Targeting bacterial virulence as a novel antibiotic and vaccine approach

在细菌毒力因子如SpyCEP单独足以致病的情况下，该毒力因子是疫苗开发和治疗方法的合适靶点。1已知纯化的重组SpyCEPSpyCEP的酶活性的详细特征是裂解和失活CXCL8和其他ELR+趋化因子，导致中性粒细胞募集到感染部位。很可能，卵裂也会影响许多其他已知的CXCL8介导的效应。由于之前缺乏足够数量的纯化酶，以前没有对酶动力学进行系统的研究，尽管开发任何抑制剂或进行抑制试验都是必需的。因此，在这项工作开始时，我将进行动力学研究，使用从气体上清液和重组生产的酶(均可在实验室获得)中纯化的SpyCEP酶，以确定SpyCEP的Km和Vmax参数，如有必要，使用ELISA监测CXCL8裂解和SDS-PAGE或基于MS的分析。SpyCEP似乎有一些额外的活动，可能与以前未知的CXCL8的影响有关，或者与其他以前未知的底物有关。应澄清这些问题，以便更系统地评估任何疫苗或治疗方法的效果。

Where a bacterial virulence factor such as SpyCEP is alone, sufficient to cause disease, that virulence factor represents a suitable target for both vaccine development and therapeutic approaches.Aim 1 Detailed characterization of the enzymatic activity of purified recombinant SpyCEPSpyCEP is known to cleave and inactivate CXCL8 and other ELR+ chemokines resulting in impaired neutrophil recruitment to the site of infection. It is likely that cleavage also impacts many other known CXCL8-mediated effects. Due to a prior lack of adequate quantities of purified enzyme, a systematic study of enzyme kinetics has not been previously undertaken, though would be required for development of any inhibitor or assay of inhibition. As such, at the start of this work I will undertake kinetic studies using purified SpyCEP enzyme from GAS supernatant and recombinantly produced enzyme (all available in the laboratory) in order to determine the KM and Vmax parameters of SpyCEP using ELISA to monitor CXCL8 cleavage and SDS-PAGE or MS-based analyses if necessary. SpyCEP appears to have a number of additional activities that may relate to previously unknown effects of CXCL8, or to additional previously unknown substrates. These should be clarified in order to more systematically assess the efficacy of any vaccine or therapeutic.