Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation

ACKNOWLEDGMENTS We acknowledge Jennifer Lodge, Woei Lam, and Rajendra Upadhya for developing and sharing the chitin and chitosan MTBH assay. We thank Todd Brennan of Duke University for providing MyD88-deficient mice. We acknowledge Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We also acknowledge Connie Nichols for critical reading of the manuscript. These experiments were supported by an NIH grant to J.A.A. and F.L.W., Jr. (R01 AI074677). C.M.L.W. was supported by a fellowship provided through the Army Research Office of the Department of Defense (no. W911NF-11-1-0136 f) (F.L.W., Jr.). J.W., L.W., and C.M. were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the MRC, Centre for Medical Mycology (MR/N006364/1). FUNDING INFORMATION MRC Centre for Medical MycologyMR/N006364/1 Carol A. Munro HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060R01 AI074677J. Andrew Alspaugh Wellcome https://doi.org/10.13039/100010269097377 Carol A. Munro DOD | United States Army | RDECOM | Army Research Office (ARO) https://doi.org/10.13039/100000183W911NF-11-1-0136 f Chrissy M. Leopold WagerPeer reviewe

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C. gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C. gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C. gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100 years. While the cause of C. gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition

Acknowledgments We acknowledge Jeanette Wagener and Louise Walker for performing the HPAEC-PAD analysis and Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We thank Mike Cook and the Duke University Cancer Center Flow Cytometry Shared Resource for assistance with the flow cytometry. We also acknowledge Michelle Plue and the Duke University Shared Materials Institute Facility for performing the transmission electron microscopy. We thank Marcel Wu¨thrich for providing the MyD88-/-and TLR2/4-/- mice, and Mari Shinohara and Elizabeth Deerhake for providing the Dectin-1-/- mice. Funding: These experiments were supported by a National Institutes of Health grant awarded to JAA and FLW, Jr. (R01 AI074677, https://grants.nih.gov/grants/oer.html). CM and colleagues Jeanette Wagener, Louise Walker, Neil Gow were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377, https://wellcome.ac.uk), Wellcome Trust Senior Investigator Award (101873) and the MRC Centre for Medical Mycology (MR/N006364/1, https://www.abdn.ac.uk/cmm/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicans erg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistanc

Fungal Morphogenesis

Morphogenesis in fungi is often induced by extracellular factors and executed by fungal genetic factors. Cell surface changes and alterations of the microenvironment often accompany morphogenetic changes in fungi. In this review, we will first discuss the general traits of yeast and hyphal morphotypes and how morphogenesis affects development and adaptation by fungi to their native niches, including host niches. Then we will focus on the molecular machinery responsible for the two most fundamental growth forms, yeast and hyphae. Last, we will describe how fungi incorporate exogenous environmental and host signals together with genetic factors to determine their morphotype and how morphogenesis, in turn, shapes the fungal microenvironment

Characterization of the PMT Gene Family in Cryptococcus neoformans

BACKGROUND:Protein-O-mannosyltransferases (Pmt's) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins. METHODOLOGY/PRINCIPAL FINDINGS:Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis. CONCLUSION/SIGNIFICANCE:These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host

The \u3ci\u3eCryptococcus neoformans\u3c/i\u3e Flc1 Homologue Controls Calcium Homeostasis and Confers Fungal Pathogenicity in the Infected Hosts

Cryptococcus neoformans, an opportunistic yeast pathogen, relies on a complex network of stress response pathways that allow for proliferation in the host. In Saccharomyces cerevisiae, stress responses are regulated by integral membrane proteins containing a transient receptor potential (TRP) domain, including the flavin carrier protein 1 (Flc1), which regulates calcium homeostasis and flavin transport. Here, we report that deletion of C. neoformans FLC1 results in cytosolic calcium elevation and increased nuclear content of calcineurin-dependent transcription factor Crz1, which is associated with an aberrant cell wall chitin overaccumulation observed in the flc1Δ mutant. Absence of Flc1 or inhibition of calcineurin with cyclosporine A prevents vacuolar fusion under conditions of combined osmotic and temperature stress, which is reversed in the flc1Δ mutant by the inhibition of TORC1 kinase with rapamycin. Flc1-deficient yeasts exhibit compromised vacuolar fusion under starvation conditions, including conditions that stimulate formation of carbohydrate capsule. Consequently, the flc1Δ mutant fails to proliferate under low nutrient conditions and displays a defect in capsule formation. Consistent with the previously uncharacterized role of Flc1 in vacuolar biogenesis, we find that Flc1 localizes to the vacuole. The flc1Δ mutant presents a survival defect in J774A.1 macrophage cell-line and profound virulence attenuation in both the Galleria mellonella and mouse pulmonary infection models, demonstrating that Flc1 is essential for pathogenicity. Thus, cryptococcal Flc1 functions in calcium homeostasis and links calcineurin and TOR signaling with vacuolar biogenesis to promote survival under conditions associated with vacuolar fusion required for this pathogen’s fitness and virulence

Interaction of Cryptococcus neoformans Rim101 and Protein Kinase A Regulates Capsule

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101Δ mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101Δ strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101Δ mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions

Remasking of Candida albicans β-Glucan in Response to Environmental pH Is Regulated by Quorum Sensing

Candida albicans is a commensal yeast of the human gut which is tolerated by the immune system but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host-derived environment cues (pH, hypoxia, and lactate). This cell wall remodeling allows C. albicans to evade or hyperactivate the host’s innate immune responses, leading to disease. Previously, we showed that adaptation of C. albicans to acidic environments, conditions encountered during colonization of the female reproductive tract, induces significant cell wall remodeling resulting in the exposure of two key fungal PAMPs (β-glucan and chitin). Here, we report that this pH-dependent cell wall remodeling is time dependent, with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking of β-glucan was mediated via the cell density-dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, nonproteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2. This dynamic cell wall remodeling influenced innate immune recognition of C. albicans, suggesting that during infection, C. albicans can manipulate the host innate immune responses

Adenylyl Cyclase Functions Downstream of the Gα Protein Gpa1 and Controls Mating and Pathogenicity of \u3ci\u3eCryptococcus neoformans\u3c/i\u3e

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen