Ene et al. 2012).

1.4.1. Carbon Metabolic Adaptation to the Host Niche

Lorenz et al. observed that upon phagocytosis of S. cerevisiae by macrophages, yeast cells induce

glyoxylate cycle associated genes, including ScICL1, ScMLS1 and ScMDH2 (Lorenz and Fink 2001,

2002). Genes in the autophagy and pexophagy pathways were also upregulated, likely providing

mechanisms for sequestering resources in a nutrient-deprived environment. Evidence of carbohydrate

depletion has been found in many systems of fungi incubated with neutrophils or macrophages: the

glyoxylate cycle, utilized during glucose deprivation, was shown to be essential for virulence in C.

albicans (Fukuda et al. 2013). In a mouse infection model, CaFOX2, the second enzyme of the β-

oxidation pathway, and CaICL1, from the glyoxylate cycle, were found to be required for C. albicans

virulence and for growth on oleic acid, a non-fermentable carbon source (Piekarska et al. 2006).

In C. glabrata response to nutrient-limiting macrophage internal milieu, occurs a wholesale shift in

carbon metabolism involving upregulation of gluconeogenesis, β-oxidation of fatty acids and glyoxylate

cycle (Kaur et al. 2007; Rai et al. 2012). Rai et al. demonstrated by C. glabrata transcript profiling that

cells used mainly fatty acids as energy source upon internalization by macrophages. The acetyl-CoA

generated during fatty acid oxidation enters the glyoxylate cycle to generate energy and intermediates

for synthesis of cellular building blocks (Rai et al. 2012). β-oxidation of odd-chain-length fatty acids also

yields propionyl-CoA as an additional product. Genes of the methylcitrate cycle, which mediate the

oxidation of propionyl-CoA to pyruvate, were also found upregulated in phagocytosed cells (Kaur et al.

2007).

When facing glucose starvation in the phagosome, C. glabrata cells use pexophagy, a specialized form

of autophagy. In a first stage, macrophage phagocytosis induces an increase in peroxisome numbers

in C. glabrata cells, likely as a strategy to switch to alternative carbon source utilization. In later stages

of phagocytosis, peroxisomes are degraded via pexophagy. Correspondingly, deletion of genes required

for pexophagy, CgATG11 and CgATG17, caused a reduction in macrophage survival (Roetzer et al.

2010).

11

The type of the carbon source of the host milieu also modulates fungal virulence. Major changes in the

biophysical and mechanical properties of the cell wall were observed when C. albicans cells grown on

lactate. The cells possessed thinner and less elastic cell walls, when compared to glucose-grown cells

(Ene et al. 2012). The available carbon source probably affects cell wall biogenesis by impacting directly

upon metabolic fluxes as well as indirectly through regulatory networks (Askew et al. 2009). Thus, by

using nonfermentable carbon sources to grow, the cell must generate by gluconeogenesis, an energy

demanding pathway, the hexose phosphates required for cell wall biosynthesis. Less carbon is probably

committed to cell wall biosynthesis, leading to the construction of a leaner but stiffer cell wall. (Ene et al.

2012) More recently, Ballou et al. showed how lactate, by inducing the masking of a major PAMP, β-

glucan, increased C. albicans ability to evade the immune system and to reduce phagocytosis, cytokine

release and neutrophil recruitment (Ballou et al. 2016). The gastro intestinal tract is highly enriched in

lactate due to its production by the local flora. This tricarboxylic acid is essential for C. glabrata to thrive

in gastro intestinal infection, in the dependency of the CgCyb2 lactate dehydrogenase, which is

responsible for lactate assimilation (Ueno et al. 2011).

1.4.2. Carbon Metabolism Regulatory Network

C. glabrata is a Crabtree-positive yeast, just like S. cerevisiae. This means it displays a respiro-

fermentative life style defined by a preference to ferment glucose even in the presence of oxygen, in

contrast to the Candida clade spp, which are respiratory yeasts and use oxidative phosphorylation in

the presence of glucose (Hagman et al. 2013).

Figure 1.3 - Expression of key carbon TFs between S. cerevisiae (Scer), C. glabrata (Cgla) and C. albicans (Calb). Shown is the

expression of selected transcription factors that regulate glucose metabolism genes (rows) in the Thompson et al. 2013 glucose

depletion data set. Figure adapted from Roy et al. 2015. For each species, the data are from five time points (columns) during

growth on glucose: Lag, Late Log, Diauxic Shift, Post Shift and Plateau (left to right). Genes are matched based on orthology and

clustered. Red: induced; green: repressed; black: no change; white: ortholog absent in species.

Still, being a pathogen, C. glabrata behavior has some disparities when compared to S. cerevisiae. The

carbohydrate sources that C. glabrata can use are mostly reduced to glucose and trehalose. As C.

12

glabrata genome does not contain the genes for galactose, lactose, sucrose, raffinose and maltose

assimilation, it is not able to grow on these carbon sources (Kurtzman et al. 2011).

C. glabrata retained duplicate genes encoding enzymes for 6 of the 10 glycolytic reactions that have

been proposed to contribute to the increased flux through this pathway (Conant and Wolfe 2007).

Accordingly, the number of glucose transporters is an important feature to an efficient glycolytic flux and

C. glabrata shares 11 HXT orthologues with S. cerevisiae (Palma et al. 2009). In paralog pairs, the

expression of one paralog is repressed when glucose is depleted from the medium, while the expression

of the other paralog is induced. The induced paralogs acquired this new regulation (similar to the

respiratory genes) partly by becoming targets of the glucose repression regulator Mig1 (Thompson et

al. 2013). In Figure 1.3 is possible to observe the differentiated expression of key carbon S. cerevisiae

transcription factors and their orthologs in C. glabrata and C. albicans during the different growth phases

on glucose, a reflection of how these two pathogens remodulated their carbon metabolism regulation to

adapt to host environment.

However, it is still unclear if the respiro-fermentative lifestyle is an advantage for human colonization

and pathogenesis in C. glabrata, given that the Candida clade yeasts are respiratory. As discussed

above, C. glabrata and C. albicans suffer a wide transcriptional change of carbon metabolism during

adaptation to the host environment (Lorenz et al. 2004; Kaur et al. 2007; Rai et al. 2012; Fukuda et al.

2013). The commensal Nakaseomyces, such as C. glabrata, may have increased glycolytic flux, so they

can compete with host cells for available glucose. Also, in glucose-limiting conditions, an efficiently

generation of glucose by gluconeogenesis may underlie the resistance to starvation. However, an

efficient respiratory pathway that produces more energy for rapid growth may be preferable depending

on the host niche (Roy and Thompson 2015).

Chromatin remodelling processes are also suggested to play a central role in reprograming the cellular

energy metabolism inside the phagosome. Chromatin modification impacts gene expression and

mutants of C. glabrata defective in chromatin organization display decreased macrophage survival.

Additionally, internalized wild-type C. glabrata cells showed a differentially modified chromatin and

elevated cellular lysine deacetylase activity, as compared to non-phagocytosed cells (Rai et al. 2012).

When comparing the transcriptional profiles of macrophage-internalized wild-type and the two chromatin

regulator mutant strains of C. glabrata, hundreds of differentially regulated genes were identified. In

contrast to wild-type cells, the functions of the genes induced in both mutant strains were involved in the

generation of carbon metabolite precursors, energy cellular respiration, respiratory electron transport

chain and cellular amino acid metabolic processes, underscoring a prominent role of carbon metabolism

when it comes to host interactions (Rai et al. 2012).

While C. glabrata carbon assimilation mechanism and their regulation remain unclear, in C. albicans,

genome-wide comparisons suggest that it displays some common features of carbon assimilation to S.

cerevisiae. But some major transcriptional rewire of its regulation have occurred, as C. albicans has the

capacity of utilizing diverse carbon sources simultaneously, while S. cerevisiae is not able to do so. In

C. albicans, the enzymes required for carboxylic and fatty acid catabolism are not degraded when

13

glucose is added to the growth medium. This occurs due to the lack of ubiquitination sites in the

gluconeogenic and glyoxylate cycle enzymes of C. albicans, which are present in S. cerevisiae

homologs. Inside the host, this trait allows C. albicans to use alternative carbon sources even if glucose

is present in the infection environment (Sandai et al. 2012). It would be interesting to observe if this trait

its exclusive to C. albicans or if it is shared with C. glabrata.

The phylogenetical proximity between C. glabrata and the model yeast S. cerevisiae assisted the

progression of research on genomic comparisons to characterize this pathogen’s genome and

metabolism. Still, to fully understand its unique virulence features, on-going research should include

characterization of sequential clinical isolates that include transcriptomic and proteomic analysis and

correlation with clinical treatment (López-Fuentes et al. 2018). A schematic overview of C. glabrata

strategies to survive and adapt to host immune response is represented in (Figure 1.4).

Figure 1.4 - Schematic overview of C. glabrata infection survival strategies. C. glabrata resides in phagosomes of

macrophages which are not fully matured and acidified. High stress resistance, efficient nutrient acquisition and

blocking of host cell activation contribute to intracellular survival of C. glabrata.

14

1.5. Predicted Role of the Transcription Factor CgTog1 (ORF CAGL0F09229g)

1.5.1. Role of the transcription factor Tog1 in S. cerevisiae

The S. cerevisiae transcription factor Tog1 is a zinc cluster protein, belonging to the Zn(II)2Cys6 sub-

family. It was first characterized as required for growth on non-fermentable carbon sources, glycerol and

lactate. Its deletion also resulted in hypersensitivity to calcofluor-white, which targets chitin, suggesting

an additional role in cell wall integrity (Akache 2001). TOG1 deletion was further found to display

defective growth on fatty acids as carbon sources, namely oleic, linoleic and palmitic acids, and to

sensitize cells to oxidative stress agents, such H2O2, diamide and menadione (Thepnok et al. 2014).

Tog1 was found to control the expression of key genes involved in this fatty acid utilization and in

oxidative stress response (Thepnok et al. 2014). Among the Tog1-regulated genes are those encoding

oleate utilizing enzymes during fatty acid β-oxidation (Table 1). These genes include POX1, FOX2 and

POT1, but also CTA1 and IDP2, whose expression is essential to cellular adaptation to the oxidative

stress generated during the fatty acid breakdown (Thepnok et al. 2014).

Expression of two genes of the glyoxylate cycle, MLS1 and ICL1, and two genes involved in carnitine

and acetyl-coA transport, YAT2 and AGP2, was also found to be under the control of Tog1 (Thepnok et

al. 2014).

Table 1.1 - Results of RT-PCR analyses showing downregulation of S. cerevisiae genes when oleate is the sole

carbon source (Thepnok et al. 2014).

Genes with decreased expression in Saccharomyces cerevisiae upon TOG1 deletion

Oleate utilizing genes in the β-Oxidation POX1, FOX2 and POT1

Cellular adaptation to oxidative stress generated during the fatty acid breakdown

CTA1 and IPD2

Glyoxylate pathway MLS1 and ICL1

Acylcarnitine transferase system YAT2

Carnitine uptake AGP2

Gluconeogenic pathway PCK1 and FBP1

15

1.6. Motivation and work outline: The possible role of transcription factor CgTog1 in C.

glabrata virulence

The role of the predicted C. glabrata CgTog1 transcription factor, encoded by ORF CAGL0F09229g,

had not been studied before this project. Preliminary results from our group suggested that TOG1 is

required for full virulence in C. glabrata, but the underlying mechanisms were not clear. The deletion of

CgTOG1 was found to decrease C. glabrata ability to kill Galleria mellonella larvae and to proliferate

inside its hemocytes (Figure 1.5). Indications so far suggest that adaptation to non-fermentable carbon

sources and oxidative stress resistance appear to be key factors in the response to phagocytosis.

Figure 1.5 - CgTOG1 deletion decreases C. glabrata virulence in the G. mellonella infection model. The survival of larvae injected

with 5×107 cells /larvae of Kchr606 wild-type (full line), and ∆cgtog1 deletion mutant (dashed line). (A) CgTOG1 expression

increases C. glabrata proliferation in G. mellonella hemocytes. The concentration of viable of Kchr606 wild-type (dark grey) and

∆cgtog1 cells (light grey) (B). These results were obtained by Pedro Pais.

Following the identification of the transcription factor CgTog1 as a determinant of C. glabrata virulence,

its role and mode of action in killing the infection model G. mellonella and surviving within macrophages

was analysed. The sub-cellular localization of CgTog1 was assessed based on the expression of a GFP

fusion protein, to try to identify the conditions that would lead to transcription factor activation, through

nuclear accumulation. Given that the predicted physiological role of Tog1 is in the control of oleate

metabolism, the link between this biological function and C. glabrata proliferation in host niches depleted

for glucose as carbon source was explored. Considering the role of Tog1 in S. cerevisiae OSR and its

importance to survival of C. glabrata during infection of the host, the role of CgTog1 in OSR was

evaluated. After determining which conditions led to CgTog1 activation, RNA sequencing was performed

to find the key target genes and biological processes that underlie the observed phenotype.

B

16

2. Materials and Methods

2.1. Yeast strains and media

The S. cerevisiae BY4741 strain (MATa, ∆ura3, ∆leu2, ∆his3, ∆met15) and the derived single deletion

mutants ∆sctog1 were obtained from the Euroscarf collection. These strains were used in phenotypic

complementation analysis by transformation with pGREG576 (Jansen et al. 2005) or pGREG576-TOG1.

The GAL1 promoter was induced supplementing 1% of galactose to minimal medium (MM) with 0,1%

glucose used for cell growth.

The wild-type C. glabrata strain Kchr606 and the derived single deletion mutant ∆cgtog1 was kindly

provided by Hiroji Chibana, Chiba University, Chiba, Japan. These strains were used for phenotypic

analysis, growth curves, RT-PCR and RNA-sequencing.

The wild-type C. glabrata strain L5U1 (ura3∆0; leu2∆0) was kindly provided by John Bennett, of the

National Institute of Allergy and Infectious Diseases, NIH, Bethesda, USA. This strain was transformed

with pGREG576 or pGREG576-MTI-TOG1 (copper-induced MTI promoter) and was used for survival

and phagocytosis assays in Galleria mellonella model and hemocytes, respectively, and for CgTOG1

sub-cellular localization. For the induction of the copper-induced MTI promoter, a concentration of

CuSO4 100µM was supplemented to the medium during cell growth in MMG.

Yeast cells were cultivated in rich yeast extract-peptone-dextrose (YPD) medium or a minimal medium

with glucose (MMG). The YPD medium contained 2% glucose (Merck), 2% bacto-peptone (Dickson)

and 1% yeast extract. The MMG contained 2% glucose, 0.27% ammonium sulphate (Merck), 0.7% yeast

nitrogen base without amino acids and ammonium sulphate (Difco). For growing L5U1 C. glabrata

transformed strains the MMG medium was supplemented with 60 mg/L of leucine, and for BY4741 S.

cerevisiae transformed strain with 60 mg/L of leucine, 20 mg/L of methionine and 20 mg/L histidine.

The yeast extract-peptone (YP) or MM plates used for the phenotype analysis were supplemented with

different carbon sources: either 2% glucose, 0.75% lactate, 1% glycerol and 0.1% oleic acid + 0.5%

Tween 80. To assess oxidative stress, menadione (1mM) or hydrogen peroxide (4, 7.5 or 25mM) were

added to the YPD or MMG medium before platting.

2.2. Phenotypic analysis

Yeast cells were grown until mid-exponential phase, collected by centrifugation, washed with sterilized-

deionized water twice and resuspended for two serially dilutions of 1:5, starting with an initial OD600nm =

0.05. Afterwards they were spotted with the three different concentrations on appropriate plates

containing different carbon sources or oxidative stress agents. Plates were then incubated at 30ºC.

17

The ability of C. glabrata Kchr606 wild-type and the deletion mutant ∆cgtog1 to grown on oleate was

additionally evaluated by cultivation in liquid growth medium YP with 0.1% of oleate and YPD as control.

The grow on the presence of peroxide hydrogen was assessed in MMG with 15 mM H2O2 and MMG as

control. Cells were grown until mid-exponential phase, filtered and washed with sterilized-deionized

water and inoculated with an initial OD600nm = 0.05 or 0.1 in triplicates. Cell growth was followed until

reaching stationary phase at 30ºC with 250 rpm agitation.

