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ABSTRACTDuring hematogenously disseminated infection, blood-borne Candida albicansinvades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicansAls3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3?/? mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicansinvasins. To elucidate the in vivofunction of Als3, we heterologously expressed C. albicans ALS3in Candida glabrata, a yeast that lacks a close ALS3ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.

ABSTRACTVaccination with the recombinant N terminus of the candidal adhesin Als3p (rAls3p-N) protects mice from lethal candidemia. Candidal Als3p also is structurally similar to the microbial surface components recognizing adhesive matrix molecule adhesin, clumping factor, from Staphylococcus aureus.To determine the potential for cross-kingdom vaccination, we immunized mice with rAls3p-N or negative control proteins and challenged them via the tail vein with S. aureusor other gram-positive or gram-negative pathogens. The rAls3p-N vaccine, but neither tetanus toxoid nor a related Als protein (Als5p), improved the survival of vaccinated mice subsequently infected with multiple clinical isolates of S. aureus, including methicillin-resistant strains. The rAls3p-N vaccine was effective against S. aureuswhen combined with aluminum hydroxide adjuvant. However, the vaccine did not improve the survival of mice infected with other bacterial pathogens. Vaccinated, infected mice mounted moderated type 1 immune responses. T lymphocyte-deficient mice were more susceptible to S. aureusinfection, but B lymphocyte-deficient mice were not. Furthermore, T but not B lymphocytes from vaccinated mice mediated protection in adoptive transfer studies. The passive transfer of immune serum was not protective. These data provide the foundation for cross-kingdom vaccine development against S. aureusand Candida, which collectively cause 200,000 bloodstream infections resulting in ≥40,000 to 50,000 deaths annually in the United States alone.

ABSTRACTRhizopus oryzaeis the most common cause of zygomycosis, a life-threatening infection that usually occurs in immunocompromised patients. A characteristic hallmark of zygomycosis is angioinvasion by the fungus, resulting in thrombosis and subsequent tissue necrosis. Interactions between R. oryzaeand vascular endothelial cells are therefore likely of central importance to the organism's pathogenetic strategy. We studied the ability of R. oryzaeto adhere to and damage human umbilical vein endothelial cells (HUVECs) in vitro. We report that R. oryzaespores and germ tubes adhere to HUVECs, whereas only spores adhere to subendothelial matrix proteins. Additionally, R. oryzaedamages endothelial cells. This endothelial cell damage requires direct contact and subsequent phagocytosis of the fungus. Surprisingly, R. oryzaeviability was not required for damage, but phagocytosis was required for dead R. oryzaeto cause damage. These results elucidate the nature of R. oryzae-endothelial cell interactions, which are likely central to the angioinvasion and tissue necrosis seen during zygomycotic infections. The fact that dead R. oryzaedamage human endothelial cells may, in part, explain the lack of efficacy of fungicidal agents during clinical disease.

ABSTRACTCandida albicansmust penetrate the endothelial cell lining of the vasculature to invade the deep tissues during a hematogenously disseminated infection. We compared 27 C. albicansmutants 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.

ABSTRACTThe ability of Candida albicansto respond to diverse environments is critical for its success as a pathogen. TheRIM101pathway controls gene expression and the yeast-to-hyphal transition in C. albicansin response to changes in environmental pH in vitro. In this study, we found that theRIM101pathway is necessary in vivo for pathogenesis. First, we show thatrim101−/rim101−andrim8−/rim8−mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, therim101−/rim101−andrim8−/rim8−mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of RIM101, RIM101-405, is a suppressor of the rim8−mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the rim8−/rim8−mutant. These results demonstrate that the RIM101pathway is required for C. albicansvirulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.

ABSTRACTThe Candida albicansgene HWP1encodes a surface protein that is required for normal hyphal development in vitro. We used mutants lacking one or both alleles of HWP1to investigate the role of this gene in virulence. Mice infected intravenously with the homozygous hwp1null mutant, CAL3, survived a median of >14 days, whereas mice infected with a control strain containing two functional alleles of HWP1survived only 3.5 days. After 1 day of infection, all strains produced similar levels of infection in the kidneys, spleen, and blood. However, after 2 and 3 days, there was a significant decrease in the number of organisms in the kidneys of the mice infected with CAL3. This finding suggests that the hwp1homozygous null mutant is normal in its ability to initiate infection but deficient in its capacity to maintain infection. CAL3 also germinated minimally in the kidneys. The ability of the heterozygous null mutant to germinate and cause mortality in mice was intermediate to CAL3, suggesting a gene dosage effect. To investigate potential mechanisms for the diminished virulence of CAL3, we examined its interactions with endothelial cells and neutrophils in vitro. CAL3 caused less endothelial cell injury than the heterozygoushwp1mutant. We conclude that the HWP1gene product is important for both in vivo hyphal development and pathogenicity of C. albicans. Also, the ability to form filaments may be critical for candidal virulence by enabling the fungus to induce cellular injury and maintain a deep-seated infection.

ABSTRACTThe disruption of a specific gene in Candida albicansis commonly used to determine the function of the gene product. We disrupted AAF1, a gene of C. albicansthat causes Saccharomyces cerevisiaeto flocculate and adhere to endothelial cells. We then characterized multiple heterozygous and homozygous mutants. These null mutants adhered to endothelial cells to the same extent as did the parent organism. However, mutants with presumably the same genotype revealed significant heterogeneity in their growth rates in vitro. This heterogeneity was not the result of the transformation procedure per se, nor was it caused by differences in the expression or function of URA3, a marker used in the process of gene disruption. The growth rate among the different heterozygous and homozygous null mutants was positively correlated with in vivo virulence in mice. It is possible that the variable phenotypes ofC. albicanswere due to mutations outside of theAAF1coding region that were introduced during the gene disruption process. These results indicate that careful phenotypic characterization of mutants of C. albicansgenerated through targeted gene disruption should be performed to exclude the introduction of unexpected mutations that may influence pathogenicity in mice.

ABSTRACTTo identify genes encoding adhesins that mediate the binding ofCandida albicansto endothelial cells, a genomic library from this organism was constructed and used to transformSaccharomyces 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. cerevisiaetransformed with the empty plasmid. This clone also exhibited enhanced adherence to epithelial cells. The C. albicansgene contained within this clone was found to beALS1. These results indicate that ALS1may encode a candidal adhesin.

ABSTRACTAdherence to the endothelial cell lining of the vasculature is probably a critical step in the egress of Candida albicansfrom the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library from C. albicanswas used to transform a nonadherent strain of Saccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared with S. cerevisiaetransformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designated CAD1. It was found to be identical to AAF1. The predicted protein encoded by CAD1/AAF1contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed with CAD1/AAF1both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined. S. cerevisiaeexpressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressing CAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced by CAD1/AAF1was similar to Flo1. Thus, CAD1/AAF1probably encodes a regulatory protein that stimulates endothelial cell adherence in S. cerevisiaeby inducing a flocculation phenotype. Whether CAD1/AAF1contributes to the adherence of C. albicansto endothelial cells remains to be determined.

ABSTRACTAlthough it is known that Candida albicanscauses 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. albicansby these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of C. albicansis 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.

The mechanisms by which Candida glabrataresists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable C. glabratato withstand these classes of stressors, a library of 215 C. glabratatranscriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin.

ABSTRACTWe have previously shown that vaccination with a vaccine based on the recombinant N-terminal domain of Als1p (rAls1p-N) protected BALB/c mice against disseminated infection caused by a single strain of Candida albicans(A. S. Ibrahim, B. J. Spellberg, V. Avenissian, Y. Fu, S. G. Filler, and J. E. Edwards, Jr., Infect. Immun. 73:999-1005, 2005, and B. J. Spellberg, A. S. Ibrahim, V. Avenissian, S. G. Filler, C. Myers, Y. Fu, and J. E. Edwards, Jr., Infect. Immun. 73:6191-6193, 2005). Here we show that the rAls1p-N vaccine also improves survival of outbred mice from disseminated candidiasis and that it is active against multiple virulent strains of C. albicansand non-C. albicansspp.

ABSTRACTWe 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.

ABSTRACTThe endothelial cell interactions of homozygous null mutants ofCandida albicansthat were deficient in secreted aspartyl proteinase 1 (Sap1), Sap2, or Sap3 were investigated. Only Sap2 was found to contribute to the ability of C. albicansto damage endothelial cells and stimulate them to express E-selectin. None of the Saps studied appears to play a role in C. albicansadherence to endothelial cells.