<i>Paracoccidioides lutzii</i> Plp43 Is an Active Glucanase with Partial Antigenic Identity with <i>P. brasiliensis</i> gp43

Background Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43. Methodology We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients' sera from Southwestern and Midwestern Brazil. Principal Findings We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species. Conclusion Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.背景 巴西副球孢子菌（Paracoccidioides brasiliensis）与卢茨副球孢子菌（Paracoccidioides lutzii）可引发副球孢子菌病（paracoccidioidomycosis, PCM）。巴西副球孢子菌的主要诊断抗原为糖蛋白gp43（glycoprotein gp43），其肽序列与本研究中命名为Plp43的卢茨副球孢子菌同源蛋白（ortholog）的同源性达81%。卢茨副球孢子菌（“Pb01样”）主要流行于巴西中西部与北部地区，近期有研究报道该区域使用巴西副球孢子菌抗原进行检测时，假阴性反应（false-negative reaction）比例较高。本研究旨在制备重组Plp43（recombinant Plp43），以探究其与gp43的抗原同源性。 方法学 本研究在毕赤酵母（Pichia pastoris）中表达rPlp43，使其作为分泌型主要组分，并利用来自巴西西南部与中西部地区的副球孢子菌病患者血清，通过免疫印迹（immunoblot）检测其反应原性。 主要发现 本研究证实rPlp43不具备糖基化修饰，且具有葡聚糖酶（glucanase）活性。免疫印迹结果显示，该蛋白无法与抗gp43单克隆抗体（anti-gp43 monoclonal antibodies）结合，提示其缺乏gp43对应的表位（epitope）。尽管如此，二者仍可能存在共同表位：gp43阳性的副球孢子菌病血清可识别rPlp43，而来自巴西中西部地区患者的gp43阴性血清（51份中有33份）同样无法结合rPlp43。2株经基因分型的卢茨副球孢子菌分离株来自gp43阴性血清对应的患者，这提示血清反应阴性的患者为卢茨副球孢子菌感染者。 结论 本研究数据表明，gp43与Plp43仅存在一个或极少数共同表位，且gp43无法用于诊断感染卢茨副球孢子菌的副球孢子菌病患者，这可能是由于Plp43在感染过程中表达水平较低所致。