Engineering Candida albicans to secrete a host immunomodulatory factor.

Gene knockout and transgenic mice are important tools that are widely used to dissect the mammalian hosts' responses to microbial invasion. A novel alternative is to engineer the pathogen itself to secrete host factors that stimulate or suppress specific immune defense mechanisms. Herein, we have described and validated an approach to facilitate the production and export of ectopic host proteins, from the most prevalent human fungal pathogen, Candida albicans. Our strategy utilized a prepropeptide from the C. albicans secreted aspartic proteinase, Sap2p. The prepeptide facilitates entry of Sap2p into the secretory pathway, while the propeptide maintains the protease as an inactive precursor, until proteolytic cleavage in the Golgi apparatus releases the mature protein. The Sap2p prepropeptide coding sequence was linked to that of two mammalian calcium-binding proteins, S100A8 and S100A9, which are associated with symptomatic vaginal candidiasis. The resulting expression constructs were then introduced into C. albicans. While the S100A8 protein is secreted into the growth medium intact, the S100A9 protein is apparently degraded during transit. Nonetheless, culture supernatants from both S100A8 and S100A9 expressing C. albicans strains acted as potent chemoattractants for a macrophage-like cell line and polymorphonuclear leukocytes. Thus, the pathogen-derived mammalian proteins possessed the expected biological activity.

Three prevacuolar compartment Rab GTPases impact Candida albicans hyphal growth.

Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in a vps21Δ/Δ ypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21Δ/Δ ypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans.

Role for endosomal and vacuolar GTPases in Candida albicans pathogenesis.

The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21Delta and ypt72Delta mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21Delta and ypt72Delta mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.

TNF induction of jagged-1 in endothelial cells is NFkappaB-dependent.

TNF-alpha is a potent proinflammatory cytokine that induces endothelial cell (EC) adhesion molecules. In addition, TNF promotes angiogenesis by inducing an EC tip cell phenotype and the expression of jagged-1, a ligand for the notch pathway. Notch signaling is critical for vascular patterning and helps to restrict the proliferation of tip cells. Here we demonstrate that TNF induction of jagged-1 in human EC is rapid and dependent upon signaling through TNFR1, but not TNFR2. A luciferase reporter construct carrying 3.7 kb of 5' promoter sequence from the human gene was responsive to both TNF and overexpression of NFkappaB pathway components. TNF-induced promoter activation was blocked by treatment with an NFkappaB inhibitor or co-expression of dominant-negative IKKbeta. Mutations in a putative NFkappaB-binding site at -3.0 kb, which is conserved across multiple species, resulted in a loss of responsiveness to TNF and NFkappaB. Electromobility shift and chromatin immunoprecipitation assays revealed binding of both p50 and p65 to the promoter in response to TNF treatment. Full promoter activity also depends on an AP-1 site at -2.0 kb. These results indicate that canonical NFkappaB signaling is required for TNF induction of the notch ligand jagged-1 in EC.

Bmh1p (14-3-3) mediates pathways associated with virulence in Candida albicans.

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of 'virulence factors' using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 degrees C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of log-phase [corrected] growth in vitro at 37 degrees C, and not at 30 degrees C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures.

TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype.

Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFkappaB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFkappaB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Relationship between Candida albicans virulence during experimental hematogenously disseminated infection and endothelial cell damage in vitro.

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.

Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei.

Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14alpha-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

Endocytosis of Candida albicans by vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins.

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.