Regulation of Copper Toxicity by Candida albicans GPA2

Copper is an essential nutrient that is toxic to cells when present in excess. The fungal pathogen Candida albicans employs several mechanisms to survive in the presence of excess copper, but the molecular pathways that govern these responses are not completely understood. We report that deletion of GPA2, which specifies a G-protein α subunit, confers increased resistance to excess copper and propose that the increased resistance is due to a combination of decreased copper uptake and an increase in copper chelation by metallothioneins. This is supported by our observations that a gpa2Δ/Δ mutant has reduced expression of the copper uptake genes, CTR1 and FRE7, and a marked decrease in copper accumulation following exposure to high copper levels. Furthermore, deletion of GPA2 results in an increased expression of the copper metallothionein gene, CRD2. Gpa2p functions upstream in the cyclic AMP (cAMP)-protein kinase A (PKA) pathway to govern hyphal morphogenesis. The copper resistance phenotype of the gpa2Δ/Δ mutant can be reversed by artificially increasing the intracellular concentration of cAMP. These results provide evidence for a novel role of the PKA pathway in regulation of copper homeostasis. Furthermore, the connection between the PKA pathway and copper homeostasis appears to be conserved in the pathogen Cryptococcus neoformans but not in the nonpathogenic Saccharomyces cerevisiae.     

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.     

Hsp21 Potentiates Antifungal Drug Tolerance in Candida albicans

Systemic infections of humans with the fungal pathogen Candida albicans are associated with a high mortality rate. Currently, efficient treatment of these infections is hampered by the relatively low number of available antifungal drugs. We recently identified the small heat shock protein Hsp21 in C. albicans and demonstrated its fundamental role for environmental stress adaptation and fungal virulence. Hsp21 was found in several pathogenic Candida species but not in humans. This prompted us to investigate the effects of a broad range of different antifungal drugs on an Hsp21-null C. albicans mutant strain. Our results indicate that combinatorial therapy targeting Hsp21, together with specific antifungal drug targets, has strong synergistic potential. In addition, we demonstrate that Hsp21 is required for tolerance to ethanol-induced stress and induction of filamentation in response to pharmacological inhibition of Hsp90. These findings might pave the way for the development of new treatment strategies against Candida infections.     

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2^-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2^-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2^-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.     

Neocentromeres Form Efficiently at Multiple Possible Loci in Candida albicans

Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-A^Cse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.     

Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans

Candida albicans, a dimorphic fungus, undergoes hyphal development in response to many different environmental cues, including growth in contact with a semi-solid matrix. C. albicans forms hyphae that invade agar when cells are embedded in or grown on the surface of agar, and the integral membrane protein Dfi1p is required for this activity. In addition, Dfi1p is required for full activation of mitogen activated protein kinase Cek1p during growth on agar. In this study, we identified a putative calmodulin binding motif in the C-terminal tail of Dfi1p. This region of Dfi1p bound to calmodulin in vitro, and mutations that affected this region affected both calmodulin binding in vitro and invasive filamentation when incorporated into the full length Dfi1p protein. Moreover, increasing intracellular calcium levels led to calcium-dependent, Dfi1p-dependent Cek1p activation. We propose that conformational changes in Dfi1p in response to environmental conditions encountered during growth allow the protein to bind calmodulin and initiate a signaling cascade that activates Cek1p.     

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.     

White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.     

N-Acetylglucosamine Induces White to Opaque Switching,a Mating Prerequisite in Candida albicans

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO₂, equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans.