Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus <em>Paracoccidioides brasiliensis</em>

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.巴西副球孢子菌（Paracoccidioides brasiliensis）是副球孢子菌病（paracoccidioidomycosis, PCM）的致病因子，而副球孢子菌病是拉丁美洲地区最流行的真菌病之一。巴西副球孢子菌的细胞壁成分可与宿主细胞相互作用，并影响副球孢子菌病的致病机制。细胞壁成分如糖基磷脂酰肌醇（glycosylphosphatidylinositol, GPI）锚定蛋白在细胞黏附与宿主组织侵袭过程中发挥关键作用。尽管GPI锚定蛋白在其他医学重要真菌致病机制中的重要性已得到学界认可，但目前对其在巴西副球孢子菌细胞及副球孢子菌病致病过程中的功能仍知之甚少。本研究从二态性致病真菌巴西副球孢子菌中克隆得到编码预测GPI锚定糖蛋白的PbPga1基因。PbPga1在散囊菌纲（Eurotiomycetes）真菌中具有保守性，其编码的蛋白在富含丝氨酸/苏氨酸的区域存在潜在糖基化位点，同时带有信号肽及推定的GPI锚定信号序列。特异性鸡抗rPbPga1抗体可将PbPga1定位于酵母菌细胞表面的母细胞与芽生孢子间的隔膜处，且芽生孢子的染色信号更强。将重组蛋白rPbPga1暴露于小鼠腹腔巨噬细胞可诱导巨噬细胞释放肿瘤坏死因子-α（TNF-α）并产生一氧化氮（NO）。此外，通过氢氧化铵处理下的β-消除反应，证实了rPbPga1存在O-糖基化位点。最后，副球孢子菌病患者的血清可通过蛋白质印迹法（Western blotting）识别rPbPga1，表明血清中存在针对rPbPga1的特异性抗体。综上，本研究结果表明，PbPga1基因编码一种细胞表面糖蛋白，可能通过GPI锚定结合于细胞膜，其可能在巴西副球孢子菌细胞壁形态发生及感染过程中发挥作用。rPbPga1可诱导炎症介质释放，且副球孢子菌病患者血清可与rPbPga1发生反应，这提示该蛋白可促进宿主天然免疫防御机制，并在巴西副球孢子菌感染过程中诱导体液免疫应答。