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

Abstract

SummaryHistoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mf). 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 Mf. 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 Mf 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 Mf, grow on ironpoor 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.

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

“…Fungi need vacuolar and trafficking functions to infect their hosts, and genetic inactivation of V-ATPase prevents virulence of C. albicans and other pathogenic fungi (52)(53)(54). We have provided evidence that V-ATPase uncoupling would be a highly effective mechanism to control virulence, because in addition to preventing proton transport, uncouplers are expected to deplete the energy reserve of the cell.…”

SRE1 Regulates Iron-Dependent and -Independent Pathways in the Fungal Pathogen Histoplasma capsulatum

“…Iron acquisition and homeostasis are critical for optimal growth of H. capsulatum in the environment and within the host (13,14,18). In this study, we used phenotypic analyses and whole-genome expression profiling to interrogate the regulon of Sre1, a conserved GATA family transcription factor whose orthologs repress the expression of iron acquisition genes in other fungi.…”

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

“…Histoplasma yeasts produce hydroxamate siderophores which can steal iron from transferrin (41,42), the most likely source of iron within the phagosome. Without siderophore production, intracellular Histoplasma growth is hampered (43,44). In addition, Histoplasma produces extracellular iron reductases (41,42), including a ␥-glutamyltransferase (Ggt1) which causes a pH-independent release of iron from transferrin that is necessary for full Histoplasma virulence in phagocytes (45).…”