Glycan microarray analysis of Candida glabrata adhesin ligand specificity

The Candida glabrata genome encodes at least 23 members of the EPA ( ep ithelial a dhesin) family responsible for mediating adherence to host cells. To better understand the mechanism by which the Epa proteins contribute to pathogenesis, we have used glycan microarray analysis to characterize their carbohydrate-binding specificities. Using Saccharomyces cerevisiae strains surface-expressing the N-terminal ligand-binding domain of the Epa proteins, we found that the three Epa family members functionally identified as adhesins in Candida glabrata (Epa1, Epa6 and Epa7) bind to ligands containing a terminal galactose residue. However, the specificity of the three proteins for glycans within this class varies, with Epa6 having a broader specificity range than Epa1 or Epa7. This result is intriguing given the close homology between Epa6 and Epa7, which are 92% identical at the amino acid level. We have mapped a five-amino-acid region within the N-terminal ligand-binding domain that accounts for the difference in specificity of Epa6 and Epa7 and show that these residues contribute to adherence to both epithelial and endothelial cell lines in vitro .     

The Candida glabrata adhesin Epa1p causes adhesion, phagocytosis, and cytokine secretion by innate immune cells

While Candida albicans is the most significant fungal pathogen for humans, Candida glabrata accounts for an increasing number of infections. Little is known about how C.?glabrata interacts with the innate immune system, the first line of defense against such organisms. The C.?glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized that Epa1p mediates unique, nonopsonic binding to macrophages, leading to induction of immune responses. We found that Epa1p mediated adhesion by both C.?glabrata (Cg) and transformed Saccharomyces cerevisiae (Sc(EPA1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S.?cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA1) induced inflammatory cytokine production (IL-8 and TNF-α) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA1) results in phagocytosis and subsequent cytokine production.     

Modular domain structure in the Candida glabrata adhesin Epa1p,a beta1,6 glucan-cross-linked cell wall protein

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI-anchored glucan-cross-linked cell wall proteins (GPI-CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N-terminal third of the protein contains the ligand-binding domain, and that the GPI anchor is essential both for cross-linking in the cell wall and for Epa1p-mediated adherence. We also found that the C-terminal Ser/Thr-rich domain, characteristic of many GPI-CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C-terminal Ser/Thr-rich region was absolutely required to project the N-terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr-rich domain of Epa1p and, presumably, of other related GPI-CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.     

Sir3 Polymorphisms in Candida glabrata Clinical Isolates

The opportunistic fungal pathogen Candida glabrata adheres tightly to epithelial cells in culture, mainly through the adhesin Epa1. EPA1 is the founding member of a family of up to 23 putative adhesin-encoding genes present in the C. glabrata genome. The majority of the EPA genes are localized close to the telomeres, where they are repressed by subtelomeric silencing that depends on the Sir, Ku, Rif1, and Rap1 proteins. EPA6 and EPA7 also encode functional adhesins that are repressed in vitro. EPA1 expression in vitro is tightly controlled both positively and negatively, and in addition, presents high cell-to-cell heterogeneity, which depends on Sir-mediated silencing. In this work, we characterized the ability to adhere to HeLa epithelial cells and the expression of several EPA genes in a collection of 79 C. glabrata clinical isolates from several hospitals in Mexico. We found 11 isolates that showed increased adherence to mammalian cells compared with our reference strain under conditions where EPA1 is not expressed. The majority of these isolates displayed over-expression of EPA1 and EPA6 or EPA7, but did not show increased biofilm formation. Sequencing of the SIR3 gene of several hyper-adherent isolates revealed that all of them contain several polymorphisms with respect to the reference strain. Interestingly, two isolates have polymorphisms in positions flanked by clusters of amino acids required for silencing in the Saccharomyces cerevisiae Sir3 protein. Our data show that there is a large variability in adhesin expression and adherence to epithelial cells among different C. glabrata clinical isolates.     

Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells

Candida glabrata owes its success as a pathogen, in part, to a large repertoire of adhesins present on the cell surface. Our current knowledge of C. glabrata adhesins and their role in the interaction between host and pathogen is limited to work with only a single family of epithelial adhesins (Epa proteins). Here, we report on the identification and characterization of a family of glycosylphosphatidylinositol-anchored cell wall proteins in C. glabrata. These proteins are absent in both Saccharomyces cerevisiae and Candida albicans, suggesting that C. glabrata has evolved different mechanism(s) for interaction with host cells. In the current study, we present data on the characterization of Pwp7p (PA14 domain containing Wall Protein) and Aed1p (Adherence to Endothelial cells) of this family in the interaction of C. glabrata with human umbilical vein endothelial cells. The deletion of C. glabrata genes PWP7 and AED1 results in a significant reduction in adherence to endothelial cells compared with the wild-type parent. These data indicate that C. glabrata utilizes these proteins for adherence to endothelial cells in vitro.     

Candida-host cell receptor-ligand interactions

The interaction of Candida species with their cognate host receptors is a key factor in the pathogenesis of different types of candidiasis. The recognition of different forms of Candida albicans by Toll-like receptors 2 and 4 on mononuclear leukocytes has recently been discovered to determine the function and activity of regulatory T-cells, determine the balance of Type 1 and Type 2 cytokines and, thereby, influence the antifungal activity of both the innate and adaptive immune response. Different forms of C. albicans are also recognized by different lectins that are expressed on the surface macrophages. C. albicans and Candida glabrata express the ALS (agglutinin-like sequence) and EPA (epithelial adhesin) families of adhesins, respectively. A key difference between C. glabrata and C. albicans is that EPA expression in C. glabrata is governed by sub-telomeric silencing, whereas ALS expression in C. albicans is regulated by other mechanisms.     

Murine model of dextran sulfate sodium-induced colitis reveals Candida glabrata virulence and contribution of β-mannosyltransferases

Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses β-1,2-linked mannosides (β-Mans), promoting its adherence to host cells and tissues. Because β-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding β-mannosyltransferases, BMT2-BMT6, were deleted simultaneously. β-Man expression was studied by Western blotting, flow cytometry, and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2-6 strains had a significant reduction in β-1,2-Man expression and a disappearance of β-1,2-mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2-6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2-6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis, where β-Mans contribute to colonization and virulence.     

Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells

Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.