Lin YangProfessor Peter Lipke’s Lab

Molecular, Cellular and Developmental Biology PhD programBrooklyn College

The City University of New York

The spatial-temporal distribution of amyloids in C. albicans biofilm

Outline • Background

• Aims of this proposal Rationale Preliminary data Experimental design

• Payoff

• Amyloids are cross beta-sheet structures formed by the assembly of a cluster of proteins or peptides.

• ways to detect amyloids:• amyloid binding dyes: Thioflavin T,

Thioflavin S, Congo Red.• electron microscopy• software algorithms: TANGO, Zipp

erDB• aggregation assay

Douglas M. Fowler et.al science direct (2007)

• Yeast cell adhesion molecules such as Als1p, Als5p of C. abicans and Flo1p, Muc1p of S. cerevisiae have functional amyloid forming sequences.

Ramsook CB et.al Eukaryotic Cell. 2010

Melissa Garcia et.al PLoS ONE (2011)

A biofilm is composed of cell aggregates, which are frequently embedded with extracellular matrix.

Jonathan S. Finkel & Aaron P. Mitchell Nature Reviews Microbiology (2011)

biofilm matrix

• Matrix components:• Mannoproteins• Carbohydrates• Lipids• eDNAs

Early, intermediate, mature phase of biofilm

Yeater et.al Microbiology (2007)

Melissa Garcia et.al PLoS ONE (2011)

Aim1: Anti-amyloids disrupt biofilm at early, intermediate and mature phase.Rationale

An Als anti-amyloid forming peptide (Als5pV326N) and high concentration of ThT inhibited biofilm formation at early phase.

D-amino acids causes the release of TasA amyloid fibers, which results in the disassembly of biofilm in Bacillus subtilis.

Figure 3. D-tyrosine causes the release of TasA fibers

Figure 2. D-amino acids break down pellicles.

Ilana Kolodkin-Gal et.al Sciece (2010)

An Als anti-amyloid forming peptide (Als5pV326N) inhibits the density of biofilm under fluid condition.

Cho Chan et.al Eukaryotic Cell (2014 )

Aim1: anti-amyloids disrupt biofilm at early, intermediate and mature phase.

• Preliminary data

ThT inhibited biofilm formation at early phase

Day185 4.5uM ThT 450uM ThT

A

B CCrystal Violet XTT assay

ThT inhibited biofilm at intermediate and mature phase

Rifamycin SV and Als5pV326N peptide released more yeast form cells from mature biofilm

Anti-amyloid Phase of biofilm affected Findings

Thioflavin T Early, Intermediate, Mature ThT caused the decrease of

biomass, metabolic rate of

biofilm at all phases. (XTT, Crystal

Violet data)

Als5 V326Npeptide Mature Als5 V326N peptide caused yeast

form cells release frommature

biofilm. (CFU calculation,microscopy)

Rifamycin SV Mature Rifamycin SV caused sparse

biofilm architecture and moreyeast form cells release at

mature phase.

Rifamycin SV and Als5pV326N peptide did not affect early and intermediate phase of biofilm based on XTT and Cyrstal violet assays.

Aim1: anti-amyloids disrupt biofilm at early, intermediate and mature phase.3. Experiments to do

• Test anti-amyloid property of rifamycin SV in C. albicans biofilm forming system: Dot blot using matrix sample extracted.

• Time course experiments to determine at which phase other amyloid binding dyes (ThS, Congo Red) affect biofilm (XTT, Crystal Violet, Microscopy)

• CFU calculation of cells released from mature biofilm upon treatment.

• Confocal microscopy: determine the architecture of biofilm upon treatment.

Aim 2: amyloids are in in vivo biofilm and are in the matrix of in vitro C. albicans biofilm.

• 1. Rationale:

Diego Romero et.al PNAS (2010)

2. Preliminary data

Aim 2: amyloids are in the biofilm matrix of C. albicans grown in vitro and in vivo. 3. Experiments to do

Table 1. A list of experiments to be applied in Aim 2 and expected resultsAssay Method Expected Result Controls

Extraction of SC 5314

biofilm matrix

Sonication,

centrifugation, dialysis,

lyophilization,

microscopyPi/Syto-9 Staining

Less than 5% of the

matrix is yeast cells

Intact cells

Detecting Als family

proteins in matrix

Dot blot, Als5p-biotin,

Avidin-HRP

Als family proteins are

in matrix sample

Als5 peptides,

Als5pV326N peptides

Staining proteins in the

matrix with ThT

fluorometer The fluorescence of ThT

increases after proteins

in the matrix is added.

ThT alone,

Als5 peptides,

Als5pV326N peptides

Discovery of amyloid

forming proteins in

matrix.

88% formic acid dissolves

matrix proteins, mass

spectrometry,TANGO/ZipperDB

algorithm

There are more proteins

that are found in ms

when the proteins aretreated with formic acid

than trypsin.

TANGO/ZipperDB

positive proteins are

found in formic acidtreated matrix.

Matrix sample

dissolved in trypsin.

In vivo biofilm staining ThT staining, confocal

microscopy

Biofilm grown on the

catheter is ThT positive

Empty catheter, in vivo

B. subtilis biofilm andtasA mutant biofilm

stained with ThT

Aim3: potential amyloid forming proteins are more highly expressed in biofilm than planktonic cell state.

• 1. Rationale

Chandra et.al Journal of Bacteriology 2001

Aim3: potential amyloid forming proteins are more highly expressed

in biofilm than planktonic cell state.

3. Experiments to doCandidate genes->proteins

•N-terminal signal peptide•GPI ancher •TANGO/ZipperDB

Proteins that meet all the requirements above are potential amyloid forming proteins

Compare with the list to see whether the potential amyloid forming proteins are more highly expressed in biofilm.

Yeater et.al Microbiology (2007)

Payoff• Aim 1 will determine when anti-amyloids affect biofilm in C.

albicans. (temporal)• Aim2 will confirm or deny whether amyloids are in C. albicans

biofilm matrix. (spatial)• Aim3 will determine whether amyloid forming proteins are biof

ilm spefic and when these proteins are differently expressed. (temporal)