UPC2 mutations and development of azole resistance in Candida albicans hospital isolates from Lebanon.

OBJECTIVES: This study evaluated the role of Upc2 in the development of azole resistance in Candida albicans isolates from Lebanese hospitalized patients and determined a correlation between resistance and virulence. METHODS: The UPC2 gene which codes for an ergosterol biosynthesis regulator was sequenced and analysed in two azole-resistant and one azole-susceptible C. albicans isolates. An amino acid substitution screening was carried out on Upc2 with a focus on its ligand binding domain (LBD) known to interact with ergosterol. Then, Upc2 protein secondary structure prediction and homology modelling were conducted, followed by total plasma membrane ergosterol and cell wall chitin quantifications. For virulence, mouse models of systemic infection were generated and an agar adhesion and invasion test was performed. RESULTS: Azole-resistant isolates harboured novel amino acid substitutions in the LBD of Upc2 and changes in protein secondary structures were observed. In addition, these isolates exhibited a significant increase in plasma membrane ergosterol content. Resistance and virulence were inversely correlated while increased cell wall chitin concentration does not seem to be linked to resistance since even though we observed an increase in chitin concentration, it was not statistically significant. CONCLUSIONS: The azole-resistant C. albicans isolates harboured novel amino acid substitutions in the LBD of Upc2 which are speculated to induce an increase in plasma membrane ergosterol content, preventing the binding of azoles to their target, resulting in resistance.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

Sequential Induction of Drug Resistance and Characterization of an Initial Candida albicans Drug-Sensitive Isolate.

BACKGROUND: The pathogenic fungus Candida albicans is a leading agent of death in immunocompromised individuals with a growing trend of antifungal resistance. METHODS: The purpose is to induce resistance to drugs in a sensitive C. albicans strain followed by whole-genome sequencing to determine mechanisms of resistance. Strains will be assayed for pathogenicity attributes such as ergosterol and chitin content, growth rate, virulence, and biofilm formation. RESULTS: We observed sequential increases in ergosterol and chitin content in fluconazole-resistant isolates by 78% and 44%. Surface thickening prevents the entry of the drug, resulting in resistance. Resistance imposed a fitness trade-off that led to reduced growth rates, biofilm formation, and virulence in our isolates. Sequencing revealed mutations in genes involved in resistance and pathogenicity such as ERG11, CHS3, GSC2, CDR2, CRZ2, and MSH2. We observed an increase in the number of mutations in key genes with a sequential increase in drug-selective pressures as the organism increased its odds of adapting to inhospitable environments. In ALS4, we observed two mutations in the susceptible strain and five mutations in the resistant strain. CONCLUSION: This is the first study to induce resistance followed by genotypic and phenotypic analysis of isolates to determine mechanisms of drug resistance.

Candida glabrata hospital isolate from Lebanon reveals micafungin resistance associated with increased chitin and resistance to a cell-surface-disrupting agent.

OBJECTIVES: This study aimed to identify the resistance mechanisms to micafungin and fluconazole in a clinical isolate of Candida glabrata. METHODS: The isolate was whole-genome sequenced to identify amino acid changes in key proteins involved in antifungal resistance, and the isolate was further characterised by pathogenicity-related phenotypic assays that supported the sequencing results. RESULTS: Amino acid substitutions were detected in 8 of 17 protein candidates. Many of these substitutions were novel, including in CHS3, CHS3B, and KRE5, which are involved in the development of micafungin resistance. Regarding fluconazole resistance, overexpression of efflux pumps was observed. Our isolate did not exhibit an increased virulence potential compared with the control strain; however, a significant increase in chitin content and potential to resist the cell surface disruptant sodium dodecyl sulphate was observed. CONCLUSIONS: This clinical Candida glabrata isolate experienced a change in cell wall architecture, which correlates with the development of micafungin resistance.

