Expression and interstrain variability of the YPS3 gene of Histoplasma capsulatum

Abstract

The YPS3 locus of the dimorphic fungus Histoplasma capsulatum encodes a secreted and surface-localized protein specific to the pathogenic yeast phase. In this study we examined this locus in 32 H. capsulatum strains and variants. Although protein production is limited to a select group of strains, the North American restriction fragment length polymorphism class 2/NAm 2 isolates, the locus was present in all the strains we examined. The YPS3 gene is well conserved in its 5' and 3' regions but displays an intragenic hypervariable region of tandem repeats that fluctuates in size between strains. This feature is similar to that seen with genes encoding several cell surface proteins in other fungi.

FIG. 1.

Alignment of the YPS3 genes from selected strains of Histoplasma capsulatum. Sequence homology is observed in the 5′ and 3′ regions of the gene, with a region of intragenic variability that is predicted to encode a series of tandem amino acid repeats. The lengths of the intragenic variable regions differ between strains and show evidence of plasticity. Hc var. duboisii, Histoplasma capsulatum var. duboisii.

FIG. 2.

The top panel (A) shows three predicted amino acid sequences with variable-length repeats. Figure 2B shows a scaled diagram of the Yps3p protein from several strains of various classifications. The black bars indicate the well-conserved N and C termini of the gene. Each hexagon represents a tandem repeat element. Gray-shaded hexagons are repeats of the consensus YPTDK sequence. White hexagons indicate a tandem repeat with one or two substitutions or an insertion, as described in the text. H.c. duboisii, Histoplasma capsulatum var. duboisii.

FIG. 3.

A. Western blotting of Histoplasma capsulatum G184AS ura5-11 yeast cells expressing the YPS3 ORF of Downs, G217B, or G186B strains by use of a heterologous H2B promoter reveals Yps3p proteins of variable sizes. The increased size of the class 1 and class 3 proteins is consistent with the larger-sized ORF due to the region of intragenic variability. B. Yps3p produced from the heterologously expressed Downs strain ORF is detectable on the surface of G184AS ura5-11 yeast cells. Left panel, immunofluorescence; right panel, differential interference contrast.

FIG. 4.

Predicted protein alignment of several putative Aspergillus species homologs. These proteins share a predicted N-terminal signal sequence and a C-terminal EGF-like domain, including the conserved spacing of six cysteines (boxed) predicted to be important for disulfide bonding. The Aspergillus species are the most minimal homologs identified and lack an intragenic tandem repeat domain. The H. capsulatum strain G217B Yps3p and B. dermatitidis Bad1p homologs are shown for comparison. The large tandem repeat region of Bad1p is indicated.