Role and mechanism of Phosphatidylinositol-specific Phospholipase C in survival and virulence of Cryptococcus neoformans. Mol Microbiol
Methee ChayakulkeereeTania C. SorrellA. Rosemary SiafakasChristabel F. WilsonNamfon PantaratKimberly J. GerikRoss BoadleJulianne T. Djordjevic
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Phospholipase D
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ABSTRACT Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1 -overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1 Δ mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1 Δ mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.
CDC42
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The protein kinase A (PKA) signal transduction pathway has been associated with pathogenesis in many fungal species. Geddes and colleagues [mBio 7(1):e01862-15, 2016, doi:10.1128/mBio.01862-15] used quantitative proteomics approaches to define proteins with altered abundance during protein kinase A (PKA) activation and repression in the opportunistic human fungal pathogen Cryptococcus neoformans. They observed an association between microbial PKA signaling and ubiquitin-proteasome regulation of protein homeostasis. Additionally, they correlated these processes with expression of polysaccharide capsule on the fungal cell surface, the main virulence-associated phenotype in this organism. Not only are their findings important for microbial pathogenesis, but they also support similar associations between human PKA signaling and ubiquitinated protein accumulation in neurodegenerative diseases.
Pathogenesis
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Abstract Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans . To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1 Δ strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.
Intracellular parasite
Virulence factor
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The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.
Antifungal drugs
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MAPK cascade
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The yeast class III phosphoinositide 3-kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol-3-phosphate, is primarily implicated in vesicle-mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit-encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy-related PI3K complex genes, we show that C. glabrata PI3K-impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.
Candida glabrata
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Summary Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans . To investigate the role of phosphatidylinositol‐specific phospholipase C (PI‐PLC) in Plb1 secretion, we identified two putative PI‐PLC‐encoding genes in C. neoformans var. grubii ( PLC1 and PLC2 ), and created Δ plc1 and Δ plc2 deletion mutants. In Δ plc1 , which expressed less PI‐PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Δ plc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen‐activated protein kinase (MAPK) in the presence of cell wall‐perturbing agents. In contrast to Δ plc2 , which was as virulent as WT, Δ plc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.
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Bacterial phosphatidylinositol‐specific phospholipases C have been shown not only to cause breakdown of phosphatidylinositol but also to release GPI‐anchored proteins from the plasma membranes of eucaryotes. Several enzymes in this group have been sequenced by cloning genomic DNA, and the enzymes of Bacillus cereus and Listeria monocytogenes were analyzed for their structures by X‐ray crystallography. In the active sites of enzymes from Bacillus genera and L. monocytogenes, the roles of component amino acid residues in catalysis have been mostly clarified. The enzyme of Bacillus thuringiensis exhibited cytotoxicity against some cultivated cells. The enzyme of L. monocytogenes was shown to contribute to listerial infection of epithelial cells and macrophages as a virulence factor cooperating with other factors such as listeriolysin O and phosphatidylcholine‐preferring phospholipase C. Recently, this enzyme proved to stimulate the signal‐transduction system of host cells in listeriosis. The requirements for effective utilization of bacterial phosphatidylinositol‐specific phospholipases C should be considered in research on GPI‐anchored proteins, cellular transduction, and so forth, given the unique properties of these enzymes.
Listeriolysin O
Phosphoinositide phospholipase C
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Virulence factor
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Listeria monocytogenes is a model intracellular pathogen which escapes from a host cell vacuole, grows intracytoplasmically, and spreads cell to cell without an extracellular phase. A number of genes necessary for pathogenicity have been discovered, two of which encode phospholipases C, a PI-PLC and a broad-range PLC. Single and double mutants were constructed with in-frame deletions in one or both PLCs. Characterization of the strains indicated that the two PLCs may have overlapping function as the double mutant was 500-fold less virulent while the single mutants had a negligible effect on virulence. The role of the PLCs appears to be multifactorial as PI-PLC has a role in escaping from the initial host vacuole and the broad-range PLC appears to have a role in cell to cell spreading.
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The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and to antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.
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