Targeting bacterial virulence as a novel antibiotic and vaccine approach

当细菌毒力因子如SpyCEP是单独的，足以引起疾病，该毒力因子代表了一个合适的目标，疫苗开发和治疗approaches.Aim 1的纯化重组SpyCEPSpyCEP的酶活性的详细表征是已知的切割和切割CXCL 8和其他ELR+趋化因子，导致受损的中性粒细胞招募到感染部位。切割也可能影响许多其他已知的CXCL 8介导的作用。由于之前缺乏足够量的纯化酶，因此之前没有进行酶动力学的系统研究，尽管开发任何抑制剂或抑制试验都需要进行酶动力学的系统研究。因此，在本工作开始时，我将使用来自GAS上清液的纯化SpyCEP酶和重组产生的酶（均在实验室中可用）进行动力学研究，以便使用ELISA监测CXCL 8切割和SDS-PAGE或MS分析（如有必要）确定SpyCEP的KM和Vmax参数。SpyCEP似乎具有许多额外的活性，这些活性可能与CXCL 8的先前未知的作用或先前未知的其他底物有关。为了更系统地评估任何疫苗或治疗剂的有效性，应澄清这些问题。

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.