Nand K. Gaur

Yeast cell adhesion molecules have functional amyloid-forming sequences.

ABSTRACT 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 β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-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.

Threonine-Rich Repeats Increase Fibronectin Binding in the Candida albicans Adhesin Als5p

ABSTRACT Commensal and pathogenic states of Candida albicans depend on cell surface-expressed adhesins, including those of the Als family. Mature Als proteins consist of a 300-residue N-terminal region predicted to have an immunoglobulin (Ig)-like fold, a 104-residue conserved Thr-rich region (T), a central domain of a variable number of tandem repeats (TR) of a 36-residue Thr-rich sequence, and a heavily glycosylated C-terminal Ser/Thr-rich stalk region, also of variable length (N. K. Gaur and S. A. Klotz, Infect. Immun. 65: 5289-5294, 1997). Domain deletions in ALS5 were expressed in Saccharomyces cerevisiae to excrete soluble protein and for surface display. Far UV circular dichroism indicated that soluble Ig-T showed a single negative peak at 212 nm, consistent with previous data indicating that this region has high β-sheet content with very little α-helix. A truncation of Als5p with six tandem repeats (Ig-T-TR6) gave spectra with additional negative ellipticity at 200 nm and, at 227 to 240 nm, spectra characteristic of a structure with a similar fraction ofβ -sheet but with additional structural elements as well. Soluble Als5p Ig-T and Ig-T-TR6 fragments bound to fibronectin in vitro, but the inclusion of the TR region substantially increased affinity. Cellular adhesion assays with S. cerevisiae showed that the Ig-T domain mediated adherence to fibronectin and that TR repeats greatly increased cell-to-cell aggregation. Thus, the TR region of Als5p modulated the structure of the Ig-T region, augmented cell adhesion activity through increased binding to mammalian ligands, and simultaneously promoted fungal cell-cell interactions.

Unfolding individual als5p adhesion proteins on live cells.

Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans . We show that the forces required to unfold individual tandem repeats of the protein are in the 150-250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally suited for cell adhesion. The single-molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells and may help us to elucidate their potential implications in diseases such as inflammation, cancer, and infection.

Candida albicans Als proteins mediate aggregation with bacteria and yeasts.

Candida albicans occupies a microniche on mucosal surfaces where diverse microbial populations interact within a biofilm. Because C. albicans is intimately involved with other microbes in this environment we studied the interactions of C. albicans with other fungi and bacteria that form mixed microbial aggregates. Once aggregation is initiated, aggregates form rapidly and incorporate fungal as well as bacterial cells. The fungus formed mixed microbial aggregates with homotypic cells (i.e., self to self, e.g., C. albicans or Als1p-expressing yeast cells aggregating with cells bearing Als1p); with heterotypic cells (i.e., self to non-self, e.g., C. albicans or Alsp-expressing yeast cells aggregating with other Candida species); and with xenotypic cells (e.g., C. albicans or Alsp-expressing yeast cells forming aggregates with bacteria). When either of the C. albicans adhesins Als1p or Als5p was displayed on the surface of non-adherent Saccharomyces cerevisiae cells, the S. cerevisiae also mediated these mixed microbial interactions. Thus the Als adhesins are potentially important for the co-adhesion of mixed microbial communities in biofilms and on mucus surfaces.

Degenerate Peptide Recognition by Candida albicans Adhesins Als5p and Als1p

ABSTRACT Candida albicans and Saccharomyces cerevisiae expressing the adhesins Als5p or Als1p adhere to immobilized peptides and proteins that possess appropriate sequences of amino acids in addition to a sterically accessible peptide backbone. In an attempt to further define the nature of these targets, we surveyed the ability of yeast cells to adhere to 90-μm-diameter polyethylene glycol beads coated with a 7-mer peptide from a library of 197 unique peptide-beads. C. albicans bound to ca. 10% of beads from the library, whereas S. cerevisiae expressing Als5p or Als1p bound to ca. 0.1 to 1% of randomly selected peptide-beads. S. cerevisiae expressing Als1p had a distinctly different adherence phenotype than did cells expressing Als5p. The former adhered in groups or clumps of cells, whereas the latter adhered initially as single cells, an event which was followed by the build up of cell-cell aggregates. Beads with adherent cells were removed, and the peptide attached to the bead was determined by amino acid sequencing. All adhesive beads carried a three-amino-acid sequence motif (τφ+) that possessed a vast combinatorial potential. Adherence was sequence specific and was inhibited when soluble peptide identical to the immobilized peptide was added. The Als5p adhesin recognized some peptides that went unrecognized by Als1p. The sequence motif of adhesive peptides identified by this method is common in proteins and offers so many possible sequence combinations that target recognition by the Als proteins is clearly degenerate. A degenerate recognition system provides the fungi with the potential of adhering to a multitude of proteins and peptides, an advantage for any microorganism attempting to establish a commensal or pathogenic relationship with a host.

