Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis

• Published in: Molecular microbiology 2008-10, Vol.70 (1), p.127-139
• Main Authors: Hilty, Jeremy, Smulian, A. George, Newman, Simon L
• Format: Article
• Language: English
• Subjects: Agrobacterium; Agrobacterium tumefaciens - genetics; Animals; Cells; DNA, Fungal - genetics; Fungal Proteins - genetics; Fungi; Genes; Genes, Fungal; Genetic Complementation Test; Histoplasma - enzymology; Histoplasma - genetics; Histoplasma - physiology; Histoplasma capsulatum; Histoplasmosis - microbiology; Homeostasis; Humans; Iron; Iron - metabolism; Lung Diseases, Fungal - microbiology; Macrophages - microbiology; Male; Mice; Mice, Inbred C57BL; Microbiology; Mutagenesis, Insertional; Mutation; Phenotype; Plasmids; Polymorphism; Reverse Transcriptase Polymerase Chain Reaction; Siderophores - metabolism; Transformation, Bacterial; Vacuolar Proton-Translocating ATPases - genetics; Virulence - genetics
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2008.06395.x

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mφ). 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 Mφ. 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 Mφ was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28°C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mφ, grow on iron-poor medium and grow as a mold at 28°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.