Histoplasma capsulatum utilizes siderophores for intracellular iron acquisition in macrophages.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (MΦ). Studies in human and murine MΦ demonstrate that the intracellular growth of H. capsulatum yeasts is exquisitely sensitive to the availability of iron. As H. capsulatum produces hydroxamate siderophores, we sought to determine if siderophores were required for intracellular survival in MΦ, and in a murine model of pulmonary histoplasmosis. The expression of SID1 (coding for L-ornithine-N(5)-monooxygenase) was silenced by RNA interference (RNAi) in H. capsulatum strain G217B, and abolished by gene targeting in strain G186AR. G217B SID1-silenced yeasts grew normally in rich medium, did not synthesize siderophores, and were unable to grow on apotransferrin-chelated medium. Their intracellular growth in human and murine MΦ was significantly decreased compared to wild type (WT) yeasts, but growth was restored to WT levels by the addition of exogenous iron, or restoration of SID1 expression. Similar results were obtained with G186AR Δsid1 yeasts. Compared to WT yeasts, G217B SID1-silenced yeasts demonstrated in C57BL/6 mice significantly reduced growth in the lungs and spleens seven days after infection, and 40% of the mice given a normally lethal inoculum of G217B SID1-silenced yeasts survived. These experiments demonstrate that: (1) SID1 expression is required for siderophore biosynthesis by H. capsulatum strain G217B, (2) SID1 expression is required for optimum intracellular growth in MΦ, and (3) inhibition of SID1 expression in vivo reduces the virulence of H. capsulatum yeasts.

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.

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.