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

Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion

Data Availability: All relevant data are available from the Gene Expression Omnibus at the following accession number: GSE97755. Funding: This work was funded by the German Research Council (DFG) Graduation College 685, Dr. Jekyll and Mr. Hyde: A systems approach to the therapy of nosocomial infections caused by Candida albicans: a commensal organism switches to a deadly pathogen/ PTJ (FKZ: 0315409BBMBF), the Dr. Manfred Plempel-foundation, the Dr. Siegried Stettendorf-Foundation, the InfectERA Program (FunComPath; BMBF FKZ 031L0001A), the Integrated Research and Treatment Center for Sepsis Control and Care (CSCC) project CanBac (BMBF, FKZ: 01EO1002), and the German Research Council (DFG) GZ:HE7565/1-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Cell biology of Candida albicans-host interactions

Acknowledgements The authors are supported by the Wellcome Trust via a Senior Investigator Award to NG, an ISST award and a Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology. The authors are also part of the MRC Centre for Medical Mycology at Aberdeen.Peer reviewedPublisher PD

Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence

We thank Jane Usher (University of Exeter) for constructive criticism of the manuscript. We thank Valmik K. Vyas and Gerald R. Fink from the Whitehead Institute for Biomedical Research, Cambridge, United Kingdom, for providing the Candida CRISPR plasmid (CaCas9 Solo system pV1200 vector). N.A.R.G. acknowledges support provided by Wellcome as a Senior Investigator Award (101873/Z/13/Z), Collaborative Award (200208/A/15/Z), and Strategic Award (097377/Z11/Z) and by the MRC Centre for Medical Mycology (MR/N006364/2). R.M.A. acknowledges support from an EPSRC/BBSRC Interface Innovation Fellowship (EP/S001352/1). K.S., N.A.R.G., S.B., and J.W. conceived the study. N.A.R.G. was awarded the grant that served as a resource for this study. K.S., S.C., D.M.M., and S.B. performed experiments. K.S., N.A.R.G., S.B., and R.M.A. analyzed and interpreted results. K.S., N.A.R.G., and S.B. wrote the paper, and all of us provided comments.Peer reviewedPublisher PD

Fungal Chitin Dampens Inflammation through IL-10 Induction Mediated by NOD2 and TLR9 Activation

Funding: JW and NARG thank the Wellcome Trust (080088, 086827, 075470), The Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the European Union ALLFUN (FP7/2007 2013, HEALTH-2010-260338) for funding. MGN was supported by a Vici grant of the Netherlands Organisation for Scientific Research. AJPB and DMM were funded by STRIFE, ERC-2009-AdG-249793 and AJPB additionally by FINSysB, PITN-GA-2008-214004 and the BBSRC [BB/F00513X/1]. MDL was supported by the MRC (MR/J008230/1). GDB and SV were funded by the Wellcome Trust (086558) and TB and MK were funded by the Deutsche Forschungsgemeinschaft (Bi 696/3-1; Bi 696/5-2; Bi 696/10-1). MS was supported by the Deutsche Forschungsgemeinschaft (Sch 897/1-3) and the National Institute of Dental and Craniofacial Research (R01 DE017514-01). TDK and RKSM were funded by the National Institute of Health (AR056296, AI101935) and the American Lebanese Syrian Associated Charities (ALSAC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Staphylococcal Peptidoglycan Co-Localizes with Nod2 and TLR2 and Activates Innate Immune Response via Both Receptors in Primary Murine Keratinocytes

In mammalian host cells staphylococcal peptidoglycan (PGN) is recognized by Nod2. Whether PGN is also recognized by TLR2 is disputed. Here we carried out PGN co-localization and stimulation studies with TLR2 and Nod2 in wild type and mutant host cells. To exclude contamination with lipoproteins, polymeric staphylococcal PGN (PGNpol) was isolated from Staphylococcus aureus Δlgt (lacking lipidated prelipoproteins). PGNpol was biotinylated (PGN-Bio) for fluorescence monitoring with specific antibodies. Keratinocytes from murine oral epithelium (MK) readily internalized PGN-Bio in an endocytosis-like process. In wt MK, PGNpol induced intracellular accumulation of Nod2 and TLR2 and co-localized with Nod2 and TLR2, but not with TLR4. In TLR2-deficient MK Nod2 and in Nod2-deficient MK TLR2 was induced, indicating that PGNpol recognition by Nod2 is independent of TLR2 and vice versa. In both mutants IL-6 and IL-1B release was decreased by approximately 50% compared to wt MK, suggesting that the immune responses induced by Nod2 and TLR2 are comparable and that the two receptors act additively in MK. In TLR2-tranfected HEK293 cells PGNpol induced NFkB-promoter fused luciferase expression. To support the data, co-localization and signaling studies were carried out with SHL-PGN, a lipase protein covalently tethered to PGN-fragments of varying sizes at its C-terminus. SHL-PGN also co-localized with Nod2 or TLR2 and induced their accumulation, while SHL without PGN did not. The results show that staphylococcal PGN not only co-localizes with Nod2 but also with TLR2. PGN is able to stimulate the immune system via both receptors

Candida albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions

The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival