The Novel Candida albicans Transporter Dur31 Is a Multi-Stage Pathogenicity Factor

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine.     

Endogenous Thrombospondin-1 Regulates Leukocyte Recruitment and Activation and Accelerates Death from Systemic Candidiasis

Disseminated Candida albicans infection results in high morbidity and mortality despite treatment with existing antifungal drugs. Recent studies suggest that modulating the host immune response can improve survival, but specific host targets for accomplishing this goal remain to be identified. The extracellular matrix protein thrombospondin-1 is released at sites of tissue injury and modulates several immune functions, but its role in C. albicans pathogenesis has not been investigated. Here, we show that mice lacking thrombospondin-1 have an advantage in surviving disseminated candidiasis and more efficiently clear the initial colonization from kidneys despite exhibiting fewer infiltrating leukocytes. By examining local and systemic cytokine responses to C. albicans and other standard inflammatory stimuli, we identify a crucial function of phagocytes in this enhanced resistance. Subcutaneous air pouch and systemic candidiasis models demonstrated that endogenous thrombospondin-1 enhances the early innate immune response against C. albicans and promotes activation of inflammatory macrophages (inducible nitric oxide synthase⁺, IL-6^high, TNF-α^high, IL-10^low), release of the chemokines MIP-2, JE, MIP-1α, and RANTES, and CXCR2-driven polymorphonuclear leukocytes recruitment. However, thrombospondin-1 inhibited the phagocytic capacity of inflammatory leukocytes in vivo and in vitro, resulting in increased fungal burden in the kidney and increased mortality in wild type mice. Thus, thrombospondin-1 enhances the pathogenesis of disseminated candidiasis by creating an imbalance in the host immune response that ultimately leads to reduced phagocytic function, impaired fungal clearance, and increased mortality. Conversely, inhibitors of thrombospondin-1 may be useful drugs to improve patient recovery from disseminated candidiasis.     

CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis

CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47^-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice. Despite normal fungal colonization at earlier times, cd47^-/- mice at four days post-infection had increased colonization of brain and kidneys accompanied by stronger inflammatory reactions. Neutrophil and macrophage numbers were significantly elevated in kidneys and neutrophils in the brains of infected cd47^-/- mice. However, no defect in phagocytic activity towards C. albicans was observed in cd47^-/- bone-marrow-derived macrophages, and neutrophil and macrophage killing of C. albicans was not impaired. CD47-deficiency did not alter the early humoral immune response to C. albicans. Th1, Th2, and Th17 population of CD4⁺ T cells were expanded in the spleen, and gene expression profiles of spleen and kidney showed stronger pro-inflammatory signaling in infected cd47^-/- mice. The chemoattractant chemokines MIP-2α and MIP-2β were highly expressed in infected spleens of cd47^-/- mice. G-CSF, GM-CSF, and the inflammasome component NLRP3 were more highly expressed in infected cd47^-/- kidneys than in infected wild-type controls. Circulating pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) were significantly elevated, but IL-17 was decreased. These data indicate that CD47 plays protective roles against disseminated candidiasis and alters pro-inflammatory and immunosuppressive pathways known to regulate innate and T cell immunity.     

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.     

Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.     

SSD1 is integral to host defense peptide resistance in Candida albicans

Candida albicans is usually a harmless human commensal. Because inflammatory responses are not normally induced by colonization, antimicrobial peptides are likely integral to first-line host defense against invasive candidiasis. Thus, C. albicans must have mechanisms to tolerate or circumvent molecular effectors of innate immunity and thereby colonize human tissues. Prior studies demonstrated that an antimicrobial peptide-resistant strain of C. albicans, 36082^R, is hypervirulent in animal models versus its susceptible counterpart (36082^S). The current study aimed to identify a genetic basis for antimicrobial peptide resistance in C. albicans. Screening of a C. albicans genomic library identified SSD1 as capable of conferring peptide resistance to a susceptible surrogate, Saccharomyces cerevisiae. Sequencing confirmed that the predicted translation products of 36082^S and 36082^R SSD1 genes were identical. However, Northern analyses corroborated that SSD1 is expressed at higher levels in 36082^R than in 36082^S. In isogenic backgrounds, ssd1Δ/ssd1Δ null mutants were significantly more susceptible to antimicrobial peptides than parental strains but had equivalent susceptibilities to nonpeptide stressors. Moreover, SSD1 complementation of ssd1Δ/ssd1Δ mutants restored parental antimicrobial peptide resistance phenotypes, and overexpression of SSD1 conferred enhanced peptide resistance. Consistent with these in vitro findings, ssd1 null mutants were significantly less virulent in a murine model of disseminated candidiasis than were their parental or complemented strains. Collectively, these results indicate that SSD1 is integral to C. albicans resistance to host defense peptides, a phenotype that appears to enhance the virulence of this organism in vivo.     

Agp2, a Member of the Yeast Amino Acid Permease Family,Positively Regulates Polyamine Transport at the Transcriptional Level

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.     

