Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.

Summary Tpk1p, Tpk2p and Efg1p are members of the Rasprotein kinase A pathway that governs the yeast-tohyphal transition in Candida albicans

SummarypH-responsive transcription factors of the Rim101/ PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1D/D mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101D/D mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.

We have shown that vaccination with the recombinant N terminus of Als1p (rAls1p-N) protects mice against disseminated and oropharyngeal candidiasis. We now report that vaccination of mice with a related candidate, rAls3p-N, induces a broader antibody response than rAls1p-N and a similar cell-mediated immune response. The rAls3p-N vaccine was equally as effective as rAls1p-N against disseminated candidiasis but was more effective than rAls1p-N against oropharyngeal or vaginal candidiasis. Antibody titers did not correlate with protection against disseminated candidiasis, but delayed-type hypersensitivity did. The rAls3p-N vaccine is a promising new vaccine candidate for further exploration to prevent systemic and mucosal candidal infections.

Candida albicans must penetrate the endothelial cell lining of the vasculature to invade the deep tissues during a hematogenously disseminated infection. We compared 27 C. albicans mutants with their wild-type parent for their capacity to damage endothelial cells in vitro and cause a lethal infection in mice following tail vein inoculation. Of 10 mutants with significantly impaired capacity to damage endothelial cells, all had attenuated virulence. Therefore, the endothelial cell damage assay can be used as a screen to identify some virulence factors relevant to hematogenously disseminated candidiasis.During the initiation of hematogenously disseminated candidiasis, blood-borne organisms must adhere to and penetrate the endothelial cell lining of the blood vessels to invade the deep tissues. One mechanism by which Candida albicans can penetrate the vascular endothelium is by damaging and eventually killing the endothelial cells. Damaged endothelial cells detach from the basement membrane, leaving gaps through which the organism can invade. Also, the exposed basement membrane can be avidly bound by additional organisms (18).C. albicans damages human vascular endothelial cells in vivo and in vitro (5,10,17,25). Maximal endothelial cell damage (ECD) occurs in vitro when C. albicans adheres to and invades the endothelial cells and then secretes lytic enzymes (2,10,11,13,16). Moreover, some C. albicans mutants with filamentation defects cause significantly less ECD than the wild-type parent strain (24). These filamentation mutants also have attenuated virulence in various experimental models of infection (reviewed in reference 23). We hypothesized that the in vitro assay for C. albicans-induced ECD (ECD assay) can serve as a model for certain aspects of host-pathogen interactions in vivo, such as the ability of the organism to adhere to, invade, and injure host cells. This hypothesis predicts that some mutants with virulence defects will be defective in the ECD assay. The goal of the present study was to investigate this prediction.C. albicans strains. The genotypes and sources of the C. albicans strains used here are listed in Tables 1 and 2. Each strain was grown overnight in yeast nitrogen base broth (Difco, Detroit, Mich.) supplemented with 2% glucose (wt/vol) at 20°C on a rotating drum. The blastospores were harvested by centrifugation, washed with phosphate-buffered saline, enumerated with a hemacytometer, and suspended in RPMI 1640 medium (Irvine Scientific, Santa Ana, Calif.).ECD assay. We used our standard 51 Cr release ECD assay to determine the abilities of mutants of C. albicans to damage endothelial cells in vitro (24). The ECD assay was performed in 96-well tissue culture plates (Corning Inc., Acton, Mass.) with endothelial cells isolated from human umbilical cord veins, as described previously (24). The inoculum was 4 ϫ 10 4 organisms per well, and the organisms were incubated with the endothelial cells for 3 h in 5% CO 2 at 37°C. At the end of the incubation period, the wells were examined with an inv...

Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33⌬ mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33⌬ mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.

We have previously shown that intraperitoneal vaccination with the recombinant N terminus of Als1p (rAls1p-N) modestly improves survival during murine disseminated candidiasis. We now report marked efficacy with subcutaneous rAls1p-N vaccination. Efficacy is retained in neutropenic and corticosteroid-treated mice. The rAls1p-N vaccine is a promising candidate for the prevention of invasive candidiasis.

In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125 FAK ), and monolayer hyperpermeability. Microscopic examination of CalAtreated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction. nonmuscle myosin; myosin light chain phosphorylation; paxillin; immunofluorescent digital imaging; size-selective solute permeability THE VASCULAR ENDOTHELIUM provides the permeability barrier that controls the passage of fluid and solutes into the perivascular space. Inflammation of this barrier initiates a hyperpermeability state characterized by the formation of large paracellular holes between adjacent endothelial cells (EC). These events occur most frequently in the systemic postcapillary venule and pulmonary arteriole circulations. The formation of paracellular gaps is the result of reorganization of the endothelial cell-cell junctional morphology that permits the enhanced leakage of plasma proteins and fluid into the tissues, thus causing edema and organ dys-