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 reviewedPublisher PD

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

Hypoglycemia and Death in Mice Following Experimental Exposure to an Extract of Trogia venenata Mushrooms

BACKGROUND: Clusters of sudden unexplained death (SUD) in Yunnan Province, China, have been linked to eating Trogia venenata mushrooms. We evaluated the toxic effect of this mushroom on mice. METHODS: We prepared extracts of fresh T. venenata and Laccaria vinaceoavellanea mushrooms collected from the environs of a village that had SUD. We randomly allocated mice into treatment groups and administered mushroom extracts at doses ranging from 500 to 3500 mg/kg and water (control) via a gavage needle. We observed mice for mortality for 7 days after a 3500 mg/kg dose and for 24 hours after doses from 500 to 3000 mg/kg. We determined biochemical markers from serum two hours after a 2000 mg/kg dose. RESULTS: Ten mice fed T. venenata extract (3500 mg/kg) died by five hours whereas all control mice (L. vinaceoavellanea extract and water) survived the seven-day observation period. All mice died by five hours after exposure to single doses of T. venenata extract ranging from 1500 to 3000 mg/kg, while the four mice exposed to a 500 mg/kg dose all survived. Mice fed 2000 mg/kg of T. venenata extract developed profound hypoglycemia (median= 0.66 mmol/L) two hours after exposure. DISCUSSION: Hypoglycemia and death within hours of exposure, a pattern unique among mushroom toxicity, characterize T. venenata poisoning

Validation of the Tetracycline Regulatable Gene Expression System for the Study of the Pathogenesis of Infectious Disease

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases

Risk Factors and Outcomes of Candidemia Caused by Biofilm-Forming Isolates in a Tertiary Care Hospital

Very few data exist on risk factors for developing biofilm-forming Candida bloodstream infection (CBSI) or on variables associated with the outcome of patients treated for this infection. METHODS AND FINDINGS: We identified 207 patients with CBSI, from whom 84 biofilm-forming and 123 non biofilm-forming Candida isolates were recovered. A case-case-control study to identify risk factors and a cohort study to analyze outcomes were conducted. In addition, two sub-groups of case patients were analyzed after matching for age, sex, APACHE III score, and receipt of adequate antifungal therapy. Independent predictors of biofilm-forming CBSI were presence of central venous catheter (odds ratio [OR], 6.44; 95% confidence interval [95% CI], 3.21-12.92) or urinary catheter (OR, 2.40; 95% CI, 1.18-4.91), use of total parenteral nutrition (OR, 5.21; 95% CI, 2.59-10.48), and diabetes mellitus (OR, 4.47; 95% CI, 2.03-9.83). Hospital mortality, post-CBSI hospital length of stay (LOS) (calculated only among survivors), and costs of antifungal therapy were significantly greater among patients infected by biofilm-forming isolates than those infected by non-biofilm-forming isolates. Among biofilm-forming CBSI patients receiving adequate antifungal therapy, those treated with highly active anti-biofilm (HAAB) agents (e.g., caspofungin) had significantly shorter post-CBSI hospital LOS than those treated with non-HAAB antifungal agents (e.g., fluconazole); this difference was confirmed when this analysis was conducted only among survivors. After matching, all the outcomes were still favorable for patients with non-biofilm-forming CBSI. Furthermore, the biofilm-forming CBSI was significantly associated with a matched excess risk for hospital death of 1.77 compared to non-biofilm-forming CBSI. CONCLUSIONS: Our data show that biofilm growth by Candida has an adverse impact on clinical and economic outcomes of CBSI. Of note, better outcomes were seen for those CBSI patients who received HAAB antifungal therapy

Fatal Disseminated Cryptococcus gattii Infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico

Clusters of Sudden Unexplained Death Associated with the Mushroom, Trogia venenata, in Rural Yunnan Province, China

INTRODUCTION: Since the late 1970's, time-space clusters of sudden unexplained death (SUD) in northwest Yunnan, China have alarmed the public and health authorities. From 2006-2009, we initiated enhanced surveillance for SUD to identify a cause, and we warned villagers to avoid eating unfamiliar mushrooms. METHODS: We established surveillance for SUD, defined as follows: sudden onset of serious, unexplained physical impairment followed by death in <24 hours. A mild case was onset of any illness in a member of the family or close socially related group of a SUD victim within 1 week of a SUD. We interviewed witnesses of SUD and mild case-persons to identify exposures to potentially toxic substances. We tested blood from mild cases, villagers, and for standard biochemical, enzyme, and electrolyte markers of disease. RESULTS: We identified 33 SUD, a 73% decline from 2002-2005, distributed among 21 villages of 11 counties. We found a previously undescribed mushroom, Trogia venenata, was eaten by 5 of 7 families with SUD clusters compared to 0 of 31 other control-families from the same villages. In T. venenata-exposed persons SUD was characterized by sudden loss of consciousness during normal activities. This mushroom grew nearby 75% of 61 villages that had time-space SUD clusters from 1975 to 2009 compared to 17% of 18 villages with only single SUD (p<0.001, Fisher's exact test). DISCUSSION: Epidemiologic data has implicated T. venenata as a probable cause of clusters of SUD in northwestern Yunnan Province. Warnings to villagers about eating this mushroom should continue

Innate Immunity against Cryptococcus, from Recognition to Elimination

Cryptococcus species, the etiological agents of cryptococcosis, are encapsulated fungal yeasts that predominantly cause disease in immunocompromised individuals, and are responsible for 15% of AIDS-related deaths worldwide. Exposure follows the inhalation of the yeast into the lung alveoli, making it incumbent upon the pattern recognition receptors (PRRs) of pulmonary phagocytes to recognize highly conserved pathogen-associated molecular patterns (PAMPS) of fungi. The main challenges impeding the ability of pulmonary phagocytes to effectively recognize Cryptococcus include the presence of the yeast’s large polysaccharide capsule, as well as other cryptococcal virulence factors that mask fungal PAMPs and help Cryptococcus evade detection and subsequent activation of the immune system. This review will highlight key phagocyte cell populations and the arsenal of PRRs present on these cells, such as the Toll-like receptors (TLRs), C-type lectin receptors, NOD-like receptors (NLRs), and soluble receptors. Additionally, we will highlight critical cryptococcal PAMPs involved in the recognition of Cryptococcus. The question remains as to which PRR–ligand interaction is necessary for the recognition, phagocytosis, and subsequent killing of Cryptococcus

Depletion of neutrophils in a protective model of pulmonary cryptococcosis results in increased IL-17A production by gamma/delta T cells

Abstract Protective responses in mice immunized with an interferon-gamma producing strain of Cryptococcus neoformans, H99γ, are associated with IL-17A production by neutrophils. Neutrophil depletion in H99γ-immunized mice did not affect pulmonary fungal burden, indicating that neutrophils are not required for clearance. However, we observed an increase in IL-17A in the lungs of neutrophil-depleted H99γ infected mice, which corresponded to an increase in IL-17A+ γδ+ T cells. Moreover, we observed increased IL-17A+/ CD3+ cells and IL-17A+/γδ+ cells, but decreased IL-17A+/Ly6G+ neutrophils in the lungs of IL-17 receptor (R)A deficient mice compared to wild-type mice. Increased production of IL-17A in neutropenic mice coincided with increased IL-6 and CXCL1, but not Th17 inducing cytokines TGF-β, IL-21 and IL-23. Concurrent depletion of neutrophils and γδ+ T cells reduced IL-17A levels. Our results suggest that γδ+ T cells mediate significant IL-17A production in neutropenic mice during the protective response to C. neoformans infection.</p