Dr. Jose F. Tort

1

2

Helminths cause chronic infections

highly prevalent worldwide

More relevant in developing regions

Effect on economy through infection of productive species

Developmental relevanceNematodes with simple cycles Trematodes with complex cycles (2 or more hosts)Programed development FlexibilityAmount of cells established Sexual & asexual phases (neoblasts)

3

Helminths cause chronic infections,highly prevalent worldwide biological models of developmentphylogenetically relevant

Importance

Difficulties

they have large genomes (108 bp/102 Mb)the relevant species are diverseComplex life cyclesCulturing and strains difficult

Developing area Different metazoan lineages with

abundant species (30K parasitic) Relatively poorly represented in

public databases

5

Rapid increase in the number of genomes and transcriptomes being sequenced due to accessibility of novel technology

Genomes Schistosoma mansoniSchistosoma japonicum

Transcriptome Fasciola hepaticaClonorchis cinensisOpistorchis viverriniEchinostoma paranaensiParagonimus westermanii

FUNCTIONAL GENOMIC TOOLSDirect mutantsReverse directed mutagenesis (specific change)

trasngenesis (added gene)knockouts (inactivated gene)antisense & RNAi (knock down)

Novel genes with unknown function

8

Generate functional genomic tools for the study of Fasciola hepatica biology and facilitate design of novel control interventions

Two approaches

1. Optimizing RNA interference as a rapid tool for gene silencing.

2. Stable Transgenesis mediated by replication incompetent pseudotyped retrovirus

A rapid method to study gene function

Knock down of a specific transcript

Well established in planaria

Optimized in Schistosomes

10

“cosuppression” in plants (Napoli et al. 90) “quelling” in Neurospora (92) C. elegans (Fire et al., 1998) Drosophila (Carthew et al., 98) Planaria (Newmark et al., 98) Trypanosomes (Ullu et al., 98) Hydra (Lohmann et al., 99) Zebrafish (Wargelius et al., 99) Ratón (Wianny & Zernicka-Goetz, 00)

Generación del ARNds

Disponibilidad del ARNds

Determinar la eficiencia

Detección del efecto

Correlacionar la eficiencia de silenciamiento con efecto

Microinyección

Soaking

Feeding

Transfección(electroporación)

16,757 clones =

86% de ORFs predichos

Fenotipos:

NONV sterility or lethality,

GRO slow growth

VPEP defects

1,722 clones con fenotipos (10.3% )

17

BASAL MACHINERY ▪ Cell autonomous

TRASMISSION & AMPLIFICATION OF THE SILENCING SIGNAL ▪ Systemic

▪ Environmental (soaking/feeding)

Genes involved in the pathways identified

Injection

Soaking

Feeding

21

22

Several genes of the RNAi and miRNApathway identified in S.mansoni

They are also present in F.hepatica

23

K Peterson et al Parasitology.(10) 137(3): 485

Caenorhabditis Schistosoma Schistosoma Fasciola

elegans japonicum mansoni hepatica

dcr-1_1 8,00E-059 6,00E-096 7,00E-027

drh-2 0,5

drh-1_1 5,00E-005 9,00E-008

Dicer Complex rde-1_1 2,00E-045 4,00E-039 2,00E-035

rde-2_1 4,2 0,056

rde-3_1 9,00E-019 1E-23 2,00E-027

rde-4_1 5,6 4,8

rde-5

RISC Complex Alg-1 0 9,00E-084

Alg-2 0 4,00E-080

dfxr

tsn-1_1 2,00E-150 6,00E-069 1,00E-112

vig-1_1 0,66 0,25

ego-1_1 0,82 2,2

RdRp ampilf complex rrf-1_1 0,81 3,6

rrf-3_1 0,79 0,4

rrf-2

Systemic RNAi rsd-2_1 5,2 1

rsd-3

rsd-6_1 0,53 0,096

sid-1_1 2,00E-015 2,00E-039 5,00E-025

sid-2_1 1,7

Required for RNAi zfp-1_1 3,00E-032 3,00E-033 6,00E-035

smg-2_1 0 0 e-104

smg-5_1 2,8 0,25

smg-6_1 2,00E-011 1,00E-012

gfl-1_1 7,00E-042 5,00E-042 2,00E-035

mut-16_1 3,5 1,1

mut-7_1 0,66 2,2

mes-3

mes-8

primary miRNA cleavage Drsh-1 1,00E-136 2,00E-012

eri-1_1 5,00E-036 2,00E-035 2,00E-033

xrn-1_1 2,00E-095 1,50E-112 6,00E-059

rha-1_1 0 0 0

Two reports so far both in juvenile stage

Soaking and electroporation as delivery methods

Exogenous reporter and protease genes as targets

24

25

LUC mRNA is incorporated predominantly through the gut

and is expressed in electroporated NEJ

siRNA also ncorporated

Silencing of an exogenous reporter(LUC)

26

ExcystementmLuc Harvest

2 days 3hs

dsMalE

mLuc

1 day 1 days 3hs

1 day 1 days

dsLuc mLuc

3hs

Silencing of an endogenous gene (LAP)

LIFE STAGE TO BE TREATEDNEJ, eggs & miracidia

MOLECULES TO BE DELIVEREDlong dsRNA & siRNA

DELIVERY METHODsoaking, electroporation, feeding

EFFICIENCYDetermining the proportion of treated worms amount of dsRNA incorporated difficult to assess

(variability in effects)

30

Control

Electrop

ElectSoak

Soak

DOSE AND CONTROLS Appropriate controls needed.

Inclusion of both a mock and an irrelevant control desirable.

Dose effects

31

1 2 3 4

327

pb202pb 405pb1 2 3 4

CL2 GAPDH

3a 3b

1a 1b

2a 2b

sense antisense

Ctll

CL2

MalE

DOSE AND CONTROLS Appropriate controls needed.

Inclusion of both a mock and an irrelevant control desirable.

Dose effects

SPECIFICITY Not affecting other genes . More than one control gene

DETECTION OF EFFECTS

Phenotypes, mRNA variation

scarce detection of efects at protein level.

32

Why pursue transgenesis for trematodes?

Well established for functional genomics in model species

To facilitate gain of function and loss of function phenotypic analysis in liver flukes

To facilitate vector-based RNA interference

Potential for insertional mutagenesis in schistosomes

Production of Murine Leukemia Virus pseudotyped with VSVG (vesicular stomatitis virus glycoprotein)

with endogenous promoters and reporters

Transfection with repilcationincopetent VSVG-MMLV virus

35

Helminths cause chronic infections,highly prevalent worldwide biological models of developmentphylogenetically relevant

Importance

Difficultiesthey have large genomes (108 bp/102 Mb)the relevant species are diverseComplex life cyclesCulturing and strains are scarceMulitcellularity

1. DNA constructs and virus production

2. Transduction of NEJs

iv Excystement

day 0

NEJ treatedday 1

NEJ harvestedday 3

mRNA & PROTEINqRT-PCR

In situ hybridization

Wblot, Immunolocalization

Activity assays

GENOMIC INTEGRATIONAnchored PCR approaches

Transgene copy number/ng of total gDNA

Direct PCR blot for transgene sequences

RETROVIRAL INFECTION Immunolocalization of virus proteins:

Anti-VSVG

3. Exploration of the

transduced parasites

MLV_30’ MLV_180’CTOL

Immunolocalization of virus protein: anti-VSVG

mRNA & PROTEIN

GENOMIC INTEGRATION

RETROVIRAL INFECTION

M

GFP

NEO

NTC

GFP

NEO

final wash

GFP

NEO

plasmid control (+)

Direct PCR and blot targeting the

transgene sequence

mRNA & PROTEIN

GENOMIC INTEGRATION

RETROVIRAL INFECTION

0

5

10

15

20

25

30

35

40

45

soaked electroporated

Luc

Co

py

nu

mb

er/n

g o

f gD

NA

Transgene copy number/ng of total gDNA

from transduced NEJ

(Absolute qPCR targeting Luciferase)

Standard: 10 fold serial dilution of pGL3Luc copy number/ng of gDNA

39

A

Kines KJ, et al. 2008, FASEB J 22(8): 2936-2948

Retroposon Anchored PCR Linker mediated PCR

mRNA & PROTEIN

GENOMIC INTEGRATION

RETROVIRAL INFECTION

Aim 1 Silencing by RNAi

• Feasible to transfer technology from S.mansoni to F.hepatica • Different delivery methods are possible in Fasciola hepatica.• Optimization underway in juveniles• Stable protocols for other stages needed • Trans-stage effects should be studied

Aim 2 Trasgensis in Fasciola hepatica

• Encouraging results suggest the presence of trangene• Endogenous F.hepatica promoters are needed to engineer specific

constructs• Genomic data needed to map integration events

• Platform for the development in other trematode models.

41

Trop. Med Hygiene Dept.

G. Washington Univ.

Paul J. Brindley

Gabriel Rinaldi

Kristine Kines

Dpto. Genetica

Facultad de Medicina

Pablo Smircich

Nicolas Dell-Oca

Ileana Corvo

Unidad de Biol. Parasitaria.

Instituto de Higiene

Carlos Carmona

Tatiana Basika

Dpto. Bioquimica

Facultad de Ciencias

Estela Castillo

Fernanda Dominguez