Disruption to de novo uridine biosynthesis alters ß-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of ß-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing ß-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of ß-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly ß-1,3-glucan. However, ß-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase ß-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase.

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCE: Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a Ki value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.

Caspofungin-induced ß(1,3)-glucan exposure in Candida albicans is driven by increased chitin levels.

To successfully induce disease, Candida albicans must effectively evade the host immune system. One mechanism used by C. albicans to achieve this is to mask immunogenic ß(1,3)-glucan epitopes within its cell wall under an outer layer of mannosylated glycoproteins. Consequently, induction of ß(1,3)-glucan exposure (unmasking) via genetic or chemical manipulation increases fungal recognition by host immune cells in vitro and attenuates disease during systemic infection in mice. Treatment with the echinocandin caspofungin is one of the most potent drivers of ß(1,3)-glucan exposure. Several reports using murine infection models suggest a role for the immune system, and specifically host ß(1,3)-glucan receptors, in mediating the efficacy of echinocandin treatment in vivo. However, the mechanism by which caspofungin-induced unmasking occurs is not well understood. In this report, we show that foci of unmasking co-localize with areas of increased chitin within the yeast cell wall in response to caspofungin, and that inhibition of chitin synthesis via nikkomycin Z attenuates caspofungin-induced ß(1,3)-glucan exposure. Furthermore, we find that both the calcineurin and Mkc1 mitogen-activated protein kinase pathways work synergistically to regulate ß(1,3)-glucan exposure and chitin synthesis in response to drug treatment. When either of these pathways are interrupted, it results in a bimodal population of cells containing either high or low chitin content. Importantly, increased unmasking correlates with increased chitin content within these cells. Microscopy further indicates that caspofungin-induced unmasking correlates with actively growing cells. Collectively, our work presents a model in which chitin synthesis induces unmasking within the cell wall in response to caspofungin in growing cells. IMPORTANCE Systemic candidiasis has reported mortality rates ranging from 20% to 40%. The echinocandins, including caspofungin, are first-line antifungals used to treat systemic candidiasis. However, studies in mice have shown that echinocandin efficacy relies on both its cidal impacts on Candida albicans, as well as a functional immune system to successfully clear invading fungi. In addition to direct C. albicans killing, caspofungin increases exposure (unmasking) of immunogenic ß(1,3)-glucan moieties. To evade immune detection, ß(1,3)-glucan is normally masked within the C. albicans cell wall. Consequently, unmasked ß(1,3)-glucan renders these cells more visible to the host immune system and attenuates disease progression. Therefore, discovery of how caspofungin-induced unmasking occurs is needed to elucidate how the drug facilitates host immune system-mediated clearance in vivo. We report a strong and consistent correlation between chitin deposition and unmasking in response to caspofungin and propose a model in which altered chitin synthesis drives increased unmasking during drug exposure.

Cek1 regulates ß(1,3)-glucan exposure through calcineurin effectors in Candida albicans.

In order to successfully induce disease, the fungal pathogen Candida albicans regulates exposure of antigens like the cell wall polysaccharide ß(1,3)-glucan to the host immune system. C. albicans covers (masks) ß(1,3)-glucan with a layer of mannosylated glycoproteins, which aids in immune system evasion by acting as a barrier to recognition by host pattern recognition receptors. Consequently, enhanced ß(1,3)-glucan exposure (unmasking) makes fungal cells more visible to host immune cells and facilitates more robust fungal clearance. However, an understanding of how C. albicans regulates its exposure levels of ß(1,3)-glucan is needed to leverage this phenotype. Signal transduction pathways and their corresponding effector genes mediating these changes are only beginning to be defined. Here, we report that the phosphatase calcineurin mediates unmasking of ß(1,3)-glucan in response to inputs from the Cek1 MAPK pathway and in response to caspofungin exposure. In contrast, calcineurin reduces ß-glucan exposure in response to high levels of extracellular calcium. Thus, depending on the input, calcineurin acts as a switchboard to regulate ß(1,3)-glucan exposure levels. By leveraging these differential ß(1,3)-glucan exposure phenotypes, we identified two novel effector genes in the calcineurin regulon, FGR41 and C1_11990W_A, that encode putative cell wall proteins and mediate masking/unmasking. Loss of either effector caused unmasking and attenuated virulence during systemic infection in mice. Furthermore, immunosuppression restored the colonization decrease seen in mice infected with the fgr41Δ/Δ mutant to wild-type levels, demonstrating a reliance on the host immune system for virulence attenuation. Thus, calcineurin and its downstream regulon are general regulators of unmasking.

Activation of Cph1 causes ß(1,3)-glucan unmasking in Candida albicans and attenuates virulence in mice in a neutrophil-dependent manner.

Masking the immunogenic cell wall epitope ß(1,3)-glucan under an outer layer of mannosylated glycoproteins is an important virulence factor deployed by Candida albicans during infection. Consequently, increased ß(1,3)-glucan exposure (unmasking) reveals C. albicans to the host's immune system and attenuates its virulence. We have previously shown that activation of the Cek1 MAPK pathway via expression of a hyperactive allele of an upstream kinase (STE11ΔN467) induced unmasking. It also increased survival of mice in a murine disseminated candidiasis model and attenuated kidney fungal burden by ≥33 fold. In this communication, we utilized cyclophosphamide-induced immunosuppression to test if the clearance of the unmasked STE11ΔN467 mutant was dependent on the host immune system. Suppression of the immune response by cyclophosphamide reduced the attenuation in fungal burden caused by the STE11ΔN467 allele. Moreover, specific depletion of neutrophils via 1A8 antibody treatment also reduced STE11ΔN467-dependent fungal burden attenuation, but to a lesser extent than cyclophosphamide, demonstrating an important role for neutrophils in mediating fungal clearance of unmasked STE11ΔN467 cells. In an effort to understand the mechanism by which Ste11ΔN467 causes unmasking, transcriptomics were used to reveal that several components in the Cek1 MAPK pathway were upregulated, including the transcription factor CPH1 and the cell wall sensor DFI1. In this report we show that a cph1ΔΔ mutation restored ß(1,3)-glucan exposure to wild-type levels in the STE11ΔN467 strain, confirming that Cph1 is the transcription factor mediating Ste11ΔN467-induced unmasking. Furthermore, Cph1 is shown to induce a positive feedback loop that increases Cek1 activation. In addition, full unmasking by STE11ΔN467 is dependent on the upstream cell wall sensor DFI1. However, while deletion of DFI1 significantly reduced Ste11ΔN467-induced unmasking, it did not impact activation of the downstream kinase Cek1. Thus, it appears that once stimulated by Ste11ΔN467, Dfi1 activates a parallel signaling pathway that is involved in Ste11ΔN467-induced unmasking.