Deletion of the Candida albicans PIR32 Results in Increased Virulence, Stress Response, and Upregulation of Cell Wall Chitin Deposition

Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.     

Roles of Cch1 and Mid1 in Morphogenesis, Oxidative Stress Response and Virulence in Candida albicans

Ca²⁺ channel Cch1, and its subunit Mid1, has been suggested as the protein complex responsible for mediating Ca²⁺ influx, which is often employed by fungal cells to maintain cell survival. The abilities of morphological switch and response to stress conditions are closely related to pathogenicity in Candida albicans. Cch1 and Mid1 activity are required for virulence of Cryptococcus neoformans and Claviceps purpurea, respectively. To investigate whether Cch1 and Mid1 also play a role in the virulence of C. albicans, we constructed cch1Δ/Δ and mid1Δ/Δ mutant strains for functional analysis of CCH1 and MID1. Although both of the mutants displayed the ability of yeast-to-hypha transition, they were defective in hyphae maintenance and invasive growth. Interestingly, deletion of CCH1 or MID1 in C. albicans led to an obvious defect phenotype in oxidative stress response. Moreover, the virulence of the mutants was reduced in a mouse model. Our results demonstrated that Cch1 and Mid1 activity are related to the virulence of C. albicans and may provide a new antifungal target.     

Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using ¹⁵N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.     

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.     

Virulence factors of Candida albicans.

Candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. Although most infections occur in patients who are immunocompromised or debilitated in some other way, the organism most often responsible for disease, Candida albicans, expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), secreted aspartyl proteases and phospholipases. Additionally, 'phenotypic switching' is accompanied by changes in antigen expression, colony morphology and tissue affinities in C. albicans and several other Candida spp. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection.     

Virulence genes in the pathogenic yeast Candida albicans

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.     

The Calcium Channel Blocker Verapamil Inhibits Oxidative Stress Response in Candida albicans

Candida albicans is a common opportunistic fungal pathogen, causing both superficial candidiasis and life-threatening systemic infections in immune-compromised individuals. Calcium signaling is responsible for this pathogen in responding to several stresses, such as antifungal drugs, alkaline pH and membrane-perturbing agents. Our recent study revealed that it is also involved in oxidative stress response. In this study, we investigated the effect of verapamil, an L-type voltage-gated calcium channel blocker, on oxidative stress response in this fungus. The addition of verapamil resulted in increased sensitivity to the oxidative agent H₂O₂, which is associated with a decrease of calcium fluctuation under the stress. Moreover, this agent caused enhanced oxidative stress, with increased levels of ROS and enhanced dysfunction of the mitochondria under the oxidative stress. Further investigations in SOD activity, GSH contents and expression of oxidative stress response-related genes indicated that the effect of verapamil is related to the repression of oxidative stress response. Our findings demonstrated that verapamil has an inhibitory effect on oxidative stress response, confirming the relationship between calcium signaling and oxidative stress in C. albicans. Therefore, calcium channels may be potential targets for therapy to enhance the efficacy of oxidative stress against C. albicans-related infections.     

Interactions of Candida albicans with epithelial cells

The fungus, Candida albicans, interacts with epithelial cells in the human host both as a normal commensal and as an invasive pathogen. It has evolved multiple complementary mechanisms to adhere to epithelial cells. Adherent C. albicans cells can invade epithelial surfaces both by penetrating into individual epithelial cells, and by degrading interepithelial cell junctions and passing between epithelial cells. Invasion into epithelial cells is mediated by both induced endocytosis and active penetration, whereas degradation of epithelial cell junction proteins, such as E‐cadherin, occurs mainly via proteolysis by secreted aspartyl proteinases. C. albicans invasion of epithelial cells results in significant epithelial cell damage, which is probably induced by lytic enzymes, such as proteases and phospholipase secreted by the organism. Future challenges include identifying the epithelial cell targets of adhesins and invasins, and determining the mechanisms by which C. albicans actively penetrates epithelial cells and induces epithelial cell damage.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.