title: Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages.
abstract: Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.
authors.affiliation: Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute Jena (HKI), Jena, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Seider
authors.forename: Franziska
authors.initials: F
authors.lastname: Gerwien
authors.forename: Lydia
authors.initials: L
authors.lastname: Kasper
authors.forename: Stefanie
authors.initials: S
authors.lastname: Allert
authors.forename: Sascha
authors.initials: S
authors.lastname: Brunke
authors.forename: Nadja
authors.initials: N
authors.lastname: Jablonowski
authors.forename: Tobias
authors.initials: T
authors.lastname: Schwarzmüller
authors.forename: Dagmar
authors.initials: D
authors.lastname: Barz
authors.forename: Steffen
authors.initials: S
authors.lastname: Rupp
authors.forename: Karl
authors.initials: K
authors.lastname: Kuchler
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hube
mesh_terms: D002118:Calcium / Q000378:metabolism; D041221:Candida glabrata / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D002460:Cell Line; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D016681:Genome, Fungal; D006801:Humans; D007501:Iron / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D009504:Neutrophils / Q000276:immunology / Q000382:microbiology; D018384:Oxidative Stress; D010587:Phagocytosis; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D007501:Iron; D002118:Calcium