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Abstract：
Infections caused by Staphylococcus aureus are increasingly prevalent, and treatment has become more difficult due to the emergence of strains that are resistant to multiple drugs, such as methicillin-resistant Staphylococcus aureus (MRSA). Penicillin-binding proteins (PBPs) are essential enzymes in peptidoglycan biosynthesis. Only found in bacteria, they are an excellent target for the development of bacterial control strategies. S. aureus has 4 PBPs, and only PBP2 has transglycosylation activity, making it a good model to evaluate whether the inactivation of the transglycosylase domain (PBP2t) could lead to bacterial death. (His6)-tagged PBP2t was purified from the E. coli cell lysate using Ni-charged resin, and ELISA and immunoblotting assays demonstrated that PBP2t is immunogenic. Flow cytometry analysis was performed to verify the binding of polyclonal antibodies to the bacterial cell surface. In order to verify the ability to provide protection, immunized mice were challenged with a sublethal dose of MRSA, and the bacterial loads in kidneys and spleen were evaluated. A reduction of 2-2.5 logs was seen in organs from immunized mice compared with the negative controls in two independent assays (p < 0.01). Our results demonstrate that the PBP2t is a promising target for the development of novel antimicrobial strategies, but further testing should be performed to validate the protection conferred by immunization with this protein.
摘要：
金黄色葡萄球菌引起的感染越来越普遍,由于对多种药物耐药的菌株的出现，治疗变得更加困难，例如耐甲氧西林金黄色葡萄球菌（MRSA）。青霉素结合蛋白（PBP）是肽聚糖生物合成中必不可少的酶。只在细菌中发现，它们是细菌控制策略发展的极好目标。金黄色葡萄球菌有4个PBPs，只有PBP2具有转糖基化活性，使其成为评估转糖基酶结构域（PBP2t）的失活是否会导致细菌死亡的良好模型。使用含镍的树脂从大肠杆菌细胞裂解物中纯化（His6）标记的PBP2t，ELISA和免疫印迹试验证明PBP2t具有免疫原性。进行流式细胞术分析以验证多克隆抗体与细菌细胞表面的结合。为了验证提供保护的能力，免疫小鼠用亚致死剂量的MRSA攻击，并评估了肾脏和脾脏中的细菌负荷。在两个独立的测定中,与阴性对照相比,在来自免疫小鼠的器官中观察到2-2.5log的减少(p<0.01)。我们的结果表明，PBP2t是开发新的抗微生物策略的有希望的目标，但是应该进行进一步的测试以验证该蛋白免疫所赋予的保护作用.
