Abstract
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.
Highlights
Paracoccidioides brasiliensis is a thermal dimorphic fungus and the etiological agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America [1] Infection occurs primarily in the lungs through inhalation of conidia or hyphal particles that switch to yeast form at the human body temperature [2]
Identification of PbPga1 in P. brasiliensis strain P. brasiliensis strain 18 (Pb18) In order to discover non-characterized GPI-proteins, we performed a tBLASTp search using the whole set of the Expressed Sequence Tags (EST) from P. brasiliensis (Marques et al, 2004) as queries against the genomes of P. brasiliensis strains (Pb03 and Pb18) and P. lutzii strain (Pb01) at the Broad Institute
Quantitative Real Time polymerase chain reaction (PCR) analysis showed that PbPga1 RNA levels are 7-fold higher in yeast than in hyphal cells
Summary
Paracoccidioides brasiliensis is a thermal dimorphic fungus and the etiological agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America [1] Infection occurs primarily in the lungs through inhalation of conidia or hyphal particles that switch to yeast form at the human body temperature [2]. The fungal cell wall is a dynamic and highly regulated structure where several molecules are important for cell wall synthesis and maintenance and in the interaction with host tissues. Extensive changes in the composition and arrangement of the cell wall may occur during fungal morphogenesis that is triggered by environmental signals [6,7,8]. Cell wall glycosylphosphatidylinositol (GPI)anchored proteins have been extensively studied in Candida albicans and Saccharomyces cerevisiae [9,10]. In these organisms, GPI-proteins are involved in cell wall integrity, as well as in pathogenic processes such as adhesion and degradation of host tissue and immune response [9,11,12,13]. Many of these proteins, adhesins e.g., have a central SerThr (serine/threonine) rich O-glycosylated domain [16]
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