2.3. Galleria mellonella survival assays

G. mellonella larvae were on a pollen grain diet at 25ºC in darkness. Larvae weighting 250 ± 25 mg

were used in the killing assays and the larvae infection was based on the protocol previously described

(Cotter et al. 2000; Mil-Homens and Fialho 2012; Santos et al. 2017). C. glabrata strains were cultivated

in YPD until stationary phase, at 30ºC with 250 rpm agitation, harvested by centrifugation and

resuspended in phosphate buffer solution (PBS) (pH 7.4). 3.5 μL of yeast cell suspension, containing

~5 × 107 cells, were injected into each caterpillar via the last left proleg. For each condition, 10 larvae

were used to follow the larval survival over a period of 72 h. Control larvae were injected with PBS (pH

7,4). The obtained results are the average of at least three independent experiments. Larvae injections

and hemocytes recovering were performed by Andreia Pimenta and Dalila Mil-Homens, under the

supervision of Prof. Arsénio Fialho, BSRG.

2.4. C. glabrata phagocytosis assays in G. mellonella hemocytes

G. mellonella hemocytes were isolated as described before (Brivio et al. 2010). Hemocytes were

suspended in Grace insect medium (GIM) (Merck) supplemented with 10% fetal bovine serum, 1%

glutamine, and 1% antibiotic (10,000 U penicillin G, 10 mg streptomycin). Cultures of C. glabrata cells

were grown until mid-exponential phase and the appropriate volume was collected to have 1,45 × 1010

cells/ mL in PBS. Galleria hemocyte monolayer medium was replaced with GIM without antimycotics,

and then cells were infected with the yeast suspensions with a final concentration of 2,0 x 105 cells/mL

in each well. After 1 h of infection at 37°C, the hemocytes were carefully washed twice with PBS and

replaced with fresh GIM medium. Viable intracellular yeast cells were measured after 1, 24 and 48 h of

infection, upon hemocyte lysis with 0.5% Triton X-100.

2.5. TOG1 sub-cellular localization assessment

The sub-cellular localization of the CgTog1 protein was determined based on the observation of L5U1

C. glabrata cells transformed with the pGREG576-MTI-TOG1 plasmid. C. glabrata cell suspensions

were grow in MMG medium overnight and then transferred to a new medium supplemented with 100

μM CuSO4, to induce protein over-expression, with an initial OD600nm = 0.03. After 6 h of incubation, for

cells duplicate at least two times, the distribution of Tog1_GFP fusion protein in C. glabrata cells were

detected by fluorescence microscopy.

18

2.6. RNA isolation

Candida cells were grown with an initial OD600nm = 0.1, during 1 h with the cells exposed to 15 mM of

H2O2 in MMG medium. The cells were then collected by centrifugation (10 min, 7000 rpm) in a Beckman

J2-MC centrifuge and the pellet was resuspended and collected in Eppendorf tubes to be stored at -

80ºC. The RNA isolation was achieved using the RiboPure™ Yeast RNA Purification Kit (Invitrogen™)

according to the manufacturer’s instructions. Two replicates of each sample were obtained from three

independent experiments, subsequently pooled together for total RNA extraction.

2.7. Quantitative RT-PCR analysis (qRT-PCR)

The isolated RNA was converted to cDNA by a step of reverse transcription PCR with TaqMan® Reverse

Transcription Reagents (Invitrogen™). The following SYBR® Green-based quantitative PCR was

performed using NZY qPCR Green Master Mix (2x), ROX (NZYTECH™). DNA sequences of the

oligonucleotides used for qRT-PCR of CTA1 gene are: Forward 5’-ATGCTCACCGTTACAGATTGG-3’

and Reverse 5’-TGTTGGAAGCGTAGTAGTTTGG-3’. qRT-PCR was performed using a 7500 RT-PCR

thermocycler block (Applied Biosystems) with the software 7500 Systems SDS Software (Applied

Biosystems). The relative quantification of each transcript was calculated by the 2∆∆𝐶𝑡 method, using the

ACT1 gene as normalizer.

2.8. RNA-Sequencing

2.8.1. Library preparation

Prior to RNA-seq analysis quality control measures was implemented. Concentration of RNA was

ascertained via fluorometric analysis on a Thermo Fisher Qubit fluorometer. Overall quality of RNA was

verified using an Agilent Tapestation instrument. Following initial QC steps sequencing libraries were

generated using the Illumina Truseq Stranded Total RNA library prep kit with ribosomal depletion via

RiboZero Gold according to the manufacturer’s protocol. Briefly, ribosomal RNA was depleted via pull

down with bead-bound ribosomal-RNA complementary oligomers. The RNA molecules were then

chemically fragmented, and the first strand of cDNA was generated using random primers. Following

RNase digestion, the second strand of cDNA was generated replacing dTTP in the reaction mix with

dUTP. Double stranded cDNA then underwent adenylation of 3' ends following ligation of Illumina-

specific adapter sequences. Subsequent PCR enrichment of ligated products further selected for those

strands not incorporating dUTP, leading to strand-specific sequencing libraries. Final libraries for each

sample were assayed on the Agilent Tapestation for appropriate size and quantity. These libraries were

then pooled in equimolar amounts as ascertained via fluorometric analyses. Final pools were absolutely

quantified using qPCR on a Roche LightCycler 480 instrument with Kapa Biosystems Illumina Library

Quantification reagents.

19

2.8.2. Gene expression analysis

Strand specific RNA-seq library preparation and sequencing was carried out as a paid service by the

NGS core from Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA. Paired-end

reads (Illumina HiSeq 3000 PE150, 2x150 bp, 2 Gb clean data) were obtained from wild type (Candida

glabrata Kchr606) and correspondent deletion mutant strain (ORF CAGL0F09229g). Sequencing reads

were trimmed using Skewer (Jiang et al. 2014) and aligned to the C. glabrata CBS138 reference

genome, obtained from the Candida Genome Database (CGD) (http://www.Candidagenome.org/), using

TopHat (Trapnell et al. 2009). HTSeq (Anders and Huber 2010) was used to count mapped reads per

gene. Differentially expressed genes were identified using DESeq2 (Love et al. 2014) with an adjusted

p-value threshold of 0.05 and -0.5 ≤ log2 fold change ≤ 0.5. Default parameters in DESeq2 were used.

Significantly differentially expressed genes were clustered using hierarchical clustering in R (Gentleman

et al. 2004). Candida albicans and Saccharomyces cerevisiae homologs were obtained from the

Candida Genome Database (CGD) (Skrzypek et al. 2017) and Saccharomyces Genome Database

(SGD) (Cherry et al. 2012), respectively.

2.9. Statistical analysis

Data from results determined in this work were analysed with a one-way ANOVA test followed by

multiple comparisons test (α=0.05, *: p<0.05, **: p<0.01, ***: p<0.001). The statistical analysis was

performed with GraphPad Prism (Graphpad Software, Inc. USA).

20

3. Results

3.1. Tog1 overexpression does not grant hypervirulence to C. glabrata

As the preliminary results demonstrated the reduced virulence of C. glabrata upon TOG1 deletion, the

effect of its overexpression was evaluated in the same infection host model, the G. mellonella larvae.

Following infection with C. glabrata cells harbouring the empty cloning vector (pGREG576) or the TOG1

expression plasmid (pGREG576-MTI-TOG1), larvae quickly developed a brown-black colouration,

indicative of the accumulation of melanin as part of the insect innate immune response (Ames et al.

2017). The survival of G. mellonella larvae was followed until 72h after infection with 5 x 107 cells/larvae

(Figure 3.1).

Figure 3.1 - TOG1 overexpression has no effect in C. glabrata virulence in the G. mellonella infection model. The

survival of larvae injected with 5×107 cells/ larvae of L5U1 wild-type strain harbouring the pGREG576 cloning vector

or the pGREG576-MTI-TOG1 expression plasmid, is displayed as Kaplan-Meier survival curves. The results and

are the average of at least three independent experiments.

Unexpectedly, the overexpression of TOG1 in L5U1 C. glabrata strain did not seem to alter its virulence,

since survival rates were similar between G. mellonella larvae infected with the Candida cells harbouring

the cloning vector pGREG576 or the expression plasmid pGREG576-MTI-TOG1 (Figure 3.1). The effect

of different cell concentration, CuSO4 concentration to induce protein over-expression, and media (YPD

and MMG) was assessed, always reaching the same conclusion: Tog1 overexpression has no effect in

C. glabrata virulence in G. mellonella larvae.

21

3.2. Tog1 is required for proliferation upon phagocytosis by G. mellonella hemocytes

One key characteristic of C. glabrata virulence is its ability to proliferate inside macrophages.

Considering the possible role of Tog1 in C. glabrata virulence and already knowing that its deletion

decreases proliferation when Candida cells are engulfed by hemocytes, the cell proliferation ability of

cells overexpressing TOG1 was evaluated by coincubation with G. mellonella hemocytes. The number

of viable cells internalized within hemocytes was accounted by assessing the colony forming units (CFU)

at 1, 24 and 48 h of internalization by hemocytes. The proliferation rate is given by the relative values

between the CFU/mL determined for each incubation time point and the CFU/mL initially placed in each

well containing a mono-layer of hemocytes.

Figure 3.2 - TOG1 expression increases C. glabrata proliferation in G. mellonella hemocytes. The concentration of

viable L5U1 wild-type harbouring the pGREG576 cloning vector or the pGREG576-MTI-TOG1 expression plasmid

cells assessed upon 1, 24 and 48 h of co-culture with G. mellonella hemocytes. The displayed results are relative

to the concentration of viable cells initially inoculated in each well containing a mono-layer of hemocytes and are

the average of at least three independent experiments.

The differences in proliferation ability upon phagocytosis were only clear after 48 h of incubation with

hemocytes. The L5U1 C. glabrata cells harbouring the plasmid pGREG576-MTI-TOG1 were capable of

proliferating within hemocytes 36% more than cells only containing the cloning vector (Figure 3.2).

Reflecting on these two results, Tog1 appears to have a role on the cell adaptation to the intracellular

milieu of phagocytic cells allowing Candida cells to proliferate effectively inside hemocytes in vitro,

although this capacity was not enough per se to increase G. mellonella larvae mortality upon C. glabrata

infection.

22

3.3. C. glabrata Tog1 is a determinant of oxidative stress resistance, but is not essential

for growth in non-fermentable carbon-sources

The transcription factor Tog1 had already been functionally characterized in S. cerevisiae, being

involved in cell growth when the carbon source is non-fermentative. Additionally, Tog1 was also implied

in cellular response to oxidative stress, which becomes more relevant when aerobic metabolism occurs

during non-fermentative growth (Thepnok et al. 2014).

To verify if Tog1 plays a similar role in C. glabrata, a phenotypic analysis was performed through spot

assays (Figure 3.3). Interestingly, the results suggest that Tog1 is not essential for C. glabrata cells to

grow in non-fermentable carbon sources, as tested for lactate, glycerol and oleate. Correspondingly,

overexpression of TOG1 did not favour cell growth in the same conditions.

Figure 3.3 - Tog1 is not essential for growth in non-fermentable carbon-sources. Kchr606 wild-type and ∆cgtog1

cells were spotted on YP plates, containing different carbon sources. The C. glabrata cells with OD600nm = 0.05 were

serially diluted on a reason of 1:5 two times before being spotted. The displayed images are representative of at

least three independent experiments.

Figure 3.4 - Expression of TOG1 is not essential for C. glabrata cells to grow on YP-Oleate. Growth of the wildtype

Kchr606 or deletion mutant ∆cgtog1 was followed by measuring optic density at 600 nm during 30 h. Growth curves

of Kchr606 in YPD (⚫), ∆cgtog1 in YPD (▲), Kchr606 in YP-Oleate 0,1% (⚫) and ∆cgtog1 in YP-Oleate 0,1% (▲).

0,01

0,1

1

10

100

0 5 10 15 20 25 30

Lo

g(O

D600nm)

Time (h)

23

Since Tog1 has a predicted role in fatty acid metabolism, specifically in oleate catabolism through fatty

acid -oxidation in the peroxisomes, the impact of TOG1 deletion on growth with oleate as carbon source

was further studied. The growth of the wild-type and ∆cgtog1 were examined in YP medium with glucose

or oleate as carbon source during 30 h (Figure 3.4). Substantiating the previous result, the ∆tog1

deletion mutant had a similar grow behaviour to the wild-type strain when oleate was used as carbon

source. However, it would be expected that a lag-phase would occur when the cells are transferred to a

different medium, due to the need to adapt to a different carbon source. This may suggest that C.

glabrata cells are capable of using other YP medium components to grow, since it has a rich and

complex composition. To address this issue, the growth of wild-type and ∆tog1 strain was also studied

in a mineral medium, MMG. In this case, both strains were uncapable of growing in MMG with oleate

(results not shown), suggesting this medium lacks essential precursors to use this fatty acid as carbon

source.

Additionally, the effect of TOG1 deletion was also tested in oxidative stress conditions, using H2O2 and

menadione as source of ROS (Figure 3.5). The ∆tog1 deletion mutant growth appeared defective when

platted in MMG with H2O2. Whereas, the L5U1 harbouring the plasmid pGREG576-MTI-TOG1 showed

increased resistance against this oxidative stress agent.

Figure 3.5 - Tog1 is a determinant of oxidative stress resistance. Kchr606 wild-type and ∆cgtog1 cells were spotted

on MMG plates supplemented with 7.5 or 25 mM H2O2 and 1 mM diamide menadione. The C. glabrata cells with

OD600nm = 0.05 were serially diluted on a reason of 1:5 two times before being spotted. The displayed images are

representative of at least three independent experiments.

To further assess the impact of TOG1 deletion in C. glabrata growth in oxidative stress conditions, the

growth of the wild-type and ∆cgtog1 were followed in MMG alone (as control) or with 15 mM H2O2 (Figure

3.6). As observable in the growth curves of Figure 3.6, exposure to hydrogen peroxide causes C.

glabrata cells to experience a long lag-phase that lasts for, at least, the first 10 hours of cultivation. After

the first 24 hours, both strains had already reached the late-exponential phase. Nonetheless, it is

possible to see in the ∆tog1 strain a delay in entering the exponential growth phase when compared to

the wild-type strain, suggesting that TOG1 is particularly important during the adaptation to growth in

the presence of H2O2, decreasing the duration of the induced lag-phase. No apparent role is played by

24

Tog1 during the exponential growth phase as both strains appear to exhibit similar growth rates (Figure

3.6).

Figure 3.6 - TOG1 expression affects C. glabrata ability to grow on MMG + 15 mM H2O2 medium. Growth of the

wildtype Kchr606 or deletion mutant ∆cgtog1 was followed by measuring optic density at 600 nm during 35 h, with

an initial OD = 0.1. Growth curves of Kchr606 in MMG (⚫), ∆cgtog1 in MMG (▲), Kchr606 in MMG + 15 mM H2O2

(⚫) and ∆cgtog1 in MMG + 15 mM (▲).

Since S. cerevisiae Tog1 was previously implicated in alternative carbon source utilization and oxidative

stress response, it was also investigated the possible role of CgTog1 in complementing the function of

its S. cerevisiae homolog. S. cerevisiae wild-type and ∆sctog1 strains harbouring an empty plasmid

(pGREG576), or a plasmid expressing Tog1 (pGREG576 + CgTOG1), were grown in the referred

conditions (Figure 3.7).

Figure 3.7 - S. cerevisiae BY4741 does not display phenotypic differences upon ScTOG1 deletion. Kchr606 wild-

type and ∆cgtog1 cells were spotted on MMG plates supplemented with 7.5 or 25 mM H2O2 and 1 mM diamide

menadione. The C. glabrata cells with OD600nm = 0.05 were serially diluted on a reason of 1:5 two times before

being spotted. The displayed images are representative of at least three independent experiments.

0,01

0,1

1

10

0 5 10 15 20 25 30 35

Lo

g(O

D600nm)

Time (h)

25

Unexpectedly, no growth differences were observed in either stress condition, not even between S.

cerevisiae wild-type and ∆sctog1 deletion mutant harbouring the empty plasmid. In this situation, it was

not possible to conclude if C. glabrata Tog1 would be able to complement the phenotypic differences in

S. cerevisiae upon TOG1 deletion, since there were none to observe. Even so, the overexpression of

CgTOG1 did not benefit the wild-type growth on the conditions tested.

Overall, these results suggest that, although Tog1 does not influence cell growth with respiratory carbon

sources, C. glabrata Tog1 plays a clear role in cellular defence against oxidative stress.

3.4. Tog1 is constitutively localized in the cell nucleus

To further access the conditions that lead to Tog1 activation, the CgTog1-GFP fusion protein was

localized by fluorescence microscopy in living cells (Figure 3.8). C. glabrata cells harbouring the plasmid

L5U1+MTI+TOG1 were grown in MMG or YPD until they reached mid-exponential phase, and then

transferred to MMG (A), YPD (B), YPD + 20 mM H2O2 (C) or YP-Oleate 0,1% (D) medium containing

100.μM CuSO4 during 5-6 h, to induce the expression of the fusion protein. In all conditions, the

transcription factor Tog1 was observed to be accumulated in the cell nucleus. Considering this result,

the specific conditions leading to Tog1 nucleus accumulation are still to be identified.

