Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
Lydia
L
Kasper
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
Katja
K
Seider
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
Franziska
F
Gerwien
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
Stefanie
S
Allert
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany; Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany.
Sascha
S
Brunke
Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University, Vienna, Austria.
Tobias
T
Schwarzmüller
College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.
Lauren
L
Ames
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
Cristina
C
Zubiria-Barrera
Massachusetts General Hospital, Department of Medicine, Division of Infectious Disease, Boston, Massachusetts, United States of America.
Michael K
MK
Mansour
Laboratoires Constant Burg, Paris, France.
Ulrike
U
Becken
Institute for Transfusion Medicine, University Hospital, Jena, Germany.
Dagmar
D
Barz
Massachusetts General Hospital, Department of Medicine, Division of Infectious Disease, Boston, Massachusetts, United States of America.
Jatin M
JM
Vyas
Division of Microbial Interface Biology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany.
Norbert
N
Reiling
Institute for Cell Biology, University of Bonn, Bonn, Germany.
Albert
A
Haas
College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.
Ken
K
Haynes
Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University, Vienna, Austria.
Karl
K
Kuchler
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany; Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany; Friedrich Schiller University, Jena, Germany.
Bernhard
B
Hube
D000818:Animals; D041221:Candida glabrata / Q000235:genetics* / Q000276:immunology*; D002177:Candidiasis / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology*; D002454:Cell Differentiation / Q000276:immunology; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D008247:Lysosomes / Q000276:immunology / Q000382:microbiology; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D010588:Phagosomes / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D015398:Signal Transduction