Effect of Delta-9-Tetrahydrocannabinol on Mouse Resistance to Systemic Candida albicans Infection

Delta-9-tetrahydrocannabinol (Δ⁹-THC), the psychoactive component of marijuana, is known to suppress the immune responses to bacterial, viral and protozoan infections, but its effects on fungal infections have not been studied. Therefore, we investigated the effects of chronic Δ⁹-THC treatment on mouse resistance to systemic Candida albicans (C. albicans) infection. To determine the outcome of chronic Δ⁹-THC treatment on primary, acute systemic candidiasis, c57BL/6 mice were given vehicle or Δ⁹-THC (16 mg/kg) in vehicle on days 1–4, 8–11 and 15–18. On day 19, mice were infected with 5×10⁵ C. albicans. We also determined the effect of chronic Δ⁹-THC (4–64 mg/kg) treatment on mice infected with a non-lethal dose of 7.5×10⁴ C. albicans on day 2, followed by a higher challenge with 5×10⁵ C. albicans on day 19. Mouse resistance to the infection was assessed by survival and tissue fungal load. Serum cytokine levels were determine to evaluate the immune responses. In the acute infection, chronic Δ⁹-THC treatment had no effect on mouse survival or tissue fungal load when compared to vehicle treated mice. However, Δ⁹-THC significantly suppressed IL-12p70 and IL-12p40 as well as marginally suppressed IL-17 versus vehicle treated mice. In comparison, when mice were given a secondary yeast infection, Δ⁹-THC significantly decreased survival, increased tissue fungal burden and suppressed serum IFN-γ and IL-12p40 levels compared to vehicle treated mice. The data showed that chronic Δ⁹-THC treatment decreased the efficacy of the memory immune response to candida infection, which correlated with a decrease in IFN-γ that was only observed after the secondary candida challenge.     

Histatin 5 Resistance of Candida glabrata Can Be Reversed by Insertion of Candida albicans Polyamine Transporter-Encoding Genes DUR3 and DUR31

Candida albicans and Candida glabrata are predominant fungi associated with oral candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with high fungicidal activity against C. albicans, however many strains of C. glabrata are resistant. Since Hst 5 requires fungal binding to cell wall components prior to intracellular translocation, reduced Hst 5 binding to C. glabrata may be the reason for its insensitivity. C. glabrata has higher surface levels of β-1,3-glucans as compared with C. albicans; however these differences did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly, the biofilm matrix of C. glabrata contained significantly higher levels of β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell through polyamine transporters Dur3p and Dur31p that are uncharacterized in C. glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate limiting step is reduced uptake that can be overcome by expression of C. albicans Dur proteins in C. glabrata.     

Histatin 5 uptake by Candida albicans utilizes polyamine transporters Dur3 and Dur31 proteins

Histatin 5 (Hst 5) is a salivary gland-secreted cationic peptide with potent fungicidal activity against Candida albicans. Hst 5 kills fungal cells following intracellular translocation, although its selective transport mechanism is unknown. C. albicans cells grown in the presence of polyamines were resistant to Hst 5 due to reduced intracellular uptake, suggesting that this cationic peptide may enter candidal cells through native yeast polyamine transporters. Based upon homology to known Saccharomyces cerevisiae polyamine permeases, we identified six C. albicans Dur polyamine transporter family members and propose a new nomenclature. Gene deletion mutants were constructed for C. albicans polyamine transporters Dur3, Dur31, Dur33, Dur34, and were tested for Hst 5 sensitivity and uptake of spermidine. We found spermidine uptake and Hst 5 mediated killing were decreased significantly in Δdur3, Δdur31, and Δdur3/Δdur31 strains; whereas a DUR3 overexpression strain increased Hst 5 sensitivity and higher spermidine uptake. Treatment of cells with a spermidine synthase inhibitor increased spermidine uptake and Hst 5 killing, whereas protonophores and cold treatment reduced spermidine uptake. Inhibition assays showed that Hst 5 is a competitive analog of spermidine for uptake into C. albicans cells, and that Hst 5 Ki values were increased by 80-fold in Δdur3/Δdur31 cells. Thus, Dur3p and Dur31p are preferential spermidine transporters used by Hst 5 for its entry into candidal cells. Understanding of polyamine transporter-mediated internalization of Hst 5 provides new insights into the uptake mechanism for C. albicans toxicity, and further suggests design for targeted fungal therapeutic agents.     

Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells

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.     

Disruption of the intestinal mucosal barrier in Candida albicans infections

Candida albicans is a common microorganism in the intestine. However, invasive C. albicans infection has emerged as a life-threatening disease in recent years. The mortality rate of invasive candidiasis is high in critically ill hosts. C. albicans can switch from the yeast to the hyphal morphology, and take advantage of the impaired intestinal mucosal barrier and insufficient immunity of the host to facilitate its colonization and penetration. Despite the availability of potent new antifungal drugs in recent years, the treatment of severe candidiasis, especially candidaemia, has not been substantially improved. In this review, the virulence factors of C. albicans, as well as the antagonistic role of the intestinal mucosal barrier will be discussed to illuminate the mechanisms of C. albicans enterogenic infections.     

Flavodoxin-Like Proteins Protect Candida albicans from Oxidative Stress and Promote Virulence

The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C. albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases. FLPs were detected at the plasma membrane in C. albicans, and the quadruple mutant was more sensitive to linolenic acid, a polyunsaturated fatty acid that can auto-oxidize and promote lipid peroxidation. These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane. In support of this, a C. albicans coq3Δ mutant that fails to synthesize ubiquinone was also highly sensitive to oxidative stress. FLPs are critical for survival in the host, as the quadruple mutant was avirulent in a mouse model of systemic candidiasis under conditions where infection with wild type C. albicans was lethal. The quadruple mutant cells initially grew well in kidneys, the major site of C. albicans growth in mice, but then declined after the influx of neutrophils and by day 4 post-infection 33% of the mice cleared the infection. Thus, FLPs and ubiquinone are important new antioxidant mechanisms that are critical for fungal virulence. The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells.     

Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.