Figure 3.8 - Tog1 is constitutively localized in the cell nucleus. Fluorescence of exponential-phase L5U1 C. glabrata

cells harbouring the pGREG576-MTI-TOG1 plasmid, after 5-6 h of copper induction in MMG (A), YPD (B), YPD +

20 mM H2O2 (C) or YP-Oleate 0,1% (D).

3.5. Oxidative stress condition induces CTA1 expression

Tog1 deletion mutant exhibited higher susceptibility to hydrogen peroxide, as demonstrated by its growth

in solid and liquid media. This suggest that genes encoding oxidative stress responsive factors may

have their expression regulated by Tog1, TOG1 deletion resulting in their down-regulation. A key

A B C D

26

enzyme of the oxidative stress response is the catalase encoded by CTA1 gene, responsible for

decomposing H2O2 into H2O and O2. To address this possibility, CTA1 gene expression was assessed

by RT-PCR in C. glabrata cells exposed to 15 mM H2O2 for 1 hour. The obtained results are expressed

in fold-change relative to transcript levels of CTA1 for wild-type in control condition (Figure 3.9).

Figure 3.9 - Expression of CTA1 in C. glabrata wild-type and ∆tog1 during oxidative stress. Analysis of gene

expression was performed by qRT-PCR from two independent experiments; The comparative Ct method was used

for quantification. Values are expressed as relative expression for CTA1 against CTA1 expression in Kchr606

control, after normalization with the endogenous reference ACT1 gene.

As expected, C. glabrata cells exposed to hydrogen peroxide induce CTA1 gene expression. After

growth for 1 hour, CTA1 transcript level was 20-fold higher in the wild-type strain. The ∆tog1 deletion

strain was also able to express the catalase gene, with a 17-fold higher expression than Kchr606 in

control.

In Roetzer et al. 2011 work, a small concentration of 0.4 mM H2O2 was found to be enough to strongly

induce expression levels of core OSR genes in C. glabrata wild-type cells, including CTA1 that was also

found up-regulated in carbon source deprivation. Although Cta1 accumulates in the peroxisome upon

phagocytosis (Roetzer et al. 2010), ∆cta1 mutant does not affect colonization in a murine model of

systemic infection (Cuéllar-Cruz et al. 2008). As evidence suggested that Tog1 does not control CTA1

expression, RNA sequencing was used to find the key targets of Tog1 that underlie the observed role

in conferring oxidative stress resistance.

27

3.6. Transcriptomic analysis of the role of Tog1 in C. glabrata oxidative stress response

A high-throughput RNA-Seq approach was used to profile transcriptional responses of C. glabrata wild-

type and ∆tog1 deletion mutant strain in control (MMG medium) or in oxidative stress conditions (MMG

medium + 15mM H2O2). Candida cells were grown for 1 h in both conditions and then collected for RNA-

sequencing. The obtained raw data was analysed computationally until reaching a list of differentially

expressed ORFs (p-value ≤ 0.05, - 0.5 ≤ log2foldchange ≥ 0.5) in each condition. The genes in each list

were manually clustered according to their biological function, annotated for C. glabrata ORFs or for

their homologs or orthologs of C. albicans in CGD (Skrzypek et al. 2017) and of S. cerevisiae in SGD

(Cherry et al. 2012). The computational tool KEGG (Kyoto Encyclopaedia of Genes and Genomes)

Mapper (Kanehisa et al. 2016) was used to visualize the metabolic pathways enriched in each dataset.

The C. glabrata genome comprises 5632 genes, being 5055 still uncharacterized ORFs, 339 non-coding

protein genes and only the remaining 238 characterized genes (as of 18th September). Thus, when

doing genome-wide analyses for this species, it is still important to rely on the information available for

orthologs and homologs genes in C. albicans, or S. cerevisiae.

In control conditions, that is 1h of cultivation in minimal medium, a total of 611 protein coding genes

were found to be differentially expressed in wild-type cells, when compared to ∆tog1 deletion mutant

cells. Among these, 261 were found to be up-regulated and 349 down-regulated in the deletion mutant

strain (Table S2).

Figure 3.10 – Genes down-regulated in ∆cgtog1 strain in control conditions, distributed according to their biological

functions.

Unknown Function22%

Aminoacids and nitrogen metabolism

15%

Carbon and Energy Metabolism

11%

Lipids Metabolism9%

Cell Cycle8%

Protein Processing5%

Mitochondrial Function5%

Cell Wall Organization5%

Transcription4%

Ion Homeostasis3%

Protein Degradation2%

Membrane Transport2%

Oxidative Stress Response2%

Autophagy2%

Adhesion2% Cellular Stress Response

2%

Translation1%

28

Figure 3.11 - Genes up-regulated in ∆cgtog1 strain in control conditions, distributed according to their biological

functions.

Considering that in control conditions the wild-type and ∆tog1 strains display no differences in terms of

growth curve, the transcriptomics results suggest that C. glabrata adapts its metabolism to maintain

growth rate in the absence of Tog1. The transcriptomic analysis revealed the main biological functions

activated by the transcription factor Tog1 in control conditions: metabolism of aminoacids and nitrogen

(15%), carbon and energy (11%), and lipids (9%) (Figure 3.10). The more enriched biological functions

among Tog1 repressed genes are protein processing (17%), protein degradation (15%) and

mitochondrial function (12%) (Figure 3.11).

3.6.1. C. glabrata transcriptional adaptation to oxidative stress: role of Tog1

C. glabrata is able to withstand high concentrations of H2O2 and although this capability is one of the

main responsible for its survival upon phagocytosis, only more recently the mechanisms of its regulation

have been reported (reviewed in Pais et al. 2016). The analysis performed revealed a total of 2595

genes differentially expressed in wild-type cells exposed to 15mM H2O2 for 1 hour. From these 2594

genes (including non-coding protein genes), 1200 were found to be up-regulated while 1394 were down-

regulated. It is interesting to note how C. glabrata suffers a remarkable transcriptome change to respond

only to this stress. In this data set, the upregulation of genes involved in the core oxidative stress

response was observed: genes encoding enzymes involved in the protein and lipids detoxification by

preserving their redox states (TSA1, TRR1, TRX2, CAGL0E00583g (ScTRX3), CAGL0C02233g

(ScMXR1), SRX1, GRX8, GRX4, GPX2), neutralization of ROS species (SOD1, CTA1) and one of the

main regulators of C. glabrata OSR, the transcription factor SKN7.

Protein Processing17%

Protein Degradation15%

Unknown Function13%

Mitochondrial Function12%

Cell Cycle8%

Transcription8%

Lipids Metabolism7%

Cell Wall Organization5%

Cellular Stress Response

Oxidative Stress Response3%

Adhesion2%

Ion Homeostasis2%

Carbon and Energy Metabolism2%

Autophagy1%

Membrane Transport1%

Aminoacids metabolism

29

Figure 3.12 – Genes down-regulated in hydrogen peroxide stressed ∆cgtog1 cells, distributed according to their

biological functions.

Figure 3.13 – Genes up-regulated in hydrogen peroxide stressed ∆cgtog1 cells, distributed according to their

biological functions.

Mitochondrial Function18%

Carbon and Energy Metabolism

16%

Unknown Function14%

Protein Processing14%

Cell Cycle12%

Lipids Metabolism10%

Aminoacids Metabolism7%

Cell Wall Organization3%

Cellular Stress Response2%

Adhesion1%

Autophagy1%

Protein Degradation1%

Oxidative Stress Resistance1%

Unknown Function24%

Cell Cycle12%

Cellular Stress Response7%

Cell Wall Organization7%

Carbon and Energy Metabolism7%

Aminoacids Metabolism6%

Translation6%

Iron Homeostasis5%

Oxidative Stress Response5%

Protein Degradation5%

Adhesion5%

Lipids Metabolism4%

Protein Processing3%

Autophagy2% Mitochondrial Function

2%

30

To understand the possible role of Tog1 in the oxidative stress response of C. glabrata, the differential

gene expression of wild-type versus ∆tog1 in the presence of hydrogen peroxide was analysed. From a

total of 261 genes whose expression was altered upon TOG1 deletion, 113 were found to be repressed

and 148 activated by Tog1 (Table S1). Genes participating in mitochondrial function (18%), carbon and

energy metabolism (16%), and protein processing (14%) were found down-regulated (Figure 3.12) in

these mutant cells. TOG1 deletion led to the up-regulation of genes whose main biological functions are

cell cycle (12%) and cellular stress response (7%) (Figure 3.13). Surprisingly, C. glabrata TOG1 deletion

barely has impact in the expression of genes involved in the oxidative stress response (1%), in the

conditions under scrutiny.

The increased sensibility of the deletion mutant strain in the presence of hydrogen peroxide appears to

be due to inadequate energy production, rather than any difference in antioxidant capacity. Many genes

of the mitochondrial function participate in the oxidative phosphorylation (OXPHOS) which couples the

electron chain transporter (ECT) with ATP synthesis (Figure 3.14). The complex I of ECT receives the

electrons of NADH generated during the catabolism of glucose and the complex II receives electrons

directly from succinate, formed during the TCA/ glyoxylate cycle. Although glycolysis was not

downregulated upon TOG1 deletion, some genes encoding enzymes of the TCA/ glyoxylate cycle had

reduced expression (Figure 3.15). The reduced activity of the mitochondrial respiratory chain could be

a consequence of the NADH and succinate reduced production, rather than absence of direct control

by Tog1. However, it has been demonstrated in S. cerevisiae that cells with respiratory ECT disrupted

and respiratory deficient cells exhibit sensitivity to hydrogen peroxide (Grant et al. 1997; Thorpe et al.

2004). Moreover, the protein processing group of Tog1 activated targets is mostly constituted by genes

involved in translation (90%), including many encoding mitochondrial ribosomal proteins.

Furthermore, five genes involved in carnitine transport encoded by the S. cerevisiae ortholog genes

CRC1 (CAGL0B04543g), AIM17 (CAGL0K08844g), CAT2 (CAGL0J11836g), YAT1 (CAGL0J11836g),

AGP2 (CAGL0C00539g) were also down-regulated upon TOG1 deletion. The mitochondrial carnitine

shuttle is required to transport acetyl-CoA produced during peroxisomal fatty acid β-oxidation for further

metabolism in the mitochondria. Deletion of CAT2 significantly decreased survival of the mutant strain

when S. cerevisiae cells were exposed to oxidative stress (Franken et al. 2008). Besides, carnitine

acetyltransferases are essential for the growth of C. albicans in non-fermentable carbon sources (Strijbis

et al. 2008), although they are not required for virulence in a mouse model of disseminated candidiasis

like are other carbon metabolism related enzymes (Zhou and Lorenz 2008).

31

Figure 3.14 - KEGG Mapper representation of oxidative phosphorylation. Blue boxes: proteins encoded by genes down-regulated in ∆cgtog1 vs wild-type strain in H2O2 conditions;

Green boxes: other proteins belonging to C. glabrata proteome; White boxes: proteins that do not exist in C. glabrata.

32

Figure 3.15 - KEGG Mapper representation of the carbon metabolism. Blue arrows: reactions catalysed by enzymes

encoded by genes down-regulated in ∆cgtog1 vs wild-type strain in H2O2 conditions; Green arrows: other reactions

belonging to C. glabrata metabolism; Black arrows: reactions that do not exist in C. glabrata.

As verified before with the growth curve of TOG1 mutant cells exposed to H2O2, after the first 24h the

∆tog1 was able to recover from the initial growth delay and catch up with the wild-type strain growth.

Thus, although its respiratory metabolism has been compromised, the mutant cells were still able to

catabolize glucose to retrieve energy. The only gene of the glycolysis pathway with differential gene

expression was the CAGL0J00451g (ScTDH3), which encodes a glyceraldehyde-3-phosphate

dehydrogenase, which was found to be repressed by Tog1. So, it was not surprising that three genes

(CAGL0G02937g (ScPDC6), CAGL0M14047g (ScADH6), CAGL0H06853g (ScADH6)) that catalyse the

last two steps of alcoholic fermentation were also found to be Tog1-repressed (Figure 3.16). Contrary

to other alcohol dehydrogenases, which use NADH for aldehyde reduction to alcohol, Adh6 is a NADPH-

dependent alcohol dehydrogenase. Interestingly, the ORF CAGL0H05137g (ScALD6), gene encoding

an aldehyde dehydrogenase, which utilizes NADP+, was found to be activated by Tog1. This step is

especially relevant in oxidative stress conditions, since it regenerates NADPH which acts as a final

electron donor in GSH or thioredoxin redox systems (Gutiérrez-Escobedo et al. 2013).

33

Figure 3.16 - Schematic representation of the fermentation enzymatic reactions, where the enzymes encoded by

S. cerevisiae ortholog PDC6 (pyruvate decarboxylase) and ADH6 (alcohol dehydrogenase) were up-regulated, and

ALD6 (aldehyde dehydrogenase) was down-regulated in ∆tog1 cells.

When ∆tog1 cells were treated with H2O2, six genes participating in iron homeostasis were up-regulated

(Table 3.1). Iron is a required element and during the synthesis of iron-containing molecules within the

mitochondrial matrix, a “transit” pool of free iron is required within this compartment (Petrat et al. 2002),

but when cells fails to keep iron homeostasis it becomes toxic at high concentrations. The toxicity of iron

is ascribed to its ability to participate in Fenton reactions generating oxygen radicals that damage a wide

range of macromolecules (Almeida et al. 2008; Lin et al. 2011).

Table 3.1 - List of genes involved in iron homeostasis which were up-regulated in ∆cgtog1 strain during oxidative

stress caused by H2O2 treatment. *Fold-change relative to differentially gene expression of ∆cgtog1 deletion mutant

against wild-type strain exposed to H2O2.

ORF / Gene Name Sc Homolog Function Definition ∆cgtog1 fold

change*

CAGL0H04279g / MT-IIB

Copper-binding metallothionein involved in sequestration of metal ions;

3.8

CAGL0G09042g AFT2 Activates genes involved in intracellular iron use; AFT1 paralog;

2.1

CAGL0H08580g TAH18 NAPDH-dependent diflavin reductase; Component of an early step in the cytosolic Fe-S protein assembly machinery;

1.6

CAGL0G06798g LSO1 Putative iron transporter; Potential role in response to iron deprivation;

1.5

CAGL0J04048g ISU2 Mitochondrial protein required for iron-sulfur protein synthesis; Performs a scaffolding function during Fe/S cluster assembly;

1.5

CAGL0L05742g MRS3 Mediates Fe2+ transport across inner mitochondrial membrane.

1.5

Among the Tog1 activated genes, 19 were also found up-regulated in the wild-type response to H2O2,

which could represent the core genes activated by Tog1 in response to oxidative stress (Table 3.2). C.

glabrata homolog of ScRCK2 (CAGL0F00649g) is the only Tog1 target that is known to be directly

involved in the response to oxidative stress.

GLYCOLYSIS PYRUVATE ACETALDEYHDE

ALCOHOL ACETATE

CO2

PDC6 NADPH

NADP+

NADP+

NADPH ADH6 ALD6

34

Table 3.2 - List of Tog1 activated genes, which are up-regulated in oxidative stress caused by H2O2 conditions.

*Fold-change relative to differentially gene expression of wild-type against ∆cgtog1 deletion mutant exposed to

H2O2.

ORF / Gene Name Sc Homolog Function Wild-type fold

change*

CAGL0A01628g / MIG1 MIG1 Transcription factor involved in glucose repression;

3.4

CAGL0G01540g / NCE103 NCE103 Beta carbonic anhydrase; 2.7

CAGL0A01199g / DIP5 DIP5 Dicarboxylic amino acid permease;

1.9

CAGL0F00649g RCK2 Response to oxidative and osmotic stress;

12.4

CAGL0E00649g PTC6 Mitophagy; 1.8

CAGL0G07931g MRPS12 Mitochondrial translation; 1.6

CAGL0A01606g HOP2 Meiosis-specific protein; 1.8

CAGL0H02491g COX7 Subunit VII of cytochrome c oxidase;

1.6

CAGL0B02079g AZR1 Plasma membrane transporter of the major facilitator superfamily;

2.9

CAGL0F05709g ATC1 Cation stress response; Establishment of bipolar budding pattern;

2.4

CAGL0I11011g

Putative adhesin; 3.0

CAGL0L01771g

Unknown function; 2.6

CAGL0K07942g

Unknown function; 1.6

CAGL0M12001g

Unknown function; 3.4

CAGL0I10224g

Unknown function; 1.6

CAGL0I00116g

Unknown function; 2.1

CAGL0E04554g

Unknown function; 30.0

CAGL0D02662g

Unknown function; 13.1

CAGL0D02640g

Unknown function. 12.5

35

3.6.2. The possible role of Tog1 in C. glabrata virulence

The transcriptome response to hydrogen peroxide stress, regulated by Tog1, as analysed in this work,

suggests that the role of this transcription factor contributes to C. glabrata virulence by modulating the

carbon and energy metabolism, although this gene was not found to be essential for this pathogen to

grow on alternative carbon sources. There are already genome-wide studies revealing genes shown to

be activated when C. glabrata is phagocytosed by murine or human macrophages. The starting

motivation for this work was to understand how the transcription factor Tog1 could affect C. glabrata

virulence. Hence, the genes showed to be activated by Tog1 where compared to those described to

partake in C. glabrata adaptation upon phagocytosis by murine and human macrophages (Kaur et al.

2007; Seider et al. 2011) (Figure 3.17).

Figure 3.17 - Venn diagram illustrating the intersection between genes up-regulated in murine and/or human

macrophages and genes found down-regulated in ∆cgtog1 vs wild-type strain in H2O2 or control conditions.

As the transcriptome profile of C. glabrata cells exposed to oxidative stress show that Tog1 do not

regulate the core OSR, and instead participates mainly in the regulation of energy and carbon

metabolism, both conditions, control and H2O2 exposure, were matched against the group of genes

upregulated in macrophages, revealing an overlap of 93 genes. The most relevant biological functions

enriched in this intersection are: aminoacids and nitrogen metabolism (18 genes), carbon and energy

(18 genes), lipid and fatty acids metabolism (8 genes) and carnitine transport (5 genes) (Table S1 and

S2). From the 34 genes up-regulated in macrophages and activated by Tog1 in response to H2O2, 6

were also up-regulated in the wild-type general response to H2O2 (not shown in the Venn diagram)

(NCE103, MIG1, DIP5, RCK2, ATC1 and CAGL0E04554g).

Tog1 Activated H2O2 Upregulated macrophages

Tog1 Activated Control

36

A gene that is up-regulated in C. glabrata transcriptional response to different stresses is NCE103.

Previous reports associated the β-carbonic anhydrase (β-CA), encoded by NCE103, to the response to

glucose starvation and H2O2 stress in vitro (Seider et al. 2011), to the engulfment by murine

macrophages (Kaur et al. 2007). In this work, NCE103 was found to be activated in the response to

H2O2 by Tog1. CAs enable CO2 fixation by enhancing its conversation to bicarbonate, which is

subsequently used in cellular metabolism (i.e. TCA cycle, aminoacids biosynthesis). The class of β-CAs

are the only family of enzymes present in many pathogenic organisms, but not the mammalian host,

which make them attractive as a target to develop new drugs (Innocenti et al. 2009).

The glucose responsive transcription factor Mig1 is also a recurrent presence in C. glabrata response

to different stresses. The MIG1 gene was induced in cells during acid to alkaline pH shift, nitrosative

stress (Linde et al. 2015), and upon phagocytosis by murine macrophages (Kaur et al. 2007). Mig1 is

the main effector in glucose repression binding to the promoters of many genes and represses their

transcription (Ng et al. 2015). CgMig1 is regulated by glucose similar to S. cerevisiae (Ng et al. 2015).

Localization of CgMig1 can be used to detect carbon stress situations: in presence of glucose, Mig1 is

located in the nucleus; upon glucose exhaustion, the transport of the repressor into the cytoplasm can

be detected, such as during engulfment (Roetzer et al. 2010).

Previous transcriptomic studies already demonstrated how nitrogen assimilation is a key trait of

pathogenic organisms after engulfment by macrophages (Lorenz et al. 2004; Kaur et al. 2007). Here,

the gene encoding the dicarboxylic amino acid permease Dip5, a nitrogen transporter, was found to be

activated by Tog1 in response to oxidative stress and in control conditions.

As deemed before, C. glabrata RCK2 is the only gene which is directly involved in OSR and seems to

be activated by Tog1. This gene encodes a mitogen-activated protein (MAP) kinase-activated protein

kinase in yeast implicated in translational regulation. The deletion of ScRCK2 induces cell death by

oxidative stress, and the causes are possible extensive effects on the state of the translation and mRNA

processing machinery as had been demonstrated with microarray analysis (Swaminathan et al. 2006).

3.6.3. In silico prediction of Tog1 binding sites

The motif discovery algorithm DREME (Discriminative Regular Expression Motif Elicitation) (Bailey

2011) was used to find short motifs enriched in the upstream promoter sequences of the genes possibly

activated by Tog1 in H2O2 (Table S1) retrieved from PathoYeastract Database (Monteiro et al. 2017).

Six possible DNA binding motifs were predicted (E-value < 0.01) (Table 3.3).

Afterwards, using the computational tool GOMO (Buske et al. 2010), which detects associations

between a DNA regulatory motif and biological roles, all promoters containing the predicted CgTog1

binding motifs were scanned for Genome Ontology (GO) term enrichment (Table 3.3).

37

Table 3.3 - Predicted binding sites for C. glabrata Tog1 and the more specific GO terms significantly associated

with these motifs.

DNA binding motif Number of promoters containing this motif

Top specific predictions

GAAGAHGA 48 Chromatin modification;

CGATGAGM 32 RNA processing;

MAATACA 88 Membrane raft;

CTCGAGR 36 Cyclin-dependent protein kinase regulator activity;

Cellular nitrogen compound biosynthetic process;

CCCYCCYC 53

Vacuolar protein catabolic process;

Plasma membrane enriched fraction;

Regulation of transcription from RNA polymerase II

promoter.

In S. cerevisiae the consensus sequences for ScTog1 are still unknown. Nonetheless, the ChIP-Seq

work performed by Thepnok et al. allowed the prevision of binding sites for this TF. Two previsions

contained the sequence GATSA: CSGATGA and CGATCAC. This sequence also can be found in two

previsions for C. glabrata: GAAGAHGA and CGATGAGM. These predictions, for both Tog1 homologs,

still require experimental validation.

38

4. Discussion

C. glabrata is a successful human secondary pathogen that has develop skilled adaptation and immune

evasion strategies (Kaur et al. 2007; Seider et al. 2014). A major feature of C. glabrata virulence is its

ability to survive macrophage phagocytosis by enduring the intracellular environmental stresses, such

as oxidative and pH stress, and nutrient starvation (Cuéllar-Cruz et al. 2008; Roetzer et al. 2010; Seider

et al. 2014). The virulence mechanisms of C. glabrata just recently started to be described. The possible

role of the transcription factor Tog1 as part of these mechanisms was analysed herein. The S. cerevisiae

homolog to this transcription factor had already been characterized as playing a role in the utilization of

alternative carbon sources and in oxidative stress tolerance (Thepnok et al. 2014). In this study, the role

of Tog1 in C. glabrata pathogenesis, as a determinant of resistance to oxidative stress, was clarified.

C. glabrata cells upon deletion of TOG1 display decreased virulence in an infection model of G.

mellonella. The larvae infected with the deletion mutant ∆cgtog1 had a higher survival rate than those

infected with the wild-type. G. mellonella as an infection host is an attractive and simple model, which

has been used to study Candida species virulence (Mesa-Arango et al. 2013; Ames et al. 2017; Santos

et al. 2017). This model presents several advantages, such as possible incubation at 37ºC, allowing

virulence to be studied at human body temperature and, importantly, some aspects of the G. mellonella

immune response show similarities with the innate immune response of mammals (Chamilos et al.

2007).

Although the deletion of TOG1 caused a higher survival of infected larvae, the overexpression of TOG1

did not improved the killing ability of C. glabrata cells. However, when determining the proliferation

capacity inside G. mellonella hemocytes, the results show that TOG1 expression is essential for the C.

glabrata proliferation upon phagocytosis, implicating the function of this transcription factor in the

adaptation and proliferation inside the phagocytotic cell.

To verify if this hypothesized function for Tog1 was related to the phenotype observed for ScTog1, spot

assays were performed in the presence of alternative carbon sources and two sources of oxidative

stress. CgTOG1 expression was found not to be essential for C. glabrata growth in the tested non-

fermentable carbon sources: lactate, glycerol and oleate. The survival inside the host depends on the

pathogen adaptation to the nutrient limitation. Some host niche are almost deprived of glucose, but may

have alternative carbon sources such as oleic acid, lactate, acetate or amino acids (Ehrström et al.

Rylander 2006; Ene et al. 2012). In addition, studies have shown how the impact of glucose and other

carbon sources affect virulence and fitness of Candida species (Ene et al. 2012; Ng et al. 2016).

Although in this work the TF Tog1 was not proven essential for C. glabrata growth on alternative carbon

sources, the transcriptome profile revealed that some genes encoding key enzymes in carbon and

energy metabolism are affected by TOG1 deletion. Contrary to S. cerevisiae, it is possible that C.

glabrata developed other regulatory mechanism to bypass Tog1 function. Work of Thepnok et al.

described ScTog1 as an activator of genes of β-oxidation and NADPH regeneration (POX1, FOX2,

39

POT1 and IDP2), the glyoxylate shunt (MLS1 and ICL1), and gluconeogenesis (PCK1 and FBP1). As

glucose was used as carbon source in this study, a shift in the metabolism of fatty acids degradation

was not expected. Nevertheless, CgTog1 was found to control the expression of S. cerevisiae orthologs

IDP2, MLS1, FBP1 (in control conditions) and PCK1 (under oxidative stress). FBP1 and PCK1 genes

encode key regulatory enzymes of gluconeogenesis. In a host environment where glucose is scarce,

gluconeogenesis allows the pathogens to generate glucose as valuable precursors of molecules like

ribose and deoxyribose, essential for cell growth.

RNA-sequencing also underscored many other genes involved in respiratory metabolism activated by

Tog1 in control and oxidative stress conditions. Worth highlighting is the group of carnitine transporter

encoding genes: in both conditions CRC1, AIM17, CAT2 and YAT1, only in control condition YAT2, and

only in H2O2 exposed cells AGP2. S. cerevisiae cannot synthesize L-carnitine de novo, and the Hnm1

transporter exclusively uptakes activity exogenous L-carnitine via the Hnm1 transporter, whose

expression is regulated by Agp2 (Aouida et al. 2013). Accordingly, ∆sctog1 mutant had reduced growth

when only carnitine was supplemented as carbon source (Thepnok et al. 2014). Contrary to S.

cerevisiae, C. albicans possesses an enzymatic pathway capable of synthesizing carnitine (Strijbis et

al. 2009). It remains unknown if C. glabrata can also synthesize carnitine. Carnitine shuttle of S.

cerevisiae appears to operate unidirectionally during growth on glucose, being Crc1 the responsible for

transporting acetyl moieties into the mitochondria matrix (van Rossum et al. 2016). Yat1 being an outer

mitochondrial transferase, shuttles carnitine to the intermembrane space (Schmalix and Bandlow 1993).

Interestingly, previous studies have specifically illustrated that carnitine supplementation results in

improved yeast growth stressed with hydrogen peroxide (Franken and Bauer 2010). Moreover, Crc1,

Cat2, Yat1 and Aim17 carnitine transporters were all found up-regulated in C. glabrata cells upon

macrophage engulfment (Kaur et al. 2007; Seider et al. 2011), enlightening the importance of the

adaptation to the nutrients available on the host niche.

In fact, Tog1 was found to activate mitochondrial function related genes specially during oxidative stress.

Thorpe et al. reported that deletion of genes involved in mitochondrial function resulted in sensitivity

towards H2O2, while for other oxidative stress agents it is not so relevant (Thorpe et al. 2013). This is

consisted with the fact that Tog1 absence did not affect C. glabrata growth on menadione stress, as

displayed herein through spot assay. The first time oxidative stress susceptibility was described for

respiratory deficient strains, it was proposed it was due to an energy requirement (Grant et al. 1997).

However, H2O2 sensitive deletants did not present deficiencies in other energy-generating reactions.

Thorpe et al. propose instead that tolerance mechanism to H2O2 is the cause for the requirement of a

functional respiratory chain (Thorpe et al. 2013). Mitochondria is responsible for intracellular ROS

generation through the ECT, being the respiratory chain complex III responsible for > 80% of the

superoxide produced. In this work, genes encoding subunits of complex III or necessary for its assembly

were found to be activated by Tog1: CYT1, CAGL0D05192g (ScQCR6), CAGL0G10153g (ScQCR7),

and CAGL0A02992g (ScBCS1). The normal response to H2O2-induced stress in wild-type cells was to

increase O2•− production, and when this was inhibited by either increased SOD levels or inhibition of

complex III function, sensitivity to H2O2 increased (Thorpe et al. 2013). Despite the O2•- conversion to

40

H2O2 during its detoxification (Morano et al. 2012), defence mechanisms for H2O2 and menadione are

distinctive different for S. cerevisiae and C. glabrata (Thorpe et al. 2004; Roetzer et al. 2011). This

highlights the fact that superoxide and H2O2 effects on the cell are fundamentally different, and although

it seems consistent with the Thorpe et al. hypothesis concerning the superoxide protective signalling

effect during H2O2-induced stress, there is still need for experimental validation. The effect on C. glabrata

virulence of petit mutations, the partial or total loss of mitochondrial DNA, has already been studied,

despite disaccording results. In a model of systemic candidiasis in immunocompetent mice, these

mutations were found to lead to a drastic reduction in virulence (Brun et al. 2005). Meanwhile,

mitochondrial dysfunction associated with azole resistance positively affected C. glabrata virulence also

in mice in a different study (Ferrari et al. 2011). Therefore, the molecular basis for this diverse phenotype

is still unclear, and the relation between mitochondrial function and virulence factors responsible for

virulence traits need yet to be identified.

Moreover, based on the transcriptomics data ∆cgtog1 cells are suggested to accumulate iron in the

mitochondria, which triggers the ROS generating Fenton reaction. Iron is present in polynuclear sulfur-

bridged iron–sulfur (Fe-S) centres. One of the major demands on mitochondrial iron is during protein

synthesis of the respiratory chain enzymatic complexes. (Atkinson and Winge 2009). Cells defective in

Fe-S cluster (ISC) biogenesis within the mitochondrial matrix, accumulated toxic amounts of iron due to

upregulation of iron transport systems via the activation of the Aft1 (Gomez et al. 2014). Iron

accumulation in yeast requires the activation of the Mrs3/ Mrs4 mitochondrial transporters, which play

essential roles in cell iron homeostasis and Fe–S clusters synthesis by shuttling iron into mitochondria

(Foury and Roganti 2002; Zhang et al. 2006). LSO1 gene is supposed to encode a protein involved in

iron transport at the cell membrane (An et al. 2015), while ISU2 encodes a protein which participates

directly in ISC assembly (Gerber et al. 2004). Both genes are regulated by AFT2 in S. cerevisiae

(MacIsaac et al. 2006).

The oxidative stress response of C. glabrata has been already characterized. The C. glabrata genome

encodes a single catalase, Cta1, whose expression is controlled by the transcription factors Skn7, Yap1,

Msn2 and Msn4 (Cuéllar-Cruz et al. 2008). The expression of CTA1 is induced in the presence of

oxidative stress and in carbon source deprivation (Roetzer et al. 2010). This catalase is essential for in

vitro resistance of C. glabrata cells to hydrogen peroxide but was not required for virulence in a mouse

model of systemic infection. Suggesting C. glabrata has an alternative pathway to cope with the lack of

Cta1 or this antioxidant enzyme is unnecessary for adaptation to the host. In this work, Tog1 resistance

to oxidative stress did not involve CTA1 activation. Regarding NADPH regeneration, Tog1 appears to

activate an acetaldehyde dehydrogenase, Ald6, supplying NADPH for cellular antioxidant systems,

while Adh6, which acts as an NADPH-dependent alcohol reductase (Larroy et al. 2002), is repressed

by Tog1. These results suggest Tog1 contributes for the cellular maintenance of NADPH/NADP+ ratio.

41

Figure 4.1 - Hypothetical model of action of Tog1 in C. glabrata cells during stress response to hydrogen peroxide:

Tog1 activates several genes encoding enzymes involved in carbon metabolism: PYC1 and CAGL0H06633g

(ScPCK1) from gluconeogenesis, CAGL0E01705g (ScMDH2) and CAGL0L09273g (CaICL1) from the glyoxylate

cycle, PYC1, LSC2 and CAGL0E03850g (ScSDH2) from TCA cycle. Sdh2 is the responsible for reducing

ubiquinone to ubiquinol during electrons transference to the ECT. Synthesis of several subunits of the OXPHOS

process are also controlled by Tog1. Malfunction during the assembly of this process can lead to a deficient iron

homeostasis regulated by Aft2, creating a pool of Fe2+ which exacerbates the Fenton reaction and generates more

ROS caused damage. Fe2+ can be transported into the cell by Lso1 and afterwards into the mitochondria by Mrs3.

