Changes in glutathione-dependent redox status and mitochondrial energetic strategies are part of the adaptive response during the filamentation process in Candida albicans

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases called candidiasis. Its ability to grow in various morphological forms, such as unicellular budding yeast, filamentous pseudohyphae and hyphae, contributes to its survival in the diverse microenvironments it encounters in the host. During infection in vivo, C. albicans is faced with high levels of reactive oxygen species (ROS) generated by phagocytes, and the thiol-dependent redox status of the cells reflects their levels of oxidative stress. We investigated the role of glutathione during the transition between the yeast and hyphal forms of the pathogen, in relation to possible changes in mitochondrial bioenergetic pathways. Using various growth media and selective mutations affecting the filamentation process, we showed that C. albicans filamentation was always associated with a depletion of intracellular glutathione levels. Moreover, the induction of hypha formation resulted in general changes in thiol metabolism, including the oxidation of cell surface − SH groups and glutathione excretion. Metabolic adaptation involved tricarboxylic acid (TCA) cycle activation, acceleration of mitochondrial respiration and a redistribution of electron transfer pathways, with an increase in the contribution of the alternative oxidase and rotenone-insensitive dehydrogenase. Changes in redox status and apparent oxidative stress may be necessary to the shift to adaptive metabolic pathways, ensuring normal mitochondrial function and adenosine triphosphate (ATP) levels. The consumption of intracellular glutathione levels during the filamentation process may thus be the price paid by C. albicans for survival in the conditions encountered in the host.     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Tight Control of Trehalose Content Is Required for Efficient Heat-induced Cell Elongation in Candida albicans

The ability to form hyphae in the human pathogenic fungus Candida albicans is a prerequisite for virulence. It contributes to tissue infection, biofilm formation, as well as escape from phagocytes. Cell elongation triggered by human body temperature involves the essential heat shock protein Hsp90, which negatively governs a filamentation program dependent upon the Ras-protein kinase A (PKA) pathway. Tight regulation of Hsp90 function is required to ensure fast appropriate response and maintenance of a wide range of regulatory and signaling proteins. Client protein activation by Hsp90 relies on a conformational change of the chaperone, whose ATPase activity is competitively inhibited by geldanamycin. We demonstrate a novel regulatory mechanism of heat- and Hsp90-dependent induced morphogenesis, whereby the nonreducing disaccharide trehalose acts as a negative regulator of Hsp90 release. By means of a mutant strain deleted for Gpr1, the G protein-coupled receptor upstream of PKA, we demonstrate that elevated trehalose content in that strain, resulting from misregulation of enzymatic activities involved in trehalose metabolism, disrupts the filamentation program in response to heat. Addition of geldanamycin does not result in hyphal extensions at 30 °C in the gpr1Δ/gpr1Δ mutant as it does in wild type cells. In addition, validamycin, a specific inhibitor of trehalase, the trehalose-degrading enzyme, inhibits cell elongation in response to heat and geldanamycin. These results place Gpr1 as a regulator of trehalose metabolism in C. albicans and illustrate that trehalose modulates Hsp90-dependent activation of client proteins and signaling pathways leading to filamentation in the human fungal pathogen.     

A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Rheb is a new member of the small G proteins of the Ras superfamily in eukaryotic organisms and controls various physiological processes. Activity of Rheb is regulated by Tsc2, a GTPase-activating protein (GAP). In this study, we have identified Candida albicans homologs of Rheb (named as Rhb1) and Tsc2. Deletion of the RHB1 gene showed enhanced sensitivity to rapamycin (an inhibitor of TOR kinase), suggesting that Rhb1 is associated with the TOR signaling pathway in C. albicans. Further analysis indicated RHB1 and TSC2 are involved in nitrogen starvation-induced filamentation, likely by controlling the expression of MEP2 whose gene product is an ammonium permease and a sensor for the nitrogen signal. Moreover, we have demonstrated that Rhb1 is also involved in cell wall integrity pathway, by transferring signals through the TOR kinase and the Mkc1 MAP kinase pathway. Together, this study brings new insights into the complex interplay of signaling and regulatory pathways in C. albicans.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.     

Soybean Toxin (SBTX) Impairs Fungal Growth by Interfering with Molecular Transport,Carbohydrate/Amino Acid Metabolism and Drug/Stress Responses

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.     

Characterization of Switch Phenotypes in <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee’s medium agar supplemented with arginine and zinc, at 25 °C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.     

An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

Inhibitors of CPH1-MAP Kinase Pathway: Ascertaining Potential Ligands as Multi-Target Drug Candidate in Candida albicans

A number of proteins contributing in pathogen’s virulence mechanisms offer a potential target for anticandidal therapeutics. CPH1 and its regulatory proteins Cst20, Hst7, Cek1 (MAPK cascade) administers filamentation and morphogenesis in human pathogenic fungus Candida albicans. These proteins are essential targets for their involvement in the successful establishment of the fungi within the host. In silico drug design using virtual screening, docking and (ADME)/Tox analysis for identification of lead compounds is an economic strategy for the development of potent anticandidal agent. The study divulged five persuasive ligands (2-O-prenyl coumaric acid, 2-nitro-4-methyl-cinnamaldehyde, 3,5-diprenyl-4-coumaric acid, VT1161, T-2307) out of 25, which collectively inhibited Cst20, Hst7, Cek1 in C. albicans. They can hence be used as natural drug leads for designing more effective inhibitors of multiple targets for C. albicans survival and progression of the disease. This study will enhance our understanding of the phenomenon “multiple targeting” and multi-target drug discovery further accelerating efficient broad-spectrum antifungal therapeutics development in near future. This study provides a good platform to eradicate the issue of “target shortage” that might facilitate the discovery of novel drugs in near future because the prolonged use of antibiotics over the years has transformed pathogenic fungus into resistant to many drugs.

     

A Candida albicans cell wall‐linked protein promotes invasive filamentation into semi‐solid medium

Growth of cells in contact with an abiotic or biological surface profoundly affects cellular physiology. In the opportunistic human pathogen, Candida albicans, growth on a semi‐solid matrix such as agar results in invasive filamentation, a process in which cells change their morphology to highly elongated filamentous hyphae that grow into the matrix. We hypothesized that a plasma membrane receptor‐type protein would sense the presence of matrix and activate a signal transduction cascade, thus promoting invasive filamentation. In this communication, we demonstrate that during growth in contact with a semi‐solid surface, activation of a MAP kinase, Cek1p, is promoted, in part, by a plasma membrane protein termed Dfi1p and results in invasive filamentation. A C. albicans mutant lacking Dfi1p showed reduced virulence in a murine model of disseminated candidiasis. Dfi1p is a relatively small, integral membrane protein that localizes to the plasma membrane. Some Dfi1p molecules become cross‐linked to the carbohydrate polymers of the cell wall. Thus, Dfi1p is capable of linking the cell wall to the plasma membrane and cytoplasm.