Global cell surface conformational shift mediated by a Candida albicans adhesin

ABSTRACT Candida albicans maintains both commensal and pathogenic states in humans. Both states are dependent on cell surface-expressed adhesins, including those of the Als family. Heterologous expression of Als5p at the surface of Saccharomyces cerevisiae results in Als5p-mediated adhesion to various ligands, followed by formation of multicellular aggregates. Following adhesion of one region of the cell to fibronectin-coated beads, the entire surface of the cells became competent to mediate cell-cell aggregation. Aggregates formed in the presence of metabolic inhibitors or signal transduction inhibitors but were reduced in the presence of 8-anilino-1-naphthalene-sulfonic acid (ANS) or Congo Red (CR), perturbants that inhibit protein structural transitions. These perturbants also inhibited aggregation of C. albicans. An increase in ANS fluorescence, which accompanied Als-dependent cellular adhesion, indicated an increase in cell surface hydrophobicity. In addition, C. albicans and Als5p-expressing S. cerevisiae showed an aggregation-induced birefringence indicative of order on the cell surface. The increase in birefringence did not occur in the presence of the aggregation disruptants ANS and CR. These results suggest a model for Als5p-mediated aggregation in which an adhesion-triggered change in the conformation of Als5p propagates around the cell surface, forming ordered aggregation-competent regions.

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

ABSTRACT Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.

Inhibition of Adherence and Killing of Candida albicans with a 23-Mer Peptide (Fn/23) with Dual Antifungal Properties

ABSTRACT Candida albicans adheres to host tissue and then proliferates in order to establish a commensal as well as a pathogenic state. Specific adherence to proteins is provided by several surface adhesins of Candida. Two well-studied proteins, Als1p and Als5p, do not require energy for adherence to occur (dead as well as living cells adhere) and have a multiplier effect of cell-cell aggregation that mediates the formation of microcolonies of Candida cells. The entire process is spontaneous, reversible, and stable for physiologically relevant chemical and physical forces. This adherence process is inhibited by the addition of free peptide ligands, including a 23-mer derived from fibronectin (Fn/23) that binds to the adhesins through H bond formation. Adherence was measured by determining the number of yeast cells that adhered to 90-μm-diameter polyethylene glycol (PEG) beads with a 7-mer peptide (KLRIPSV) synthesized on the surfaces of the beads. The concentration of the Fn/23 peptide that inhibited the adherence of cells to the peptide-coated beads by 50% was 4 to 5 μM, and the magnitudes of adherence were similar regardless of the presence or absence of physiologic salt concentrations. The minimum fungicidal concentration of Fn/23 was 2 to 4 μM in water, but there was no killing in physiologic salt concentrations. Peptides from the C and N termini or the center sequence of Fn/23 had no effect on inhibition of adherence and little effect on fungal viability. The fungicidal effect was similar to that seen with 23-, 19-, and 18-mer peptides derived from porcine myeloid cells, a Helicobacter pylori ribosomal protein, and a hybrid of cecropin and magainin, respectively. However, these fungicidal peptides did not inhibit C. albicans adherence to the peptide-coated PEG beads. This dual property of Fn/23, i.e., inhibition of adherence and killing of C. albicans, may provide important adjuvant effects in the treatment of disease caused by this fungus.

Postmortem candidaemia: marker of disseminated disease

Aim The significance of finding Candida species in heart blood cultures obtained at postmortem examination has never been studied. This article describes the findings of autopsy patients with postmortem candidaemia and it compares them with findings in autopsy patients with antemortem candidaemia. Method 23 patients with Candida species isolated from heart blood at autopsy were identified over a 10-year period. These patients were compared with 10 autopsy patients found during the same time period with antemortem blood cultures isolating Candida species, but not positive postmortem heart blood cultures. Antemortem and postmortem records were reviewed. Results All 23 patients with Candida species isolated from postmortem blood culture had one or more antemortem risk factors for disseminated candidiasis, such as positive antemortem blood cultures, isolation of Candida from sterile internal sites, neutropenia, recent abdominal surgery, broad-spectrum antibiotic administration or the use of central venous catheters or other invasive devices. Eight patients showed histological proof of invasive candidiasis in addition to the positive heart blood cultures. This group did not differ with respect to risk factors from 10 autopsy patients with disseminated candidiasis and antemortem blood cultures with Candida species. However, all the patients with antemortem candidaemia had histological evidence of disseminated candidiasis at autopsy. Conclusion Candidaemia, when documented by heart blood culture performed at autopsy or by antemortem blood culture, is an insensitive, but highly specific, indicator of disseminated candidiasis.