Cell Wall Proteome Profiling of a Candida albicans Fluconazole-Resistant Strain from a Lebanese Hospital Patient Using Tandem Mass Spectrometry-A Pilot Study.

Candida albicans is an opportunistic pathogenic fungus responsible for high mortality rates in immunocompromised individuals. Azole drugs such as fluconazole are the first line of therapy in fungal infection treatment. However, resistance to azole treatment is on the rise. Here, we employ a tandem mass spectrometry approach coupled with a bioinformatics approach to identify cell wall proteins present in a fluconazole-resistant hospital isolate upon drug exposure. The isolate was previously shown to have an increase in cell membrane ergosterol and cell wall chitin, alongside an increase in adhesion, but slightly attenuated in virulence. We identified 50 cell wall proteins involved in ergosterol biosynthesis such as Erg11, and Erg6, efflux pumps such as Mdr1 and Cdr1, adhesion proteins such as Als1, and Pga60, chitin deposition such as Cht4, and Crh11, and virulence related genes including Sap5 and Lip9. Candidial proteins identified in this study go a long way in explaining the observed phenotypes. Our pilot study opens the way for a future large-scale analysis to identify novel proteins involved in drug-resistance mechanisms.

Molecular mechanism of fluconazole resistance and pathogenicity attributes of Lebanese Candida albicans hospital isolates.

Hospital infections caused by the opportunistic fungus Candida albicans are increasingly common and life threatening. The first line of defense consists of administering antifungal drugs such as azoles including fluconazole that prevent ergosterol biosynthesis. C. albicans is rapidly developing resistance towards antifungal drugs through various mechanisms including mutations in ERG11 which is a gene involved in the ergosterol biosynthesis pathway. These mutations prevent the binding of the drug and inactivate ergosterol synthesis. Alternatively, upregulation of cell membrane ergosterol content generates resistance by countering the effect of the drug. In this study we sequenced the ERG11 gene in 6 fluconazole sensitive and 8 fluconazole resistant C. albicans isolates recovered from clinical settings in Lebanon and quantified the ergosterol content of their plasma membranes to identify mechanisms linked to fluconazole resistance. A number of pathogenicity attributes were also analyzed to determine any correlation with fluconazole resistance. Our results revealed an increase in ergosterol content in the fluconazole resistant isolates. In addition, we identified both novel and previously reported amino acid substitutions in ERG11 as well as frameshift mutations that might contribute to resistance. The fluconazole resistant isolates did not exhibit an increased virulence potential in a mouse model of systemic infection and showed decreased in vitro potential to form biofilms. No discrepancy between drug resistant and sensitive isolates to cell surface disrupting agents was observed. This approach is the first of its kind to be carried out in Lebanon to identify possible mechanisms and phenotypes of drug resistant C. albicans isolates.

Whole genome sequencing of a clinical drug resistant Candida albicans isolate reveals known and novel mutations in genes involved in resistance acquisition mechanisms.

Candida albicans is an opportunistic pathogen accounting for the majority of cases of Candida infections. Currently, C. albicans are developing resistance towards different classes of antifungal drugs and this has become a global health burden that does not spare Lebanon. This study aims at determining point mutations in genes known to be involved in resistance acquisition and correlating resistance to virulence and ergosterol content in the azole resistant C. albicans isolate CA77 from Lebanon. This pilot study is the first of its kind to be implemented in Lebanon. We carried out whole genome sequencing of the azole resistant C. albicans isolate CA77 and examined 18 genes involved in antifungal resistance. To correlate genotype to phenotype, we evaluated the virulence potential of this isolate by injecting it into BALB/c mice and we quantified membrane ergosterol. Whole genome sequencing revealed that eight out of 18 genes involved in antifungal resistance were mutated in previously reported and novel residues. These genotypic changes were associated with an increase in ergosterol content but no discrepancy in virulence potential was observed between our isolate and the susceptible C. albicans control strain SC5314. This suggests that antifungal resistance and virulence potential in this antifungal resistant isolate are not correlated and that resistance is a result of an increase in membrane ergosterol content and the occurrence of point mutations in genes involved in the ergosterol biosynthesis pathway.

