Bom Ana Paula D Ano, Corrêa Izabella B S, Argondizzo Ana Paula C, Medeiros Marco Alberto, Santos Roger B Dos, Souza Theo Luiz F de, Silva Junior José G da
Lab. of Macromolecules, Bio-Manguinhos, Fiocruz, Av. Brasil, 4365, Room 205, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil.
Lab. of Recombinant Technologies, Bio-Manguinhos, Fiocruz, Av. Brasil, 4365, Room 209, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil.
An Acad Bras Cienc. 2018 Aug;90(2 suppl 1):2299-2310. doi: 10.1590/0001-3765201820170151. Epub 2018 Jun 25.
PsaA (pneumococcal surface antigen A) is a S. pneumoniae virulence factor that belongs to the metal transport system. The Manganese PsaA binding has been associated with oxidative stress resistance becoming a pivotal element in the bacteria virulence. It has been shown that Zinc inhibits the Manganese acquisition and promotes bacteria toxicity. We have performed a PsaA conformational analysis both in the presence (Zn-rPsaA) and in the absence of Zinc (free-rPsaA). We performed experiments in the presence of different Zinc concentrations to determine the metal minimum concentration which induced a conformational change. The protein in free and Zn-binding condition was also studied in pH ranging 2.6-8.0 and in temperature ranging 25oC-85oC. pH experiments showed a decrease of fluorescence intensity only in acidic medium. Analysis of the heat-induced denaturation demonstrated that Zinc-binding promoted an increase in melting temperature from 55oC (free-rPsaA) to 78.8oC (Zn-rPsaA) according to fluorescence measurements. In addition, the rPsaA stabilization by Zinc was verified through analysis of urea and guanidine hydrochloride denaturation. Data showed that Zinc promoted an increase in the rPsaA stability and its removal by EDTA can lead to a PsaA intermediate conformation. These findings can be considered in the development of vaccines containing PsaA as antigen.
肺炎球菌表面抗原A（PsaA）是一种属于金属转运系统的肺炎链球菌毒力因子。锰与PsaA的结合与抗氧化应激相关，成为细菌毒力的关键因素。研究表明，锌会抑制锰的摄取并增强细菌毒性。我们对存在锌（锌结合型重组PsaA，Zn-rPsaA）和不存在锌（游离型重组PsaA，free-rPsaA）的情况下的PsaA进行了构象分析。我们在不同锌浓度下进行实验，以确定诱导构象变化的最低金属浓度。还研究了游离状态和锌结合状态下的蛋白质在pH值范围为2.6 - 8.0以及温度范围为25℃ - 85℃时的情况。pH实验表明，仅在酸性介质中荧光强度会降低。热诱导变性分析表明，根据荧光测量结果，锌结合使熔解温度从55℃（游离型重组PsaA）提高到78.8℃（锌结合型重组PsaA）。此外，通过对尿素和盐酸胍变性的分析验证了锌对重组PsaA的稳定作用。数据表明，锌促进了重组PsaA稳定性的提高，用乙二胺四乙酸（EDTA）去除锌会导致PsaA呈现中间构象。在开发以PsaA为抗原的疫苗时可考虑这些发现。