The expression of these proteins is repressed in Tog1 response to H2O2 and are represented in grey. Several

proteins (Agp2, Yat1, Crc1 and Cat2) involved in carnitine transportation of acyl /acetyl groups are also activated

by Tog1. Acetyl molecules can enter directly on the TCA cycle to generate more reducing power in the form of

NADH or succinate. NCE103 encodes a carbonic anhydrase that catalyses CO2 hydration to bicarbonate, an

important metabolic substrate. Rck2 is a MAP kinase-activated protein kinase required for ribosomes

reprogramming during oxidative stress. The activation of ALD6 by Tog1 can help the cellular NAPDH regeneration

to act as a co-factor of antioxidant enzymes. Between the CgTog1 predicted binding sites, GAAGAHGA and

CGATGAGM were the most similar to the predicted binding sites of ScTog1.

This work shed light on the role of Tog1 as virulence determinant in C. glabrata. Tog1 was implicated in

resistance to oxidative stress caused by H2O2, not because, as initially thought, it participates in the core

42

OSR of C. glabrata (Figure 4.1), but because Tog1 seems to activate several genes involved in normal

mitochondrial function, and energy and carbon metabolism. C. glabrata resistance to H2O2 in vitro is

known to be very high, but the estimated concentration that C. glabrata experiences inside mammalian

host is only 0.4 mM. This highlights how inside phagocytic cells, oxidative stress is not enough, on in its

own, to kill C. glabrata cells, but rather require a combination of stresses to inhibit their growth (Kaloriti

et al. 2012). The reduced virulence of the ∆cgtog1 deletion mutant observed in G. mellonella hemocytes

could be an amplified effect of this strain sensitivity to H2O2 in combination with other stresses felt inside

phagosomes: pH and nitrosative stress, and carbon starvation. Actually, the only oxidative stress

responding gene that appears relevant in macrophage up-regulation and seems to be activated by Tog1

is the RCK2 gene. Since this ortholog of S. cerevisiae gene encoding MAP kinase-activated protein

kinase is still uncharacterized in C. glabrata, would be interest to see if its phenotype underlies any

virulence trait. Specially due to responses to osmotic, oxidative, nitrosative, and cell wall stresses being

regulated by a range of different MAPK pathways (Monge et al. 2006; Brown, Haynes and Quinn 2009;

Haynes et al. 2012). Nevertheless, was observed that adaptation to oxidative stress and glucose

starvation causes a simultaneous upregulation of a set of genes, beneficial for both oxidative stress and

glucose starvation (Roetzer et al. 2011). The transcriptomic dataset obtained with this work, may

suggest that Tog1 during oxidative stress exposure, could simultaneously control response to glucose

starvation.

An interesting fact is that the homology and synteny of ScTOG1 is only shared by C. glabrata among all

yeasts, as verified by the online tool Yeast Gene Order Browser (Byrne 2005), yet the identity shared is

only 39.5% which could illustrate the different phenotypes observed between ScTOG1 and CgTOG1.

Still, only C. glabrata genome conserves this TF, that could be one of its unique virulence traits.

Future work should consistent on the identification direct target genes activated in vivo through

Chromatine ImmunoPrecipitation (ChIP) assays. In positive cases, in vitro TF-DNA binding assays may

be carried out through Surface Plasma Resonance (SPR), in order to elucidate the precise nucleotide

sequences recognized by Tog1, verifying, or not, the in silico prediction. It will also be interesting to

assess the effect of Tog1 in mitochondrial function and in carbon and energy metabolism.

43

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6. Supplemental Material

Table S1 – List of genes down-regulated in ∆cgtog1 versus wild-type strain after exposure to H2O2 for 1 hour. ORFs enlighten in light grey: genes found upregulated in C. glabrata

upon phagocytosis (Kaur et al. 2007; Seider et al. 2011) . ORF enlighten in dark grey: genes upregulated in macrophages and downregulated in ∆cgtog1 versus wild-type strain

in control condition.

ORF log2FoldChange Name

Sc homolog Name

Definition

Adhesio

n

CAGL0H00110g -0.8806 Adhesin-like protein with internal repeats;

CAGL0I11011g -1.6025 Putative adhesin;

Am

inoacid

s M

eta

bolis

m

CAGL0M00880g -0.5838 CAR2 CAR2 L-ornithine transaminase (OTAse); catalyses the second step of arginine degradation;

CAGL0D00176g -0.5932 GDH3 NADP(+)-dependent glutamate dehydrogenase; synthesizes glutamate from ammonia and alpha-ketoglutarate; expression regulated by nitrogen and carbon sources;

CAGL0I04994g -0.6548 MET6 MET6 Cobalamin-independent methionine synthase; involved in methionine biosynthesis and regeneration;

CAGL0A01199g -0.8908 DIP5 DIP5 Dicarboxylic amino acid permease;

CAGL0E05632g -0.8987 PUT4 Proline permease;

CAGL0L12012g -0.9991 TMT1 Trans-aconitate methyltransferase; cytosolic enzyme that catalyses the methyl esterification of 3-isopropylmalate, an intermediate of the leucine biosynthetic pathway, and trans-aconitate, which inhibits the citric acid cycle;

CAGL0L03267g -1.0101 GAP1 GAP1 General amino acid permease; Gap1p senses the presence of amino acid substrates to regulate localization to the plasma membrane when needed;

CAGL0J08184g -1.0254 ALP1 Arginine transporter;

CAGL0C04917g -1.1269 CPA2 Large subunit of carbamoyl phosphate synthetase; carbamoyl phosphate synthetase catalyses a step in the synthesis of citrulline, an arginine precursor;

CAGL0A03212g -1.2050 ATO3 Plasma membrane protein, putative ammonium transporter;

Auto

phagy

CAGL0M01848g -0.8659 SLM4 Component of the EGO and GSE complexes; the vacuolar/endosomal membrane associated EGO/GSE complex regulates exit from rapamycin-induced growth arrest;

CAGL0E00649g -0.8829 PTC6 Mitochondrial type 2C protein phosphatase (PP2C); has similarity to mammalian PP1Ks; involved in mitophagy;

51

CAGL0H06633g -0.5730 PCK1 Phosphoenolpyruvate carboxykinase; key enzyme in gluconeogenesis;

CAGL0E01705g -0.5996 MDH2 Cytoplasmic malate dehydrogenase; one of three isozymes that catalyze interconversion of malate and oxaloacetate; involved in the glyoxylate cycle and gluconeogenesis during growth on two-carbon compounds;

Carb

on a

nd E

nerg

y M

eta

bolis

m

CAGL0G04081g -0.6416 THI73 Putative plasma membrane permease; proposed to be involved in carboxylic acid uptake and repressed by thiamine;

CAGL0B02717g -0.7509 ACS2 Acetyl-CoA ligase with roles in acetyl-CoA synthesis, histone acetylation and replicative cell aging;

CAGL0F08107g -0.7635 LSC2 LCS2 Beta subunit of succinyl-CoA ligase; succinyl-CoA ligase is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate;

CAGL0K00913g -0.7826 ADE3 Cytoplasmic trifunctional enzyme C1-tetrahydrofolate synthase;

CAGL0F09053g -0.8374 MNL1 Alpha-1,2-specific exomannosidase of the endoplasmic reticulum;

CAGL0L09273g -0.9036 ICL2 2-methylisocitrate lyase of the mitochondrial matrix;

CAGL0K10736g -0.9387 CYB2 CYB2 Cytochrome b2 (L-lactate cytochrome-c oxidoreductase); component of the mitochondrial intermembrane space, required for lactate utilization;

CAGL0L06116g -0.9638 YGL185C Putative protein with sequence similar to hydroxyacid dehydrogenases;

CAGL0L06842g -0.9947 THI3 THI3 Regulatory protein that binds Pdc2p and Thi2p transcription factors;

CAGL0H00847g -1.0322 HUT1 Protein with a role in UDP-galactose transport to the Golgi lumen;

CAGL0F06941g -1.1134 PYC1 PYC1 Pyruvate carboxylase isoform; cytoplasmic enzyme that converts pyruvate to oxaloacetate;

CAGL0E05610g -1.1530 PYK2 Pyruvate kinase;

CAGL0F04719g -1.1740 GSY2 Glycogen synthase;

CAGL0M03377g -1.2049 GLC3 Glycogen branching enzyme involved in glycogen accumulation;

CAGL0A01089g -1.3566 PBI1 predicted alcohol O-acetyltransferase activity and role in alcohol metabolic process;

CAGL0G01540g -1.4504 NCE103 NCE103 Carbonic anhydrase; metalloenzyme that catalyses CO2 hydration to bicarbonate, which is an important metabolic substrate, and protons;

CAGL0H05137g -1.6306 ALD6 Cytosolic aldehyde dehydrogenase;

52

CAGL0I05148g -1.7030 DLD1 DLD1 Major mitochondrial D-lactate dehydrogenase;

CAGL0A01628g -1.7852 MIG1 MIG1 Transcription factor involved in glucose repression;

CAGL0G04763g -1.9742 RGS2 Negative regulator of glucose-induced cAMP signalling; directly activates the GTPase activity of the heterotrimeric G protein alpha subunit Gpa2p;

CAGL0D02640g -3.6383 HTX2 High-affinity glucose transporter of the major facilitator superfamily;

CAGL0D02662g -3.7082 HTX2 High-affinity glucose transporter of the major facilitator superfamily;

CAGL0J06622g -0.5453 PML39 Protein required for nuclear retention of unspliced pre-mRNAs;

Cell

Cycle

CAGL0M00902g -0.5822 DIF1 Protein that regulates nuclear localization of Rnr2p and Rnr4p; phosphorylated by Dun1p in response to DNA damage and degraded;

CAGL0G02739g -0.6193 XBP1 Transcriptional repressor; binds promoter sequences of cyclin genes, CYS3, and SMF2;

CAGL0H01331g -0.6692 HDA2 Subunit of the HDA1 histone deacetylase complex;

CAGL0L02453g -0.6846 MIT1 Transcriptional regulator of pseudohyphal growth;

CAGL0L10428g -0.6931 MSA1 Activator of G1-specific transcription factors MBF and SBF

CAGL0A04543g -0.6996 AAR2 Component of the U5 snRNP complex; required for splicing of U3 precursors;

CAGL0D02310g -0.7324 MSC6 Multicopy suppressor of HER2 involved in mitochondrial translation;

CAGL0C02519g -0.7677 MIG3 Transcriptional regulator;

CAGL0J02948g -0.7765 FCY2 FCY22 Putative purine-cytosine permease;

CAGL0D06512g -0.8146 CDC25 Membrane bound guanine nucleotide exchange factor;

CAGL0A01606g -0.8523 HOP2 Meiosis-specific protein that localizes to chromosomes;

CAGL0H03047g -0.9212 SPC105 Subunit of a kinetochore-microtubule binding complex;

CAGL0I05060g -1.1522 DOT6 Protein involved in rRNA and ribosome biogenesis;

CAGL0F05709g -1.2436 ATC1 Nuclear protein; possibly involved in regulation of cation stress responses and/or in the establishment of bipolar budding pattern;

CAGL0M11990g -1.2861 CLN3 G1 cyclin involved in cell cycle progression

53

CAGL0F00605g -1.6832 GLK1 EMI2 Required for transcriptional induction of the early meiotic-specific transcription factor IME1; C

ell

Wall

Org

aniz

ation CAGL0A01221g -0.5257 AQY1 Spore-specific water channel; mediates the transport of water across cell membranes,

CAGL0K01133g -0.5836 TWF1 Twinfilin; highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton; coordinates actin filament severing and monomer sequestering at sites of rapid actin turnover;

CAGL0K10164g -0.6268 SED1 Major stress-induced structural GPI-cell wall glycoprotein; associates with translating ribosomes, possible role in mitochondrial genome maintenance;

CAGL0E04620g -1.2902 PST1 PST1 Cell wall protein that contains a putative GPI-attachment site; secreted by regenerating protoplasts; up-regulated by activation of the cell integrity pathway, as mediated by Rlm1p;

CAGL0L01771g -1.3959 PHD1 Transcriptional activator that enhances pseudohyphal growth;

Str

ess

Response CAGL0D05170g -0.5419 PHO4 PHO4

Sequence specific DNA-binding RNA polymerase II transcription factor that activates expression of genes involved in the response to phosphate starvation such as PHO5;

CAGL0G03289g -0.7386 SSA3 SSA4 Heat shock protein that is highly induced upon stress;

CAGL0B02079g -1.5591 AZR1 Plasma membrane transporter of the major facilitator superfamily

Lip

ids M

eta

bolis

m

CAGL0I02134g -0.5072 PEX21B PEX18 Peroxin required for targeting of peroxisomal matrix proteins containing PTS2; primarily responsible for peroxisomal import during growth on oleate, and expression is induced during oleate growth;

CAGL0K12210g -0.5205 YMC2 Putative mitochondrial inner membrane transporter; proposed role in oleate metabolism and glutamate biosynthesis;

CAGL0H04081g -0.5351 ERG13 ERG13 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase;

CAGL0C03069g -0.5502 CHO1 Phosphatidylserine synthase; functions in phospholipid biosynthesis;

CAGL0B04543g -0.6071 CRC1 Mitochondrial inner membrane carnitine transporter;

CAGL0J04136g -0.6887 MCT1 Predicted malonyl-CoA:ACP transferase;

CAGL0M04367g -0.6959 CKI1 Choline kinase;

CAGL0D05940g -0.7676 ERG1 ERG1 Squalene epoxidase; catalyses the epoxidation of squalene to 2,3-oxidosqualene;

CAGL0J04004g -0.8229 MCP1 Mitochondrial protein of unknown function involved in lipid homeostasis;

CAGL0J11836g -0.8919 CAT2 Carnitine acetyl-CoA transferase; present in both mitochondria and peroxisomes;

CAGL0C05027g -1.0152 YAT1 Outer mitochondrial carnitine acetyltransferase;

54

CAGL0C00539g -1.0268 AGP2 Plasma membrane regulator of polyamine and carnitine transport;

CAGL0K08844g -1.0715 AIM17 Mitochondrial inner membrane carnitine transporter;

CAGL0H07227g -0.5583 OMS1 Protein integral to the mitochondrial membrane; has a conserved methyltransferase motif and is predicted to be an RNA methyltransferase;

CAGL0G04521g -0.5605 PAM16 Protein binding protein involved in protein import into mitochondrial matrix; localizes to presequence translocase-associated import motor and mitochondrial inner membrane presequence translocase complex;

CAGL0A02992g -0.5668 BCS1 Protein translocase and chaperone required for Complex III assembly;

CAGL0H03531g -0.5763 MNP1 MNP1 Mitochondrial ribosomal protein of the large subunit; required for normal respiratory growth;

CAGL0M09581g -0.5853 ATP1 ATP1 Alpha subunit of the F1 sector of mitochondrial F1F0 ATP synthase; which is a large, evolutionarily conserved enzyme complex required for ATP synthesis;

CAGL0J09790g -0.5853 GGC1 Mitochondrial GTP/GDP transporter; essential for mitochondrial genome maintenance; has a role in mitochondrial iron transport;

Mitochondrial F

unctio

n

CAGL0J00847g -0.5858 YJL045W Minor succinate dehydrogenase isozyme;

CAGL0E03850g -0.5875 SDH2 Iron-sulfur protein subunit of succinate dehydrogenase; the complex couples the oxidation of succinate to the transfer of electrons to ubiquinone as part of the TCA cycle and the mitochondrial respiratory chain;

CAGL0G10153g -0.5935 QCR7 Subunit 7 of ubiquinol cytochrome-c reductase (Complex III);

CAGL0L06204g -0.6573 COX13 Subunit VIa of cytochrome c oxidase; present in a subclass of cytochrome c oxidase complexes that may have a role in minimizing generation of reactive oxygen species;

CAGL0H02491g -0.6784 COX7 Subunit VII of cytochrome c oxidase (Complex IV); Complex IV is the terminal member of the mitochondrial inner membrane electron transport chain;

CAGL0J03300g -0.6827 ICP55 Mitochondrial aminopeptidase;

CAGL0L08426g -0.6944 SUE1 SUE1 Protein required for degradation of unstable forms of cytochrome c;

CAGL0M09317g -0.7026 AIM24 Protein with a role in determining mitochondrial architecture;

CAGL0A03542g -0.7798 COX12 COX12 Subunit VIb of cytochrome c oxidase;

CAGL0D05192g -0.7818 QCR6 Subunit 6 of the ubiquinol cytochrome-c reductase complex; also known as the cytochrome bc(1) complex or Complex III, is a component of the mitochondrial inner membrane electron transport chain;

55

CAGL0L10406g -0.8001 CYT1 CYT1 Cytochrome c1; component of the mitochondrial respiratory chain;