Phenotypic and Cell Wall Proteomic Characterization of a DDR48 Mutant Candida albicans Strain.

Candida albicans is an opportunistic fungus possessing multiple virulence factors controlling pathogenicity. Cell wall proteins are the most important among these factors, being the first elements contacting the host. Ddr48 is a cell wall protein consisting of 212 amino acids. A DDR48 haploinsufficient mutant strain was previously found necessary for proper oxidative stress response and drug resistance. In this study, we aimed to further elucidate the role of Ddr48 by performing additional phenotypic characterization assays. A combinatory proteomic and bioinformatics approach was also undertaken to determine differentially expressed cell wall proteins. Results showed that the mutant strain exhibited a 10% decrease in adhesion mirrored by a 20% decrease in biofilm formation, and slight sensitivity to menadione, diamide, and SDS. Both strains showed similar hyphae formation, virulence, temperature tolerance, and calcofluor white and Congo red sensitivities. Furthermore, a total of 8 and 10 proteins were identified exclusively in the wild-type strain grown under filamentous and nonfilamentous conditions respectively. Results included proteins responsible for superoxide stress resistance (Sod4 and Sod6), adhesion (Als3, Hyr4, Pmt1, and Utr2), biofilm formation (Hsp90, Ece1, Rim9, Ipp1, and Pra1) and cell wall integrity (Utr2 and Pga4). The lack of detection of these proteins in the mutant strain correlates with the observed phenotypes.

Genotypic and phenotypic characterization of Candida albicans Lebanese hospital isolates resistant and sensitive to caspofungin.

The fungus Candida albicans is both a commensal and an opportunistic human pathogen, present as part of the normal human microflora causing serious mucosal, and systemic life threatening infections. The antifungal drug caspofungin of the echinocandin family is the latest generation of antifungal drugs to be developed. It functions by inhibiting glucan synthase thus weakening the fungal cell wall leading to death. Recently reports of resistance to caspofungin have been reported mainly through mutations in the FKS encoded subunits of glucan synthase at hot spot 1 (amino acids 641 to 649, FSTLSLRDP) and hot spot 2 (amino acids 1357 to 1364, DWIRRYTL). Our study aimed at sequencing both hot spots from 16 C. albicans Lebanese hospital isolates resistant and sensitive to caspofungin to determine whether mutations in these hot spots are present, and whether such mutations also impart resistance to our isolates. In addition, we wanted to determine any relationship between resistance and pathogenicity related attributes such as virulence, resistance to cell wall disrupting agents, biofilm formation, and cell wall chitin deposition. Five isolates were found to contain mutations with the mutations restricted to resistant strains. Within hot spot 1 substitution at positions S642, T643, L644, R647, and D648 were found, while within hot spot 2 substitutions at positions L1364, T1363, and R1360, W1358 and R1361 were identified with some of the mutations not previously documented. Strains that were resistant to caspofungin also showed increased resistance to Congo red but decreased biofilm formation and attenuated virulence in a mouse model of infection. Caspofungin sensitive strains showed decreased resistance to Congo red yet increased virulence and biofilm formation. Chitin content analysis showed that caspofungin resistant strains had elevated levels of chitin resulting in cell wall thickening that counters the effect of caspofungin, while sensitive strains showed decreased chitin content. Our results demonstrate an inverse correlation between resistance and virulence whereby resistance is due to thickening of the cell wall preventing the cell from gaining virulence attributes, while a more susceptible cell wall increases susceptibility to drugs but allows increased virulence.

Proteomic analysis of a Candida albicans pga1 Null Strain.

