Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis.

Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

Histoplasma Responses to Nutritional Immunity Imposed by Macrophage Activation.

The fungal pathogen Histoplasma capsulatum resides within the phagosome of host phagocytic cells. Within this intracellular compartment, Histoplasma yeast replication requires the acquisition of several essential nutrients, including metal ions. Recent work has shown that while iron, zinc, and copper are sufficiently abundant in resting macrophages, cytokine activation of these host cells causes restriction of these metals from intracellular yeasts as a form of nutritional immunity. Faced with limited iron availability in the phagosome following macrophage activation by IFN-γ, Histoplasma yeasts secrete iron-scavenging siderophores and employ multiple strategies for reduction of ferric iron to the more physiologically useful ferrous form. IFN-γ activation of macrophages also limits availability of copper in the phagosome, forcing Histoplasma reliance on the high affinity Ctr3 copper importer for copper acquisition. GM-CSF activation stimulates macrophage production of zinc-chelating metallothioneins and zinc transporters to sequester zinc from Histoplasma yeasts. In response, Histoplasma yeasts express the Zrt2 zinc importer. These findings highlight the dynamics of phagosomal metal ion concentrations in host-pathogen interactions and explain one mechanism by which macrophages become a less permissive environment for Histoplasma replication with the onset of adaptive immunity.