CAGL0F04807g -0.8552 OM45 Mitochondrial outer membrane protein of unknown function;

CAGL0G06402g -0.8606 CIR2 Putative ortholog of human ETF-dH; found in a large supramolecular complex with other mitochondrial dehydrogenases; may have a role in oxidative stress response;

CAGL0C01325g -0.8668 COX5B Subunit Vb of cytochrome c oxidase;

CAGL0L06160g -0.8728 COX4 Subunit IV of cytochrome c oxidase; the terminal member of the mitochondrial inner membrane electron transport chain; precursor N-terminal 25 residues are cleaved during mitochondrial import;

CAGL0J11330g -0.8900 MRPL49 Component of the large subunit of the mitochondrial ribosome, which mediates translation in the mitochondrion;

CAGL0G03201g -0.9290 Predicted FMN binding, oxidoreductase activity, riboflavin reductase (NADPH) activity and role in oxidation-reduction process;

CAGL0G01166g -0.9333 DIC1 Mitochondrial dicarboxylate carrier; integral membrane protein, catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane, transports cytoplasmic dicarboxylates into the mitochondrial matrix;

CAGL0I04796g -0.9399 SCO2 Protein anchored to mitochondrial inner membrane; may have a redundant function with Sco1p in delivery of copper to cytochrome c oxidase;

CAGL0A03784g -1.0182 TIM18 Component of the mitochondrial TIM22 complex; involved in insertion of polytopic proteins into the inner membrane; may mediate assembly or stability of the complex;

CAGL0J06380g -1.3960 INH1 Protein that inhibits ATP hydrolysis by the F1F0-ATP synthase;

OS

R

CAGL0F00649g -3.6293 RCK2 Protein kinase involved in response to oxidative and osmotic stress;

Pro

tein

Degra

dation

CAGL0K04653g -0.8374 UBX2 Bridging factor involved in ER-associated protein degradation (ERAD);

CAGL0M12749g -1.3681 DOA4 Ubiquitin hydrolase; deubiquitinates intralumenal vesicle (ILVs) cargo proteins;

Pro

tein

Pro

cessin

g

CAGL0M07513g -0.5197 MRPL3 Mitochondrial ribosomal protein of the large subunit;

CAGL0F04411g -0.5335 MRPL16 Mitochondrial ribosomal protein of the large subunit;

CAGL0E00715g -0.5347 RSM24 Mitochondrial ribosomal protein of the small subunit;

CAGL0J11858g -0.5455 RML2 RML2 Mitochondrial ribosomal protein of the large subunit (L2);

56

Pro

tein

Pro

cessin

g

CAGL0J06930g -0.5466 MRP51 Mitochondrial ribosomal protein of the small subunit;

CAGL0J02376g -0.5481 FAF1 Protein required for pre-rRNA processing;

CAGL0H07029g -0.5523 MRPL35 Mitochondrial ribosomal protein of the large subunit;

CAGL0L10494g -0.6307 GYP1 Essential t-SNARE that mediates fusion of vesicles with the late Golgi;

CAGL0K01375g -0.6350 MRP10 MRP10 Mitochondrial ribosomal protein of the small subunit;

CAGL0K06501g -0.6594 TLG1 Phosphatidylinositol-3-phosphate binding protein involved in mitochondrial and nuclear inheritance; tethers endoplasmic reticulum to the vacuole;

CAGL0K01243g -0.6650 MRPL25 Mitochondrial ribosomal protein of the large subunit;

CAGL0G04675g -0.6793 RSM28 Mitochondrial ribosomal protein of the small subunit;

CAGL0M08382g -0.6855 MRP49 Mitochondrial ribosomal protein of the large subunit;

CAGL0I01430g -0.6908 ANB1 Translation elongation factor eIF-5A;

CAGL0G07931g -0.6990 MRPS12 Mitochondrial protein;

CAGL0I02706g -0.7178 MRPL33 Component of the large subunit of the mitochondrial ribosome, which mediates translation in the mitochondrion;

CAGL0C01331g -0.7384 HPM1 AdoMet-dependent methyltransferase; involved in a novel 3-methylhistidine modification of ribosomal protein Rpl3p; seven beta-strand MTase family member;

CAGL0E04400g -0.9441 DIA4 Probable mitochondrial seryl-tRNA synthetase;

CAGL0A01760g -1.0902 MSR1 Arginyl-tRNA synthetase;

CAGL0K01529g -1.1621 SLM3 tRNA-specific 2-thiouridylase;

Un

know

n F

unction CAGL0M08536g -0.5437 YKL162C Putative protein of unknown function;

CAGL0C03311g -0.6364 Putative protein of unknown function;

CAGL0B05137g -0.6439 Putative protein of unknown function;

CAGL0K07942g -0.6599 Putative protein of unknown function;

CAGL0K04279g -0.6731 SCM4 SCM4 Mitochondrial outer membrane protein of unknown function;

57

Unknow

n F

unction

CAGL0I10224g -0.6818 Putative protein of unknown function;

CAGL0D02794g -0.7523 Putative protein of unknown function;

CAGL0J05830g -0.7612 YNL144C Putative protein of unknown function;

CAGL0A04367g -0.7628 YBL059W Putative protein of unknown function

CAGL0G02849g -0.7790 UIP4 Protein of unknown function; Interacts with Ulp1p;

CAGL0A02002g -0.8107 YOL024W Putative protein of unknown function

CAGL0C01793g -0.8830 Putative protein of unknown function;

CAGL0J03190g -0.9035 TDA2 Protein of unknown function;

CAGL0H03619g -1.0225 YNL011C Putative protein of unknown function

CAGL0I00116g -1.0569 Putative protein of unknown function;

CAGL0L01573g -1.3131 YML053C Putative protein of unknown function

CAGL0M00792g -1.3764 Putative protein of unknown function;

CAGL0K05390g -1.4734 Putative protein of unknown function;

CAGL0D01254g -1.4986 Putative protein of unknown function;

CAGL0M12001g -1.7615 Putative protein of unknown function;

CAGL0E04554g -4.9081 Putative protein of unknown function;

58

Table S2 - List of genes down-regulated in ∆cgtog1 versus wild-type strain in control condition. ORFs enlighten in light grey: genes found upregulated in C. glabrata upon

phagocytosis (Kaur et al. 2007; Seider et al. 2011). ORF enlighten in dark grey: genes upregulated in macrophages and downregulated in ∆cgtog1 versus wild-type strain exposed

to H2O2.

ORF log2FoldChange Name

Sc ortholog Name

Definition

Adhesin

s

CAGL0J11176g -1.06254 TDA7 Putative adhesin-like protein;

CAGL0I11011g -1.19962 Putative adhesin-like protein;

CAGL0H08844g -1.30869 DDR48 Putative adhesin;

CAGL0J02530g -1.39175 Putative adhesin-like protein;

CAGL0J11990g -1.70864 AWP4 Putative adhesion protein;

CAGL0H07469g -4.42751 ICS2 Putative adhesin-like protein with internal repeats;

Am

inoacid

s a

nd n

itro

gen m

eta

bolis

m

CAGL0J09240g -0.99857 LYS21 LYS21 Putative homocitrate synthase involved in lysine biosynthesis; localizes to the nucleus;

CAGL0G06028g -1.11233 ARO9 ARO9 Aromatic aminotransferase II; catalyses the first step of tryptophan, phenylalanine, and tyrosine catabolism;

CAGL0J11308g -1.11462 NPR1 Protein kinase; stabilizes several plasma membrane amino acid transporters by antagonizing their ubiquitin-mediated degradation;

CAGL0K07634g -1.11954 GAT1 GAT1 Transcriptional activator of nitrogen catabolite repression genes;

CAGL0L12254g -1.12202 ALT1 Alanine transaminase (glutamic pyruvic transaminase); involved in alanine biosynthesis and catabolism;

CAGL0J11484g -1.23034 DUG3 DUG3 Component of glutamine amidotransferase (GATase II); forms a complex with Dug2p to degrade glutathione (GSH);

CAGL0I10747g -1.27301 MEP1 Ammonium permease;

CAGL0G01254g -1.31622 ARO8 ARO8 Aromatic aminotransferase I; expression is regulated by general control of amino acid biosynthesis;

CAGL0L05588g -1.33879 NIT2 Has domain(s) with predicted hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds activity and role in nitrogen compound metabolic process;

CAGL0C01595g -1.36502 HIS7 Imidazole glycerol phosphate synthase; glutamine amidotransferase:cyclase that catalyses the fifth step of histidine biosynthesis;

CAGL0H03795g -1.38612 LEU2 LEU2 Beta-isopropylmalate dehydrogenase (IMDH); catalyses the third step in the leucine biosynthesis pathway;

59

Am

ino

acid

s a

nd n

itro

gen m

eta

bolis

m

CAGL0H00396g -1.39493 LEU3 Sequence-specific DNA binding RNA Polymerase II transcription factor that acts as both an activator and a repressor of genes involved in leucine biosynthesis;

CAGL0J02398g -1.42757 HIS6 Enzyme that catalyses the fourth step in the histidine pathway;

CAGL0M12837g -1.46056 SER33 3-phosphoglycerate dehydrogenase and alpha-ketoglutarate reductase;

CAGL0G06732g -1.46634 LEU9 Alpha-isopropylmalate synthase II (2-isopropylmalate synthase);

CAGL0E05632g -1.49483 PUT4 Proline permease;

CAGL0B00902g -1.50095 HIS4 HIS4 Multifunctional enzyme with phosphoribosyl-ATP pyrophosphatase; catalyses several steps in the biosynthesis of histidine;

CAGL0B00286g -1.50651 CHA1 Catabolic L-serine (L-threonine) deaminase; catalyses the degradation of both L-serine and L-threonine; required to use serine or threonine as the sole nitrogen source;

CAGL0M04059g -1.53446 PHA2 Prephenate dehydratase; catalyses the conversion of prephanate to phenylpyruvate, which is a step in the phenylalanine biosynthesis pathway;

CAGL0L00429g -1.57231 GVC2 P subunit of the mitochondrial glycine decarboxylase complex; glycine decarboxylase is required for the catabolism of glycine to 5,10-methylene-THF;

CAGL0M03465g -1.65558 ADY2 Putative transmembrane protein involved in export of ammonia;

CAGL0L00759g -1.66124 HIS1 HIS1 ATP phosphoribosyltransferase; a hexameric enzyme, catalyses the first step in histidine biosynthesis;

CAGL0B02651g -1.67347 MET32 Zinc-finger DNA-binding transcription factor; involved in transcriptional regulation of the methionine biosynthetic genes;

CAGL0A01199g -1.6808 DIP5 DIP5 Dicarboxylic amino acid permease; mediates high-affinity and high-capacity transport of L-glutamate and L-aspartate;

CAGL0G04389g -1.69505 GZF3 GATA zinc finger protein; negatively regulates nitrogen catabolic gene expression by competing with Gat1p for GATA site binding;

CAGL0K05357g -1.70818 GLN1 GLN1 Glutamate-ammonia ligase involved in glutamine biosynthesis; localizes to nuclear periphery;

CAGL0I09009g -1.72921 HIS2 Histidinolphosphatase;

CAGL0G09691g -1.78705 SER1 3-phosphoserine aminotransferase; catalyses the formation of phosphoserine from 3-phosphohydroxypyruvate, required for serine and glycine biosynthesis;

CAGL0I02530g -1.79888 FMO1 Flavin-containing monooxygenase; catalyses oxidation of biological thiols to maintain the ER redox buffer ratio for correct folding of disulfide-bonded proteins;

CAGL0J03762g -1.88179 MET7 MET7 Folylpolyglutamate synthetase; catalyses extension of the glutamate chains of the folate coenzymes, required for methionine synthesis and for maintenance of mitochondrial DNA;

CAGL0M00880g -1.89974 CAR2 CAR2 L-ornithine transaminase (OTAse); catalyses the second step of arginine degradation;

60

Am

ino

acid

s a

nd n

itro

gen m

eta

bolis

m

CAGL0B01012g -1.91499 AGP1 Low-affinity amino acid permease with broad substrate range; involved in uptake of asparagine, glutamine, and other amino acids;

CAGL0A03212g -1.95265 ATO3 Plasma membrane protein, putative ammonium transporter;

CAGL0G04741g -1.96844 LEU4 Alpha-isopropylmalate synthase involved in leucine biosynthesis; catalyses the conversion of 2-keto-isovalerate to 2-isopropylmalate;

CAGL0G01210g -2.02768 NIT3 NIT3 Nit protein;

CAGL0L03157g -2.09495 DAL80 Negative regulator of genes in multiple nitrogen degradation pathways

CAGL0M12188g -2.10399 GCV3 H subunit of the mitochondrial glycine decarboxylase complex; also required for all protein lipoylation; expression is regulated by levels of 5,10-methylene-THF;

CAGL0L12012g -2.18056 TMT1 Trans-aconitate methyltransferase; cytosolic enzyme that catalyses the methyl esterification of 3-isopropylmalate, an intermediate of the leucine biosynthetic pathway, and trans-aconitate, which inhibits the citric acid cycle;

CAGL0M04499g -2.31365 PUT1 Proline oxidase; nuclear-encoded mitochondrial protein involved in utilization of proline as sole nitrogen source;

CAGL0L03267g -2.33187 GAP1 GAP1 General amino acid permease; Gap1p senses the presence of amino acid substrates to regulate localization to the plasma membrane when needed;

CAGL0L02937g -2.35933 HIS3 HIS3 Imidazoleglycerol-phosphate dehydratase; catalyses the sixth step in histidine biosynthesis;

CAGL0F09207g -2.44714 BAT1 BAT1 Mitochondrial branched-chain amino acid (BCAA) aminotransferase;

CAGL0C01243g -2.48498 HIS5 Histidinol-phosphate aminotransferase; catalyses the seventh step in histidine biosynthesis;

CAGL0I09284g -2.50301 SHM1 Mitochondrial serine hydroxymethyltransferase; converts serine to glycine plus 5,10 methylenetetrahydrofolate;

CAGL0L06094g -2.51332 STR3 STR3 Peroxisomal cystathionine beta-lyase; converts cystathionine into homocysteine; may be redox regulated by Gto1p;

CAGL0J06028g -2.51811 MEP2 MEP2 Ammonium permease involved in regulation of pseudohyphal growth;

CAGL0J04554g -2.53393 AAT2 Cytosolic aspartate aminotransferase involved in nitrogen metabolism; localizes to peroxisomes in oleate-grown cells;

CAGL0M05533g -2.69869 DUR1,2 DUR1,2 Urea amidolyase; contains both urea carboxylase and allophanate hydrolase activities, degrades urea to CO2 and NH3;

CAGL0D04356g -3.13106 GVC1 T subunit of the mitochondrial glycine decarboxylase complex; glycine decarboxylase is required for the catabolism of glycine to 5,10-methylene-THF;

CAGL0D04026g -3.4952 UGA1 Gamma-aminobutyrate (GABA) transaminase; 4-aminobutyrate aminotransferase; involved in the 4-aminobutyrate and glutamate degradation pathways;

CAGL0K03157g -4.47333 DUR31 DUR3 Plasma membrane transporter for both urea and polyamines; expression is highly sensitive to nitrogen catabolite repression;

61

Auto

phagy CAGL0M13651g -1.33045 DCK1

Signaling protein involved in mitophagy; localizes to the plasma membrane, and shifts to mitochondria under oxidative stress

CAGL0H08261g -1.49677 PCR1 Serine-type carboxypeptidase involved in vacuolar protein catabolism, vacuolar zymogen activation, phytochelatin biosynthesis and macroautophagy;

CAGL0J10846g -1.66616 PCL5 Cyclin; interacts with and phosphorylated by Pho85p cyclin-dependent kinase (Cdk), induced by Gcn4p at level of transcription, specifically required for Gcn4p degradation;

CAGL0H00704g -1.6959 ATG41 Required for selective and nonselective autophagy, and mitophagy;

CAGL0M01848g -1.72022 SLM4 Role in lysosomal microautophagy; Component of the EGO and GSE complexes;

CAGL0M02915g -2.69714 ICY2 Ortholog(s) have role in CVT pathway, autophagosome assembly, mitophagy;

Carb

on a

nd E

nerg

y M

eta

bolis

m

CAGL0H05445g -0.93442 PGI1 PGI6 Phosphoglucose isomerase; catalyses the interconversion of glucose-6-phosphate and fructose-6-phosphate; involved in gluconeogenesis and glycolysis;

CAGL0M14025g -0.99104 YMR315W Protein with NADP(H) oxidoreductase activity; transcription is regulated by Stb5p in response to NADPH depletion induced by diamide;

CAGL0L10538g -1.07095 PAN5 2-dehydropantoate 2-reductase; part of the pantothenic acid pathway;