We previously characterized Pga1, a Candida albicans (C. albicans) cell wall protein necessary for proper virulence, adhesion, and resistance to oxidative stress. By utilizing tandem mass spectrometry coupled with bioinformatics to investigate cell wall proteome expression in a pga1 null fourteen and 36 proteins were identified in the wild type grown under filamentous and non-filamentous conditions respectively, but were not detected in the mutant, including members of the PGA GPI anchored family. Virulence and adhesion proteins such as Hsp 90, Sap10, Cdc11, Int 3 and members of the lipase family were also identified exclusively in the wild type.

Tandem Mass Spectrometric Cell Wall Proteome Profiling of a Candida albicans hwp2 Mutant Strain.

BACKGROUND: Candida albicans is present as part of the normal gut flora and detected in the oral cavities and GI tracts of around fifty percent of adults. Benign colonization can turn pathogenic causing a variety of mild to severe infections. In a pathogen, the cell wall and cell surface proteins are major antigenic determinants and drug targets as they are the primary structures that contact the host. Cell surface proteins perform a variety of functions necessary for virulence such as adhesion, host degradation, resistance to oxidative stress, and drug resistance. We have previously characterized Hwp2, a C. albicans cell wall adhesin shown to play a major role in the cell wall architecture and function as hwp2 mutants were deficient in chitin deposition, filamentation, adhesion and invasive growth, virulence, and resistance to oxidative stress. OBJECTIVE/METHOD: Here, we utilized tandem mass spectrometry coupled with a bioinformatics approach to differentially profile the cell wall proteome of a wild-type strain compared to an hwp2 null mutant to determine key differentially expressed proteins. RESULT: Many proteins identified exclusively in the wild-type go a long way in explaining the abovementioned phenotypes. These include virulence factors such as members of the SAP family including Sap4, Sap5, and Sap10, as well as several lipases involved in host degradation. We also identified members of the PGA family of proteins Pga28, Pga32, Pga41 and Pga50, which function in adhesion, Cht2 a chitinase involved in chitin remodeling, and several proteins that function in promoting filamentation such as Phr1, Mts1, and Rbr1.

Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence.

We have previously characterized Pir32, a Candia albicans cell wall protein that we found to be involved in filamentation, virulence, chitin deposition, and resistance to oxidative stress. Other than defining the cell shape, the cell wall is critical for the interaction with the surrounding environment and the point of contact and interaction with the host surface. In this study, we applied tandem mass spectrometry combined with bioinformatics to investigate cell wall proteome changes in a pir32 null strain. A total of 16 and 25 proteins were identified exclusively in the null mutant strains grown under non-filamentous and filamentous conditions. These proteins included members of the PGA family with various functions, lipase and the protease involved in virulence, superoxide dismutases required for resisting oxidative stress, alongside proteins required for cell wall remodeling and synthesis such as Ssr1, Xog1, Dfg5 and Dcw1. In addition proteins needed for filamentation like Cdc42, Ssu81 and Ucf1, and other virulence proteins such as Als3, Rbt5, and Csa2 were also detected. The detection of these proteins in the mutant and their lack of detection in the wild type can explain the differential phenotypes previously observed.

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non-filamentous conditions.

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface-disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild-type strain. The strains were grown under filamentous and non-filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI-TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild-type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI-TOF MS is a reliable technique in identifying mutant-specific protein expression in C. albicans.

Identification, typing, antifungal resistance profile, and biofilm formation of Candida albicans isolates from Lebanese hospital patients.

As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer) and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness.

The Candida albicans Hwp2p can complement the lack of filamentation of a Saccharomyces cerevisiae flo11 null strain.

The opportunistic fungal pathogen Candida albicans is one of the leading agents of life-threatening infections affecting immunocompromised individuals. Many factors make C. albicans a successful pathogen. These include the ability to switch between yeast and invasive hyphal morphologies in addition to an arsenal of cell wall virulence factors such as lipases, proteases, dismutases and adhesins that promote the attachment to the host, a prerequisite for invasive growth. We have previously characterized Hwp2, a C. albicans cell wall protein which we found necessary for proper oxidative stress, biofilm formation and adhesion to host cells. Baker's yeast Saccharomyces cerevisiae also possesses adhesins that promote aggregation and flocculence. Flo11 is one such adhesin that has sequence similarity to Hwp2. Here we determined that transforming an HWP2 cassette can complement the lack of filamentation of an S. cerevisiae flo11 null strain and impart on S. cerevisiae adhesive properties similar to those of a pathogen.

