The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis.
Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.
Hilty
Jeremy
J
Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Smulian
A George
AG
Newman
Simon L
SL
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006658:Histoplasma / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D006660:Histoplasmosis / Q000382:microbiology*; D006706:Homeostasis; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008172:Lung Diseases, Fungal / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017262:Siderophores / Q000378:metabolism; D014169:Transformation, Bacterial; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics*; D014774:Virulence / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D017262:Siderophores; D007501:Iron; D025262:Vacuolar Proton-Translocating ATPases