CAGL0D00770g -1.08261 IDP1 IDP1 Mitochondrial NADP-specific isocitrate dehydrogenase; catalyzes the oxidation of isocitrate to alpha-ketoglutarate;

CAGL0D02156g -1.13259 GNA1 Glucosamine 6-phosphate N-acetyltransferase involved in UDP-N-acetylglucosamine biosynthesis; localizes to nucleus and cytoplasm in high-throughput studies

CAGL0J02904g -1.15601 GIP2 Putative regulatory subunit of protein phosphatase Glc7p

CAGL0L06116g -1.22989 YGL185C Putative protein with sequence similar to hydroxyacid dehydrogenases

CAGL0B04917g -1.24676 IDP2 Cytosolic NADP-specific isocitrate dehydrogenase; catalyses oxidation of isocitrate to alpha-ketoglutarate;

CAGL0E06358g -1.26785 GPM1 GPM1 Tetrameric phosphoglycerate mutase; mediates the conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and the reverse reaction during gluconeogenesis;

CAGL0K07480g -1.30903 PGM1 PGM1 Phosphoglucomutase, minor isoform; catalyses the conversion from glucose-1-phosphate to glucose-6-phosphate, which is a key step in hexose metabolism;

CAGL0K01705g -1.32233 GPM2 Non-functional homolog of Gpm1p phosphoglycerate mutase; if functional, would convert 3-phosphoglycerate to 2-phosphoglycerate in glycolysis;

CAGL0D00198g -1.32305 BDH1 NAD-dependent (R,R)-butanediol dehydrogenase;

CAGL0B01727g -1.3259 YDR109C D-ribulokinase; carbohydrate kinase that specifically converts D-ribulose to D-ribulose 5-phosphate during pentose metabolism;

CAGL0M06721g -1.34655 CAB2 Subunit of the CoA-Synthesizing Protein Complex (CoA-SPC);

62

Carb

on a

nd E

nerg

y M

eta

bolis

m

CAGL0A02816g -1.41813 YPR1 NADPH-dependent aldo-keto reductase; reduces multiple substrates including 2-methylbutyraldehyde and D,L-glyceraldehyde, expression is induced by osmotic and oxidative stress;

CAGL0F07007g -1.44879 PKP2 Mitochondrial protein kinase;

CAGL0L03982g -1.47666 MLS1 Malate synthase, enzyme of the glyoxylate cycle; involved in utilization of non-fermentable carbon sources; expression is subject to carbon catabolite repression; localizes in peroxisomes during growth on oleic acid, otherwise cytosolic;

CAGL0I02178g -1.47314 MPC2 Highly conserved subunit of the mitochondrial pyruvate carrier (MPC);

CAGL0F08085g -1.51739 MPC3 Highly conserved subunit of the mitochondrial pyruvate carrier (MPC); expressed during growth on nonfermentable carbon sources, and heterodimerizes with Mpc1p to form the respiratory isoform of MPC;

CAGL0H08327g -1.53014 TPI1 TPI4 Triose phosphoisomerase involved in the glycolytic breakdown of carbohydrates into pyruvate; localizes to mitochondria and plasma membranes;

CAGL0L00803g -1.60428 PIG2 Putative type-1 protein phosphatase targeting subunit;

CAGL0A01089g -1.61545 PBI1 Predicted alcohol O-acetyltransferase activity and role in alcohol metabolic process;

CAGL0L06138g -1.61588 TPN1 TPN1 Plasma membrane pyridoxine (vitamin B6) transporter;

CAGL0M11242g -1.70775 YMR226C NADP(+)-dependent serine dehydrogenase and carbonyl reductase;

CAGL0J07084g -1.77561 YPL113C Glyoxylate reductase; acts on glyoxylate and hydroxypyruvate substrates;

CAGL0D02662g -1.84889 HTX2 High-affinity glucose transporter of the major facilitator superfamily;

CAGL0K00913g -2.0503 ADE3 Cytoplasmic trifunctional enzyme C1-tetrahydrofolate synthase;

CAGL0E01529g -2.06709 PFK27 6-phosphofructo-2-kinase; catalyses synthesis of fructose-2,6-bisphosphate; inhibited by phosphoenolpyruvate and sn-glycerol 3-phosphate;

CAGL0H04037g -2.07618 GAC1 Regulatory subunit of the Glc7p protein phosphatase type 1 complex; involved in glycogen metabolic process, mitotic spindle assembly checkpoint, meiotic nuclear division and response to heat;

CAGL0K05687g -2.07971 OYE2 Conserved NADPH oxidoreductase containing flavin mononucleotide (FMN); responsible for geraniol reduction into citronellol during fermentation;

CAGL0G09064g -2.16283 YIG1 Protein that interacts with glycerol 3-phosphatase;

63

Carb

on a

nd E

nerg

y M

eta

bolis

m

CAGL0M12551g -2.28076 RGI2 Protein of unknown function;

CAGL0J03080g -2.79239 RGI1 Protein of unknown function; involved in energy metabolism under respiratory conditions;

CAGL0C05555g -2.79664 GUD1 Guanine deaminase; a catabolic enzyme of the guanine salvage pathway producing xanthine and ammonia from guanine;

CAGL0L00649g -3.09351 ACS1 Acetyl-coA synthetase isoform; along with Acs2p, acetyl-coA synthetase isoform is the nuclear source of acetyl-coA for histone acetylation; expressed during growth on nonfermentable carbon sources and under aerobic conditions;

CAGL0B03663g -3.46621 CIT2 Citrate synthase, peroxisomal isozyme involved in glyoxylate cycle;

CAGL0L09086g -3.48199 CIT3 Dual specificity mitochondrial citrate and methylcitrate synthase;

CAGL0L09273g -3.54552 ICL2 2-methylisocitrate lyase of the mitochondrial matrix;

CAGL0H04939g -3.61317 FBP1 Fructose-1,6-bisphosphatase; key regulatory enzyme in the gluconeogenesis pathway, required for glucose metabolism;

CAGL0K04609g -4.39037 VHT High-affinity plasma membrane H+-biotin (vitamin H) symporter; mutation results in fatty acid auxotrophy; 12 transmembrane domain containing major facilitator subfamily member;

Cell

Cycle

CAGL0H02343g -0.95505 SAP30 Component of Rpd3L histone deacetylase complex; involved in silencing at telomeres, rDNA, and silent mating-type loci; involved in telomere maintenance

CAGL0M04565g -1.10489 ACF2 Intracellular beta-1,3-endoglucanase; expression is induced during sporulation; may have a role in cortical actin cytoskeleton assembly;

CAGL0I04444g -1.13133 ADE1 N-succinyl-5-aminoimidazole-4-carboxamide ribotide synthetase; required for 'de novo' purine nucleotide biosynthesis;

CAGL0L07898g -1.14149 MAK32 Protein necessary for stability of L-A dsRNA-containing particles;

CAGL0L00671g -1.14305 FCY21 FCY2 Purine-cytosine permease; mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation;

CAGL0M07447g -1.14321 YMR027W A metal-dependent phosphatase, part of the DUF89 protein family; dephosphorylates fructose-1-phosphate; human ortholog, C6orf211 is involved in response to DNA damage;

CAGL0K07111g -1.19479 DAD3 Essential subunit of the Dam1 complex (aka DASH complex);

CAGL0I05852g -1.20416 CLN2 Cyclin-dependent protein kinase (CDK) regulatory subunit involved in regulating passage through the cell cycle, as well as cell cycle re-entry after pheromone-induced arrest;

CAGL0I03564g -1.20968 CLB3 B-type cyclin involved in cell cycle progression;

CAGL0F00605g -1.24307 GLK1 EMI2 Non-essential protein of unknown function; required for transcriptional induction of the early meiotic-specific transcription factor IME1;

64

Cell

Cycle

CAGL0J09328g -1.25799 PCL2

Cyclin interacts with cyclin-dependent kinase Pho85p; member of the Pcl1,2-like subfamily, involved in the regulation of polarized growth and morphogenesis and progression through the cell cycle;

CAGL0H05203g -1.29675 RGL1 Regulator of Rho1p signalling, cofactor of Tus1p;

CAGL0J08822g -1.34956 CLB4 Cyclin-dependent kinase regulatory subunit involved in the regulation of the G2/M phase transition of the mitotic cell cycle;

CAGL0I03080g -1.55494 URA3 URA3 Orotidine-5'-phosphate decarboxylase involved in the 'de novo' biosynthesis of pyrimidine nucleobases; localized to the cytosol;

CAGL0M06765g -1.576 SIW14 Tyrosine phosphatase involved in actin organization and endocytosis; localized to the cytoplasm;

CAGL0F06589g -1.80116 KAR5 Protein required for nuclear membrane fusion during karyogamy;

CAGL0I09856g -1.84046 SFG1 Nuclear protein putative transcription factor;

CAGL0F02167g -1.89837 MSH4 Protein involved in meiotic recombination;

CAGL0F05841g -1.92467 CSN9 Subunit of the Cop9 signalosome;

CAGL0M09207g -1.9432 STE18 G protein gamma subunit; forms a dimer with Ste4p to activate the mating signaling pathway, forms a heterotrimer with Gpa1p and Ste4p to dampen signaling;

CAGL0M08184g -2.08595 STE3 STE3 Receptor for a factor pheromone; couples to MAP kinase cascade to mediate pheromone response; transcribed in alpha cells and required for mating by alpha cells, ligand bound receptors endocytosed and recycled to the plasma membrane;

CAGL0H02959g -2.14609 TOS8 TOS8 Homeodomain-containing protein and putative transcription factor; found associated with chromatin; target of SBF transcription factor;

CAGL0A01716g -2.1882 PNC1 Nicotinamidase that converts nicotinamide to nicotinic acid; part of the NAD(+) salvage pathway; required for life span extension by calorie restriction; lacks a peroxisomal targeting signal but is imported into peroxisomes via binding to Gpd1p;

CAGL0L06512g -2.24257 BNS1 Orthologs have role in meiotic cell cycle;

CAGL0M10153g -2.35249 STE20 Cdc42p-activated signal transducing kinase; involved in pheromone response, pseudohyphal/invasive growth, vacuole inheritance, down-regulation of sterol uptake;

CAGL0L02761g -3.29114 STE4 Signaling protein for pheromone-dependent signal transduction involved in conjugation with cellular fusion; contributes to invasive growth in response to glucose limitation, chemotropism, regulation of RNA-mediated transposition;

CAGL0H00594g -3.39272 BBP1 Protein required for the spindle pole body (SPB) duplication;

CAGL0F03883g -0.87505 GAS4 GAS3 Putative 1,3-beta-glucanosyltransferase; has similarity go other GAS family members; low abundance, possibly inactive member of the GAS family of GPI-containing proteins;

65

Cell

Wall

Org

aniz

ation

CAGL0I07953g -0.94111 AVO1 Component of a membrane-bound complex containing the Tor2p kinase;

CAGL0E01419g -0.95355 YPS2 MCK7 GPI-anchored aspartyl protease; member of the yapsin family of proteases involved in cell wall growth and maintenance;

CAGL0K02277g -1.2972 DSE1 Daughter cell-specific protein; may regulate cross-talk between the mating and filamentation pathways;

CAGL0H07997g -1.36152 KNH1 KNH1 Protein with similarity to Kre9p; Kre9p is involved in cell wall beta 1,6-glucan synthesis;

CAGL0K00715g -1.41519 RTA1 RTA1 Protein involved in 7-aminocholesterol resistance; has seven potential membrane-spanning regions; expression is induced under both low-heme and low-oxygen conditions; member of the fungal lipid-translocating exporter (LTE) family of protein;

CAGL0E01793g -1.53935 YPS6 YPS1 Aspartic protease; hyperglycosylated member of the yapsin family of proteases, attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor;

CAGL0D00286g -1.63019 BMT1 Beta-mannosyltransferase;

CAGL0A04081g -1.65269 NCW2 Structural constituent of the cell wall;

CAGL0G09955g -1.77126 DPM1 Dolichyl-phosphate mannose synthase that catalyses the formation of the mannosyl donor required for the production of dolichol-linked oligosaccharides used in glycosylation, as well as the glycolipid core required for GPI anchor biosynthesis;

CAGL0L01771g -1.97531 PHD1 Transcriptional activator that enhances pseudohyphal growth;

CAGL0I00484g -2.04468 EXG1 Major exo-1,3-beta-glucanase of the cell wall; involved in cell wall beta-glucan assembly; exists as three differentially glycosylated isoenzymes;

CAGL0J01463g -2.21064 CWP1 A structural constituent of the cell wall involved in both fungal-type cell wall organization and ascospore-type prospore membrane assembly;

CAGL0H04983g -2.55872 PSA1 PSA1 GDP-mannose pyrophosphorylase (mannose-1-phosphate guanyltransferase); synthesizes GDP-mannose from GTP and mannose-1-phosphate in cell wall biosynthesis;

CAGL0G09515g -1.12324 LDS1 Protein Involved in spore wall assembly;

CAGL0H06237g -1.28641 SPR3 Septin protein involved in sporulation;

CAGL0C05577g -2.73036 ADY3 Protein required for spore wall formation;

Str

ess R

esponse

CAGL0C02321g -0.98948 PHM8 PHM8 Lysophosphatidic acid (LPA) phosphatase, nucleotidase;

CAGL0E06380g -1.22987 NNR2 Widely-conserved NADHX dehydratase; converts (S)-NADHX to NADH in ATP-dependent manner; YKL151C promoter contains STREs (stress response elements);

CAGL0J08613g -1.45039 YVC1 Vacuolar cation channel; mediates release of Ca2+ from the vacuole in response to hyperosmotic shock;

CAGL0G08019g -1.97134 ILT1 Protein of unknown function; deletion confers sensitivity to cationic compounds;

66

CAGL0M07293g -2.38988 PDR12 Plasma membrane ATP-binding cassette (ABC) transporter; weak-acid-inducible multidrug transporter required for weak organic acid resistance;

CAGL0D00154g -3.3439 AQY1 AQY1 Spore-specific water channel; mediates the transport of water across cell membranes, developmentally controlled;

Ion H

om

eosta

sis

CAGL0D04708g -1.06227 CTR1 Copper ion transmembrane transporter that has role in copper ion import; localizes to plasma membrane;

CAGL0F06413g -1.1677 FET3 FET3 Ferro-O2-oxidoreductase; multicopper oxidase that oxidizes ferrous (Fe2+) to ferric iron (Fe3+) for subsequent cellular uptake by transmembrane permease Ftr1p;

CAGL0K07392g -1.44138 ZRC1 Vacuolar zinc ion transmembrane transporter involved in cellular zinc homeostasis;

CAGL0G04631g -1.59891 IZH1 Membrane protein involved in zinc ion homeostasis;

CAGL0I06743g -1.66854 FTR1 FTR1 High-affinity iron transporter of the plasma membrane involved in iron assimilation; also implicated in arsenate transport;

CAGL0J04048g -2.06475 ISU2 Mitochondrial protein required for iron-sulfur protein synthesis;

CAGL0F00187g -2.11202 FET4 FET4 Low-affinity Fe(II) transporter of the plasma membrane;

CAGL0K12100g -2.13942 HEM13 Coproporphyrinogen III oxidase; oxygen-requiring enzyme that catalyses sixth step in heme biosynthetic pathway; transcription is repressed by oxygen and heme (via Rox1p and Hap1p);

CAGL0E02519g -2.63458 IZH2 Plasma membrane receptor for plant antifungal osmotin;

CAGL0I07491g -2.7757 IZH4 Membrane protein involved in zinc ion homeostasis;

CAGL0F05929g -1.97557 RCH1 Putative transporter; localizes to the plasma membrane in response to high levels of extracellular calcium;

Lip

ids M

eta

bolis

m

CAGL0D02618g -1.0454 PEX11 PEX11 Peroxisomal protein required for medium-chain fatty acid oxidation; also required for peroxisome proliferation, possibly by inducing membrane curvature;

CAGL0K07458g -1.19471 YPK1 Protein kinase active on serine and threonine residues; participates in cellular sphingolipid homeostasis by negatively regulating sphingolipid biosynthesis; also involved in glycerophospholipid metabolism and response to mating pheromone;

CAGL0E06424g -1.20283 MCR1 Mitochondrial NADH-cytochrome b5 reductase; involved in ergosterol biosynthesis;

CAGL0J02684g -1.24932 ERG28 Endoplasmic reticulum membrane protein;

CAGL0H09460g -1.36484 FAA2 Medium chain fatty acyl-CoA synthetase; activates imported fatty acids; accepts a wide range of fatty acid chain lengths with a preference for medium chains, C9:0-C13:0; localized to the peroxisome;

CAGL0K08844g -1.36745 AIM17 Role in carnitine biosynthetic process;

