Exposure of Candida albicans ß (1,3)-glucan is promoted by activation of the Cek1 pathway.

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of ß (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of ß (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in ß (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of ß (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased ß (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis.

Candida albicans mutants for phosphatidylserine (PS) synthase (cho1ΔΔ) and PS decarboxylase (psd1ΔΔ psd2ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the de novo pathway, but these mutants are still capable of growth in culture media, as they can import ethanolamine from media to synthesize PE through the Kennedy pathway. Given that the host has ethanolamine in its serum, the exact mechanism by which virulence is lost in these mutants is not clear. There are two competing hypotheses to explain their loss of virulence. (i) PE from the Kennedy pathway cannot substitute for de novo-synthesized PE. (ii) The mutants cannot acquire sufficient ethanolamine from the host to support adequate PE synthesis. These hypotheses can be simultaneously tested if ethanolamine availability is increased for Candida while it is inside the host. We accomplish this by transcomplementation of C. albicans with the Arabidopsis thaliana serine decarboxylase gene (AtSDC), which converts cytoplasmic serine to ethanolamine. Expression of AtSDC in either mutant restores PE synthesis, even in the absence of exogenous ethanolamine. AtSDC also restores virulence to cho1ΔΔ and psd1ΔΔ psd2ΔΔ strains in systemic and OPC infections. Thus, in the absence of de novo PE synthesis, C. albicans cannot acquire sufficient ethanolamine from the host to support virulence. In addition, expression of AtSDC restores PS synthesis in the cho1ΔΔ mutant, which may be due to causing PS decarboxylase to run backwards and convert PE to PS.

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

Lrg1 Regulates ß (1,3)-Glucan Masking in Candida albicans through the Cek1 MAP Kinase Pathway.

Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide ß (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes ß (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and ß (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1ΔΔ mutant exhibited increased activation of Cdc42 and Ras1 but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1ΔΔ mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1ΔΔ mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11ΔN467 allele was expressed in C. albicans In the absence of doxycycline, this allele overexpressed STE11ΔN467 , which induced production of proinflammatory tumor necrosis factor alpha (TNF-α) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11ΔN467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11ΔN467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture.IMPORTANCECandida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer ß (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during ß (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes ß (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.

A Student's Guide to Giant Viruses Infecting Small Eukaryotes: From Acanthamoeba to Zooxanthellae.

The discovery of infectious particles that challenge conventional thoughts concerning "what is a virus" has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning "giant viruses", with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host-virus systems that fall into this "giant" category, demonstrating that this field of inquiry presents great opportunities for future research.

Candida albicans OPI1 regulates filamentous growth and virulence in vaginal infections, but not inositol biosynthesis.

ScOpi1p is a well-characterized transcriptional repressor and master regulator of inositol and phospholipid biosynthetic genes in the baker's yeast Saccharomyces cerevisiae. An ortholog has been shown to perform a similar function in the pathogenic fungus Candida glabrata, but with the distinction that CgOpi1p is essential for growth in this organism. However, in the more distantly related yeast Yarrowia lipolytica, the OPI1 homolog was not found to regulate inositol biosynthesis, but alkane oxidation. In Candida albicans, the most common cause of human candidiasis, its Opi1p homolog, CaOpi1p, has been shown to complement a S. cerevisiae opi1∆ mutant for inositol biosynthesis regulation when heterologously expressed, suggesting it might serve a similar role in this pathogen. This was tested in the pathogen directly in this report by disrupting the OPI1 homolog and examining its phenotypes. It was discovered that the OPI1 homolog does not regulate INO1 expression in C. albicans, but it does control SAP2 expression in response to bovine serum albumin containing media. Meanwhile, we found that CaOpi1 represses filamentous growth at lower temperatures (30 °C) on agar, but not in liquid media. Although, the mutant does not affect virulence in a mouse model of systemic infection, it does affect virulence in a rat model of vaginitis. This may be because Opi1p regulates expression of the SAP2 protease, which is required for rat vaginal infections.

The Wsc1p cell wall signaling protein controls biofilm (Mat) formation independently of Flo11p in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains of the ∑1278b background generate biofilms, referred to as mats, on low-density agar (0.3%) plates made with rich media (YPD). Mat formation involves adhesion of yeast cells to the surface of the agar substrate and each other as the biofilm matures, resulting in elaborate water channels that create filigreed patterns of cells. The cell wall adhesion protein Flo11p is required for mat formation; however, genetic data indicate that other unknown effectors are also required. For example, mutations in vacuolar protein sorting genes that affect the multivesicular body pathway, such as vps27Δ, cause mat formation defects independently of Flo11p, presumably by affecting an unidentified signaling pathway. A cell wall signaling protein, Wsc1p, found at the plasma membrane is affected for localization and function by vps27Δ. We found that a wsc1 mutation disrupted mat formation in a Flo11p-independent manner. Wsc1p appears to impact mat formation through the Rom2p-Rho1p signaling module, by which Wsc1p also regulates the cell wall. The Bck1p, Mkk1/Mkk2, Mpk1p MAP kinase signaling cascade is known to regulate the cell wall downstream of Wsc1p-Rom2p-Rho1p but, surprisingly, these kinases do not affect mat formation. In contrast, Wsc1p may impact mat formation by affecting Skn7p instead. Skn7p can also receive signaling inputs from the Sln1p histidine kinase; however, mutational analysis of specific histidine kinase receiver residues in Skn7p indicate that Sln1p does not play an important role in mat formation, suggesting that Skn7p primarily acts downstream of Wsc1p to regulate mat formation.