Role of hyphal formation in interactions of Candida albicans with endothelial cells.

The ability to change from yeast to hyphal morphology is a major virulence determinant of Candida albicans. Mutants with defined defects in filamentation regulatory pathways have reduced virulence in mice. However, is it poorly understood why hyphal formation is critical for C. albicans to cause hematogenously disseminated infections. We used recently constructed mutants to examine the role of hyphal formation in the interactions of C. albicans with endothelial cells in vitro. These interactions included the ability of the mutants to invade and injure endothelial cells. Because the formation of hyphae may influence the host inflammatory response to C. albicans, we also investigated the capacity of these mutants to stimulate endothelial cells to express E-selectin and intercellular adhesion molecule 1. We infected endothelial cells with C. albicans strains containing homozygous null mutations in the following filamentation regulatory genes: CLA4, CPH1, EFG1, and TUP1. Whereas the wild-type strain formed true hyphae on endothelial cells, we found that neither the Deltaefg1 nor the Deltacph1 Deltaefg1 double mutant germinated. The Deltatup1 mutant formed only pseudohyphae. We also found that the Deltaefg1, Deltacph1 Deltaefg1, and Deltatup1 mutants had significantly reduced capacities to invade and injure endothelial cells. Therefore, Efg1p and Tup1p contribute to virulence by regulating hyphal formation and the factors that enable C. albicans to invade and injure endothelial cells. With the exception of the Deltacph1 Deltaefg1 mutant, all other mutants stimulated endothelial cells to express at least one of the leukocyte adhesion molecules. Therefore, the combined activities of Cph1p and Efg1p are required for C. albicans to stimulate a proinflammatory response in endothelial cells.

The effect of the new triazole, voriconazole (UK-109,496), on the interactions of Candida albicans and Candida krusei with endothelial cells.

In this study, we investigated how voriconazole affects specific endothelial cell interactions utilizing both fluconazole-susceptibles and resistantR Candida albicans strains (C. albicansS and C. albicansR, respectively) as well as Candida krusei. Our data show that exposing C. albicansS to voriconazole significantly reduced its adherence to endothelial cells (p <0.001). The adherence of C. albicansR to endothelial cells was not affected by treatment with either antifungal agent. Exposure of C. albicans to both agents inhibited germ tube formation; however, voriconazole showed higher ability in inhibiting germination as compared with fluconazole. The effect of antifungals on germination was also tested during co-incubation of yeast cells with endothelial cells. Pretreated C. albicansS cells germinated on endothelial cells in the presence of voriconazole or fluconazole. However, the degree of germination was reduced by 81% and 16%, respectively. Similar results were observed with C. albicansR. Our data demonstrate that voriconazole treatment reduced the median germ tube length of C. albicansS and C. albicansR by approximately 60%, whereas fluconazole reduced the germ tube length of these strains by 27% and 63%, respectively (P < 0.0001 for each comparison). We compared the efficacy of voriconazole and fluconazole in protecting endothelial cells against damage caused by C. albicansS, C. albicansR, and C. krusei. Voriconazole and fluconazole reduced C. albicans-mediated endothelial cell injury by about 90% and 40%, respectively (P < 0.01 for each comparison). Additionally, voriconazole treatment significantly reduced C. krusei-mediated injury to endothelial cells by 69% (P < 0.01), whereas fluconazole did not exhibit significant protection (P < 0.6). These results demonstrate that voriconazole, in addition to its direct inhibitory activity against fungi, may act against Candida spp. by interfering with critical host/parasite interactions, such as adherence and endothelial cell damage, as well as germination. Therefore, this triazole represents a new and promising agent for the treatment of disseminated candidal infections caused by both fluconazole-susceptible and -resistant species.

Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells.

To identify genes encoding adhesins that mediate the binding of Candida albicans to endothelial cells, a genomic library from this organism was constructed and used to transform Saccharomyces cerevisiae. These transformed organisms were screened for adherence to endothelial cells, and a highly adherent clone was identified. The adherence of this clone to endothelial cells was over 100-fold greater than that of control S. cerevisiae transformed with the empty plasmid. This clone also exhibited enhanced adherence to epithelial cells. The C. albicans gene contained within this clone was found to be ALS1. These results indicate that ALS1 may encode a candidal adhesin.

Endothelial cell injury caused by Candida albicans is dependent on iron.

Although it is known that Candida albicans causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in Candida-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated and grew normally. Loading endothelial cells with iron reversed the cytoprotective effects of iron chelation. Moreover, chelation of endothelial cell iron significantly reduced phagocytosis of C. albicans by these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of C. albicans is required for endothelial cell injury to occur, inhibition of phagocytosis is likely the principal mechanism of the cytoprotective effects of iron chelation. The production of toxic reactive oxygen intermediates by host cells is known to be inhibited by iron chelation. Therefore, we investigated whether treating endothelial cells with antioxidants could mimic the cytoprotective effects of iron chelation. Neither extracellular nor membrane-permeative antioxidants reduced candidal injury of endothelial cells. Furthermore, depleting endothelial cells of the endogenous antioxidant glutathione did not render them more susceptible to damage by C. albicans. These results suggest that candidal injury of endothelial cells is independent of the production of reactive oxygen intermediates and that the cytoprotective effects of iron chelation are not due to inhibition of the synthesis of these toxic intermediates.