Surface Localization of the Yps3p Protein of Histoplasma capsulatum

Abstract

The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.

The interface between virulence and host response to the pathogenic fungus Histoplasma capsulatum

“…YPS3 , which encodes a protein that is both localized to the cell wall and secreted, has been shown to be a virulence determinant [ 17 ]. Originally identifi ed as a yeast-phase specifi c gene by subtractive DNA technology, it was considered to be a hallmark of yeast cells.…”

Expression and Interstrain Variability of the YPS3 Gene of Histoplasma capsulatum

“…N-terminal sequencing. We collected Yps3p by the use of a previously described polysaccharide binding assay (1). Briefly, we washed 50 mg of crab shell chitin (Sigma Aldrich, Saint Louis, MO) once with phosphate-buffered saline (PBS) and incubated it for 1 h with filtered, concentrated supernatant from mid-logarithmic-phase G217B cultures.…”

RNA Interference-Mediated Silencing of theYPS3Gene ofHistoplasma capsulatumReveals Virulence Defects

“…Following growth on plates, individual colonies were selected and grown in liquid medium. Three-day liquid cultures of 72 transformants were screened and assayed via Western blotting for secreted protein by using our previously described chitin binding assay (3). Briefly, a portion of the culture supernatant was mixed with the polysaccharide chitin (Sigma-Aldrich, St. Louis, MO), and the mixture was incubated for 1 h at room temperature.…”

“…The encoded protein is found both on the H. capsulatum cell wall and is also secreted from cells (21). On the cell wall, it is surface exposed, and it gets to the surface via secretion from the cell and binding to the polysaccharide chitin (3). Beyond its yeast-phase specificity, it is also expressed only in a subset of H. capsulatum strains, and these strains are typically North American isolates that have the highest virulence levels (13).…”