Deletion of the Candida albicans PIR32 results in increased virulence, stress response, and upregulation of cell wall chitin deposition.

Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.

The Candida albicans Dse1 Protein Is Essential and Plays a Role in Cell Wall Rigidity, Biofilm Formation, and Virulence.

The fungal pathogen Candida albicans is one of the leading causative agents of death in immunocompromised individuals. It harbors an arsenal of cell wall anchored factors that are implicated in virulence such as filamentation inducing factors, adhesins, lipases, proteases, and superoxide dismutases. Dse1 is a cell wall protein involved in cell wall metabolism. The purpose of this study is to characterize the role Dse1 plays in virulence. Dse1 appears to be an essential gene as no homozygous null mutant was possible. The heterozygote mutant exhibited increased susceptibility to calcofluor white, a cell wall disrupting agent, with a subsequent reduction in cell wall chitin content, decreased oxidative stress tolerance, a 30% reduction in biofilm formation, and a delay in adhesion that was mirrored by a reduction in virulence in a mouse model of infection. Dse1 thus appears to be an important protein involved in cell wall integrity and rigidity.

The Candida albicans Hwp2 is necessary for proper adhesion, biofilm formation and oxidative stress tolerance.

Candida albicans is an important fungal pathogen of humans that is responsible for the majority of mucosal and systemic candidiasis. The host-pathogen interaction in C. albicans has been the subject of intense investigation as it is the primary step that leads to establishment of infection. Hwp2 is a cell wall GPI-anchored cell wall protein that was previously shown to be necessary for hyphal and invasive growth on solid media. The purpose of the current study is to further characterize the protein as far as its role in oxidative stress, sensitivity to cell wall disrupting agents, adhesion to human epithelial and endothelial cells, biofilm formation and chitin content. It appears that Hwp2 is necessary for proper oxidative stress tolerance, adhesion and biofilm formation as an hwp2 null is more susceptible to increasing doses of hydrogen peroxide, unable to adhere efficiently to epithelial and endothelial cell lines and unable to form wild type biofilm levels.

Characterisation of Pga1, a putative Candida albicans cell wall protein necessary for proper adhesion and biofilm formation.

The fungal pathogen Candida albicans is a leading causative agent of death in immunocompromised individuals. Many factors have been implicated in virulence including filamentation-inducing transcription factors, adhesins, lipases and proteases. Many of these factors are glycosylphosphatidylinositol-anchored cell surface antigenic determinant proteins. Pga1 is one such protein shown to be upregulated during cell wall regeneration. The purpose of this study was to characterise the role Pga1 plays by creating a homozygous pga1 null strain and comparing the phenotype with the parental strain. It was observed that the mutant strain showed less oxidative stress tolerance and an increased susceptibility to calcofluor white, a cell surface disrupting agent that inhibits chitin fibre assembly which translated as a 40% decrease in cell wall chitin content. Furthermore, the mutant exhibited a 50% reduction in adhesion and a 33% reduction in biofilm formation compared with the parental strain, which was reflected as a slight reduction in virulence. Our data suggest that Pga1 plays an important role in cell wall rigidity and stability. It was also observed that the pga1 null was over filamentous on both liquid and solid media and exhibited increased resistance to SDS suggesting upregulation of filamentation-inducing genes and cell surface components to partially compensate for the deletion.

Yeast cell adhesion molecules have functional amyloid-forming sequences.

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776-782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The beta-aggregation predictor TANGO found highly beta-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were beta-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High beta-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.