Phenotypic variation and intracellular parasitism by histoplasma Capsulatum

Abstract

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: alpha-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

Figure 1

Growth-dependent modulation of α-(1,3)-glucan in H. capsulatum yeast cell walls. H. capsulatum G186AR yeasts were washed and used as an inoculum (Left) for a culture initiated at low density (2 × 10⁶ cells per ml). At 24 h (Center) and 96 h (Right) after inoculation, an aliquot of yeasts was removed and monitored for α-(1,3)-glucan by comparing immunofluorescence (Upper) and differential interference contrast (Lower) images (magnification ×1,700). Murine monoclonal IgM antibody MOPC104E was used to detect α-(1,3)-glucan, and FITC-tagged goat anti-murine IgM was used as a fluorescent secondary antibody.

Figure 2

CBP and calcium dependency of H. capsulatum growth. Cultures of H. capsulatum were grown for 4 days in normal medium or in medium with EGTA added to chelate calcium. Cumulative growth was measured (by absorbance at 600 nm) for the cbp1-null strain (cbp1∷hph), the trans-complemented strain cbp1∷hph(CBP1), and the wild-type parental strain G186AR. Each bar represents the mean for triplicate samples, with error bars indicating standard deviations from the mean. Growth inhibition by EGTA could be reversed by adding equimolar calcium chloride to the cultures (data not shown).

Figure 3

Role of CBP in parasitism of macrophages. P388D1 macrophage-like cells were inoculated with strains of H. capsulatum at a multiplicity of one yeast per five macrophages and then cocultured for 5 days as described (7). (A) cbp1-null strain cbp1∷hph; (B) trans-complemented strain cbp1∷hph(CBP1); and (C) wild-type parental strain G186AR (magnification ×800).

Figure 4

Regulation of CBP1 during transition from yeast to mycelial forms. H. capsulatum strain G186ASura5 carrying the CBP1 promoter-gfp fusion was grown as yeast at 37°C and then transferred to 25°C at time 0 h. (A) Differential interference contrast microscopy. (B) Fluorescence microscopy (magnification ×1,200).