Structural Features Responsible for the Biological Stability of Histoplasma’s Virulence Factor CBP

Abstract

The virulence factor CBP is the most abundant protein secreted by Histoplasma capsulatum, a pathogenic fungus that causes histoplasmosis. Although the biochemical function and pathogenic mechanism of CBP are unknown, quantitative Ca 2+ binding measurements indicate that CBP has a strong affinity for calcium (K D = 6.45 ± 0.4 nM). However, no change in structure was observed upon binding of calcium, prompting a more thorough investigation of the molecular properties of CBP with respect to self-association, secondary structure, and stability. Over a wide range of pH values and salt concentrations, CBP exists predominantly as a stable, noncovalent homodimer in both its calcium-free and-bound states. Solution-state NMR and circular dichroism (CD) measurements indicated that the protein is largely α-helical, and its secondary structure content changes little over the range of pH values encountered physiologically. ESI-MS revealed that the six cysteine residues of CBP are involved in three intramolecular disulfide bonds that help maintain a highly protease resistant structure. Thermally and chemically induced denaturation studies indicated that unfolding of disulfide-intact CBP is reversible and provided quantitative measurements of protein stability. This disulfide-linked, protease resistant, homodimeric α-helical structure of CBP is likely to be advantageous for a virulence factor that must survive the harsh environment within the phagolysosomes of host macrophages.The dimorphic fungal pathogen Histoplasma capsulatum is a major causative agent of respiratory and systemic disease, especially in immunocompromised individuals. Once inside the host, the yeast form of Histoplasma proliferates in what should be a potent antimicrobial environment, the macrophage phagolysosome (1). We previously showed that Histoplasma interferes with normal acidification of this intracellular compartment (2), but intracellular yeasts must also cope with lysosomal enzymes, reactive oxygen and nitrogen intermediates, iron limitation, and nutrient deprivation. H. capsulatum is one of only a few organisms known to survive this harsh environment; consequently, the unique mechanisms employed by H. capsulatum to replicate within the phagolysosomes of mammalian cells are of particular interest. Currently, only two virulence factors have been genetically proven, and the molecular mechanisms of H. capsulatum pathogenesis are not well understood (3,4). † This work was supported by National Institutes of Health Grants AI25584 to W.E.G., DK48046 to D.P.C., DK52574 to the Protein Structure Core of the Washington University Digestive Diseases Research Core Center, and 2P41RR00954 for the Mass Spectrometry Research Resource at Washington University. * To whom correspondence should be addressed. Telephone: (314) 362-2742. Fax: (314) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe first characterized virulence determinant, CBP (calcium binding protein),1 was identified as a yeast phase-specific secreted protein that s...

NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B

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