CAGL0J02970g -1.41857 CEM1 Putative 3-ketoacyl-acyl carrier protein synthase involved in fatty acid biosynthesis; localized to mitochondria

67

Lip

ids M

eta

bolis

m

CAGL0K04125g -1.44264 ACB1 Acyl-CoA-binding protein; transports newly synthesized acyl-CoA esters from fatty acid synthetase (Fas1p-Fas2p) to acyl-CoA-consuming processes;

CAGL0C03487g -1.46679 YNR048W Potential noncatalytic subunit for phospholipid translocase Dnf3p;

CAGL0F05071g -1.50999 ECI1 Peroxisomal delta3,delta2-enoyl-CoA isomerase; hexameric protein that converts 3-hexenoyl-CoA to trans-2-hexenoyl-CoA, essential for the beta-oxidation of unsaturated fatty acids, oleate-induced;

CAGL0H05005g -1.55751 CSR1 Phosphatidylinositol transfer protein; has a potential role in regulating lipid and fatty acid metabolism under heme-depleted conditions; interacts specifically with thioredoxin peroxidase;

CAGL0L09493g -1.60445 EGH1 Steryl-beta-glucosidase with broad specificity for aglycones;

CAGL0F01793g -1.66559 ERG3 ERG3 C-5 sterol desaturase, catalyzes formation of C-5(6) double bond in episterol that yields 5,7,24(28)-ergostatrienol in third to last step of ergosterol biosynthesis pathway;

CAGL0H01375g -1.72326 SUR2 SUR2 Sphinganine C4-hydroxylase; catalyses the conversion of sphinganine to phytosphingosine in sphingolipid biosynthesis;

CAGL0I10923g -1.76774 LIP5 Protein involved in biosynthesis of the coenzyme lipoic acid;

CAGL0F01111g -1.8136 OPI10 Protein with a possible role in phospholipid biosynthesis;

Lip

ids M

eta

bolis

m

CAGL0I04070g -1.87389 SLC1 1-acyl-sn-glycerol-3-phosphate acyltransferase; catalyses the acylation of lysophosphatidic acid to form phosphatidic acid, a key intermediate in lipid metabolism;

CAGL0E04334g -2.07211 ERG11 ERG11 Lanosterol 14-alpha-demethylase; catalyses the demethylation of lanosterol during the biosynthesis of ergosterol;

CAGL0D00946g -2.13224 CSR1 Phosphatidylinositol transfer protein; has a potential role in regulating lipid and fatty acid metabolism under heme-depleted conditions; interacts specifically with thioredoxin peroxidase; may have a role in oxidative stress resistance;

CAGL0I08305g -2.22972 YAT2 Carnitine acetyltransferase; has similarity to Yat1p,

CAGL0J11836g -2.28484 CAT2 Carnitine acetyl-CoA transferase; present in both mitochondria and peroxisomes;

CAGL0H08712g -2.31275 Predicted phosphatidylinositol binding activity;

CAGL0J03916g -2.40261 HES1 Protein implicated in the regulation of ergosterol biosynthesis;

CAGL0K12210g -2.41006 YMC2 Putative mitochondrial inner membrane transporter; proposed role in oleate metabolism and glutamate biosynthesis;

CAGL0I00418g -2.48976 OLE1 Delta(9) fatty acid desaturase; required for monounsaturated fatty acid synthesis and for normal distribution of mitochondria;

CAGL0K01771g -2.56931 YAT1 Outer mitochondrial carnitine acetyltransferase;

CAGL0B04543g -2.71771 CRC1 Mitochondrial inner membrane carnitine transporter;

68

CAGL0C05027g -2.86221 YAT1 Outer mitochondrial carnitine acetyltransferase;

CAGL0I02134g -3.93383 PEX21B PEX18 primarily responsible for peroxisomal import during growth on oleate, and expression is induced during oleate growth;

Mem

bra

ne T

ransport

CAGL0L10868g -1.17576 FSF1 Putative protein; predicted to be an alpha-isopropylmalate carrier; belongs to the sideroblastic-associated protein family;

CAGL0G05093g -1.41344 YDR061W Protein with similarity to ABC transporter family members;

CAGL0I09108g -1.4464 ESBP6 Protein with similarity to monocarboxylate permeases;

CAGL0L03696g -1.71386 ECM3 Predicted role in transmembrane transport and integral component of membrane localization;

CAGL0M14113g -2.55841 TNR2 THI7 Plasma membrane transporter responsible for the uptake of thiamine;

CAGL0B04213g -3.23335 RGC1 Putative regulator of the Fps1p glycerol channel;

Mitochondrial F

unction

CAGL0K03047g -0.91715 ABF2 Mitochondrial DNA-binding protein; involved in mitochondrial DNA replication and recombination;

CAGL0K07436g -1.03849 YHM2 Citrate and oxoglutarate carrier protein; exports citrate from and imports oxoglutarate into the mitochondrion, causing net export of NADPH reducing equivalents;

CAGL0K07876g -1.09082 MDM36 Mitochondrial protein; required for normal mitochondrial morphology and inheritance; component of the mitochondria-ER-cortex-ancor (MECA);

CAGL0K10274g -1.12583 CAT5 Protein required for ubiquinone (Coenzyme Q) biosynthesis;

CAGL0M12210g -1.14721 BOL1 Mitochondrial matrix protein involved in Fe-S cluster biogenesis;

CAGL0J09900g -1.24321 POR1 Voltage-gated anion channel; involved in ion transport, cellular redox homeostasis, and mitochondrial organization; integral membrane protein of the outer mitochondrial membrane;

CAGL0K11616g -1.24908 OAC1 Mitochondrial inner membrane transporter; transports oxaloacetate, sulfate, thiosulfate, and isopropylmalate; member of the mitochondrial carrier family;

CAGL0G06402g -1.52254 ISA1 Mitochondrial iron-sulfur cluster binding protein involved in iron-sulfur cluster assembly and biosynthesis of biotin;

CAGL0G03905g -1.77587 CIR2 Putative ortholog of human ETF-dH; found in a large supramolecular complex with other mitochondrial dehydrogenases; may have a role in oxidative stress response;

CAGL0E03850g -1.95815 MSS18 Nuclear encoded protein needed for splicing of mitochondrial intron;

CAGL0K04279g -2.00875 SDH2 Iron-sulfur protein subunit of succinate dehydrogenase;

CAGL0D04840g -2.26155 SCM4 SCM4 Mitochondrial outer membrane protein of unknown function;

CAGL0J09262g -2.52461 CRD1 Cardiolipin synthase; responsible for biosynthesis of cardiolipin, which is required for normal mitochondrial function;

69

CAGL0I03784g -3.17851 STF1 Protein involved in regulation of the mitochondrial F1F0-ATP synthase;

CAGL0L09108g -3.25633 ADH3 Mitochondrial alcohol dehydrogenase isozyme III; involved in the shuttling of mitochondrial NADH to the cytosol under anaerobic conditions and ethanol production;

CAGL0J01441g -3.394 PDH1 Putative 2-methylcitrate dehydratase; mitochondrial protein that participates in respiration; induced by diauxic shift;

OS

R

CAGL0J03608g -1.00709 HCM1 Sequence-specific DNA binding transcription factor involved in spindle pole body organization, mitochondrion organization, and the cellular response to oxidative stress;

CAGL0I04554g -1.03338 GRX7 GRX7 Cis-golgi localized monothiol glutaredoxin; more similar in activity to dithiol than other monothiol glutaredoxins; involved in the oxidative stress response;

CAGL0M13189g -1.5682 MSN4 MSN4 Stress-responsive transcriptional activator;

CAGL0F08745g -1.76402 STF2 Protein involved in resistance to desiccation stress; Stf2p exhibits antioxidant properties, and its overexpression prevents ROS accumulation and apoptosis;

CAGL0H03971g -2.17579 YPC4 Protein of unknown function; has sequence and structural similarity to flavodoxins; predicted to be palmitoylated;

CAGL0L11990g -2.6196 GRX4 Glutathione-dependent oxidoreductase; hydroperoxide and superoxide-radical responsive; monothiol glutaredoxin subfamily member along with Grx3p and Grx5p; protects cells from oxidative damage;

Pro

tein

Degra

datio

n

CAGL0J07282g -0.97602 GPB1 Multistep regulator of cAMP-PKA signalling;

CAGL0L05038g -1.13698 MUD2 Protein involved in early pre-mRNA splicing;

CAGL0I05522g -1.40413 UBP9 Ubiquitin-specific protease that cleaves ubiquitin-protein fusions;

CAGL0A02024g -1.48622 LAG2 Protein that negatively regulates the SCF E3-ubiquitin ligase;

CAGL0K12254g -1.55469 VID24

GID Complex regulatory subunit; binds GID Complex in response to glucose through interactions with complex member Vid28p; regulates fructose-1,6-bisphosphatase (FBPase) targeting to the vacuole; promotes proteasome-dependent catabolite degradation of FBPase; peripheral membrane protein located at Vid (vacuole import and degradation) vesicles;

CAGL0E02651g -1.75106 YSP3 Putative precursor of the subtilisin-like protease III;

CAGL0M03971g -1.92847 SKP2 F-box protein predicted to be part of an SCF ubiquitin protease complex; involved in regulating protein levels of sulfur metabolism enzymes;

CAGL0K08536g -2.17568 APE1 APE1 A metalloaminopeptidase involved in vacuolar protein catabolysis;

CAGL0G02563g -3.1535 UBP1 Ubiquitin-specific protease; cleaves ubiquitin from ubiquitinated proteins; UBP11 has a paralog, UBP7, that arose from the whole genome duplication;

CAGL0I05742g -0.98768 CSE2 Subunit of the RNA polymerase II mediator complex;

70

Tra

nscription

CAGL0C02519g -1.06576 MIG3 Transcriptional regulator;

CAGL0D01430g -1.1584 HOS1 Class I histone deacetylase (HDAC) family member;

CAGL0J03806g -1.2031 WTM1 Transcriptional modulator;

CAGL0I00902g -1.20359 GAT2 Protein containing GATA family zinc finger motifs;

CAGL0H02101g -1.33229 RTC3 Protein of unknown function involved in RNA metabolism;

CAGL0K04697g -1.35647 STP4 Protein containing a Kruppel-type zinc-finger domain; similar to Stp1p, Stp2p; predicted transcription factor;

CAGL0E02315g -1.61124 HTZ1 Histone variant H2AZ; exchanged for histone H2A in nucleosomes by the SWR1 complex; involved in transcriptional regulation through prevention of the spread of silent heterochromatin;

CAGL0K02585g -1.72537 YAP3 YAP3 Basic leucine zipper (bZIP) transcription factor;

CAGL0H02497g -1.77015 GFD1 Coiled-coiled protein of unknown function

CAGL0G02739g -1.91363 XBP1 Transcriptional repressor; binds promoter sequences of cyclin genes, CYS3, and SMF2; not expressed during log phase of growth, but induced by stress or starvation during mitosis, and late in meiosis;

CAGL0B03421g -1.97003 HAP1 Zinc finger transcription factor; involved in the complex regulation of gene expression in response to levels of heme and oxygen; localizes to the mitochondrion as well as to the nucleus;

CAGL0L06776g -2.88687 GAT2 Protein containing GATA family zinc finger motifs;

CAGL0J03960g -3.30357 WTM2 Transcriptional modulator;

Unknow

n F

unction

CAGL0M04939g -0.92153 TRI1 Protein of unknown function;

CAGL0D01210g -0.97272 Protein of unknown function;

CAGL0A02882g -1.00815 Protein of unknown function;

CAGL0J11110g -1.01471 MRX12 Protein of unknown function;

CAGL0F04125g -1.0419 YBL029W Protein of unknown function;

CAGL0B03861g -1.08636 YJR011C Protein of unknown function;

CAGL0I10224g -1.12659 Protein of unknown function;

71

Unknow

n F

unction

CAGL0H08305g -1.13147 Protein of unknown function;

CAGL0F01969g -1.1319 YLR049C Protein of unknown function;

CAGL0L00205g -1.13297 Protein of unknown function;

CAGL0K06105g -1.14445 BOP2 Protein of unknown function;

CAGL0C03311g -1.22963 Protein of unknown function;

CAGL0J08547g -1.23929 LPX1 Protein of unknown function;

CAGL0E03366g -1.25978 Protein of unknown function;

CAGL0J09416g -1.27351 SNA4 Protein of unknown function;

CAGL0F04697g -1.31747 YLR257W Protein of unknown function;

CAGL0L02343g -1.32615 IRC22 Protein of unknown function;

CAGL0M05995g -1.33365 PET10 Protein of unknown function;

CAGL0I08591g -1.35668 YER185C Putative protein of unknown function;

CAGL0M04620g -1.35904 Protein of unknown function;

CAGL0M11000g -1.37075 EGO2 Protein of unknown function;

CAGL0H00825g -1.41883 YPL245W Putative protein of unknown function

CAGL0H06831g -1.42806 FRM2 Putative protein of unknown function;

CAGL0H09966g -1.43102 FMP23 Protein whose biological role is unknown;

CAGL0B03883g -1.44161 Protein of unknown function;

CAGL0E06072g -1.44804 YPL229W Putative protein of unknown function

CAGL0G05962g -1.45762 YHR140W Protein whose biological role is unknown;

CAGL0G01474g -1.47107 BOP3 Protein of unknown function;

72

Unknow

n F

unction

CAGL0B03817g -1.50401 MHO1 Protein of unknown function;

CAGL0K08206g -1.51066 YGL140C Protein of unknown function;

CAGL0I07315g -1.52885 YOR131C Protein of unknown function;

CAGL0H03201g -1.5357 LCL3 Protein of unknown function;

CAGL0K06380g -1.56091 Protein of unknown function;

CAGL0M03245g -1.56805 PXP1 Protein of unknown function;

CAGL0M11902g -1.57131 YOR338W Protein of unknown function;

CAGL0K05665g -1.57797 Protein of unknown function;

CAGL0B04191g -1.66725 YPR114W Protein of unknown function;

CAGL0E04554g -1.66844 Protein of unknown function;

CAGL0E03267g -1.70132 RTS3 Protein of unknown function;

CAGL0A02981g -1.71703 Protein of unknown function;

CAGL0D02761g -1.74415 Protein of unknown function;

CAGL0J07876g -1.84644 RTC4 Protein of unknown function;

CAGL0G08954g -1.88765 YOL019W Putative protein of unknown function;

CAGL0L06864g -1.93364 SIP5 Putative protein of unknown function;

CAGL0K08338g -1.97125 Protein of unknown function

CAGL0D02728g -2.05078 AIM46 Protein of unknown function;

CAGL0B01595g -2.07844 Protein of unknown function

CAGL0C02387g -2.1407 YER034W Protein of unknown function

CAGL0K07678g -2.17687 Protein of unknown function

73

Unknow

n F

unction

CAGL0L08547g -2.21251 Protein of unknown function;

CAGL0I00374g -2.2957 GEP7 Protein of unknown function

CAGL0L12518g -2.30734 Putative protein of unknown function;

CAGL0G02057g -2.33703 YOR062C Putative protein of unknown function;

CAGL0K11946g -2.34025 Protein of unknown function;

CAGL0A02299g -2.38338 Protein of unknown function;

CAGL0K05390g -2.45913 Protein of unknown function;

CAGL0J12034g -2.47316 Protein of unknown function;

CAGL0B00116g -2.53226 Putative protein of unknown function;

CAGL0M05401g -2.65877 YBR201C-

A Protein of unknown function;

CAGL0A02255g -2.69676 Protein of unknown function;

CAGL0K07205g -2.69914 YGL117W Putative protein of unknown function

CAGL0I01276g -2.79025 YHR112C Protein whose biological role is unknown;

CAGL0E05456g -2.91073 Protein of unknown function;

CAGL0D00990g -2.91871 YDL057W Protein of unknown function;

CAGL0A02277g -3.05777 YCL049C Protein of unknown function

CAGL0H07337g -3.14667 Protein of unknown function

CAGL0I05610g -3.14971 YNR014W Protein of unknown function

CAGL0L09999g -3.29354 BSC2 Putative protein of unknown function

CAGL0L13392g -3.34489 Putative protein of unknown function

CAGL0M13266g -3.52083 Protein of unknown function

74

CAGL0G06050g -3.66053 Protein of unknown function CAGL0A02002g -3.83133 Protein of unknown function;

CAGL0D00869g -4.50172 Putative protein of unknown function;

CAGL0M08558g -4.6186 Putative protein of unknown function;

CAGL0F07953g -4.6375 SPG1 Protein whose biological role is unknown;

CAGL0F00116g -4.77997 Protein of unknown function;

CAGL0H00572g -5.08133 TDA4 Putative protein of unknown function;