1. Lin Yang
Professor Peter Lipke’s Lab
Molecular, Cellular and Developmental Biology PhD program
Brooklyn College
The City University of New York
The spatial-temporal distribution of
amyloids in C. albicans biofilm
3. • 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,
ZipperDB
• aggregation assay
Douglas M. Fowler et.al science direct (2007)
4. • 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)
5. A biofilm is composed of cell aggregates, which
are frequently embedded with extracellular
matrix.
Jonathan S. Finkel & Aaron P. Mitchell Nature
Reviews Microbiology (2011)
8. 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.
9. 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)
10. An Als anti-amyloid forming peptide (Als5pV326N
) inhibits the density of
biofilm under fluid condition.
Cho Chan et.al Eukaryotic
Cell (2014 )
14. Rifamycin SV and Als5pV326N
peptide released more yeast
form cells from mature biofilm
15.
16. 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 V326N
peptide Mature Als5 V326N
peptide caused yeast
form cells release from mature
biofilm. (CFU calculation,
microscopy)
Rifamycin SV Mature Rifamycin SV caused sparse
biofilm architecture and more
yeast 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.
17. 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.
18. 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)
20. Aim 2: amyloids are in the biofilm matrix of C. albicans grown in
vitro and in vivo.
3. Experiments to doTable 1. A list of experiments to be applied in Aim 2 and expected results
Assay Method Expected Result Controls
Extraction of SC 5314
biofilm matrix
Sonication,
centrifugation, dialysis,
lyophilization,
microscopy
Pi/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 are
treated with formic acid
than trypsin.
TANGO/ZipperDB
positive proteins are
found in formic acid
treated 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 and
tasA mutant biofilm
stained with ThT
21. Aim3: potential amyloid forming proteins are more
highly expressed in biofilm than planktonic cell state.
• 1. Rationale
Chandra et.al Journal of Bacteriology 2001
22. Aim3: potential amyloid forming
proteins are more highly expressed
in biofilm than planktonic cell state.
3. Experiments to do
Candidate 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)
23. 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
biofilm spefic and when these proteins are differently
expressed. (temporal)
Editor's Notes
a. aB fiber structure from NMR cross b sheet structure. b. transmission electron micrograph of islet amyloid polypeptide. c. x ray fiber diffraction pattern of ab 1-42 associated with alzheimer's disease
In our lab, our focus is amyloid forming proteins. the monomers are covalently anchored on the cell wall of yeast cells. After amyloid regions are exposed, the monomers are clustered through the interaction of amyloid forming sequences.
| Scanning electron micrograph (SEM) of an in vitro Candida albicans biofilm. The biofilm sample was sliced to show three layers in a cross-sectional view. The basal layer primarily includes yeast cells, as is evident in the lower enlarged inset. The central layer is mainly hyphae. The upper layer has yeast cells budding from the hyphae. The upper enlarged inset shows the extracellular matrix material, which seems fibrous in this preparation. b | SEM of an in vivo C. albicans biofilm from the rat catheter model11. Yeast cells, hyphae and some pseudohyphal cells are evident, along with extracellular matrix material. Images courtesy of J. Suhan (Carnegie Mellon University, Pittsburgh, Pennsylvania, USA), and J. Nett and D. Andes (University of Wisconsin–Madison, USA).
Matrix: immobilize biofilm cells and keep them in close proximity,help cell-cell interaction.
proteins: structural protein stablize matrix, interation with polysaccharides. Enzymes. Food resources.
DNAs: structural(treat with nucleolytic enzymes, lose of flocculation). adhesion. antimicrobial activity.
Lipids: adherence to surface. "surfactants"
Fig. 3.Summary of the cellular processes that are associated with genes upregulated at the different time points duringC.
albicansbiofilm development. The time-course of biofilm development is shown, highlighting the time points (6, 12 and 48 h)
studied by microarray analysis. Descriptions of cellular processes are summarized from the detailed data presented in
Supplementary Table S1. Categories of genes upregulated at 6 vs 12 h are summarized under the 6 h heading. Data from both
the 12 vs 6 h and 12 vs 48 h comparisons are placed under the 12 h heading. Few genes are upregulated at 48 h compared to
12 h, suggesting that initiation of new metabolic activity is relatively low in the mature biofilm. The individual genes upregulated
at 48 vs 12 h are listed
In her C. albicans model, biofilm is incubated at 30 oC with YPD medium, under which condition hyphae are not formed. However, this in vitro model is not appropriate for the study of systemic candidiasis, because this disease occurs at 37oC and hyphal formation is necessary for systemic infections (31, 31). Therefore, I will mimic the natural state of biofilm and induce hyphae formation in this project. I will grow C. albicans biofilm at 37 oC with RPMI-MOPs, which specifically induce hyphae. This system can mimic the physiological temperature in human body and is accepted by National Committee for Clinical Laboratory Standards of antifungal susceptibility study
yqxM6 is a A:T base pair mutation at 569 mutant cells. these cells are resistant to D amino acids treatment. wild type yqxM gene is requied for the association of tas A with cells
2 Biofilm growth for S. cerevisiae strains at two shear stresses. Biofilms of
S. cerevisiae expressing Als5p or Als5pV326N or harboring an empty vector.
Adherent cells were grown under laminar flow for 48
Propidium iodide
gold labeled anti-tasA antibodies. biofilm formation is impaired by tasA and eps mutants.
OC (recognizing the cross-stacked β-sheet structure of fibrillar oligomers, protofibrils, and fibrils [51] Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers
