The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. kruseiwas 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes fromC. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content ofC. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm forC. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that ofC. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicansand C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.

Endothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection with Candida albicans by secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined that C. albicans stimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-␣), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-␣ but also by IL-1␣ and IL-1␤, all of which were synthesized by endothelial cells in response to C. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-␣, IL-1␣, and IL-1␤. These observations demonstrate that different proinflammatory endothelial cell responses to C. albicans are induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response to C. albicans and perhaps other opportunistic pathogens that disseminate hematogenously.