Regulation of SsrA-mediated proteolysis of S. aureus

 描述（由申请人提供）：与其他原核生物相比，人类病原体金黄色葡萄球菌中SsrA降解基序的蛋白水解调节通过看似独特的途径发生。在大多数情况下，ATP依赖性蛋白酶ClpP在被伴侣ClpX或ClpA识别后识别并降解SsrA标记的蛋白质。S.相反，金黄色葡萄球菌使用伴侣ClpC和衔接子TrfA来协助ClpP降解SsrA。通过观察到SsrA分解在缺乏TrfA的菌株中在硫醇或氧化应激期间继续发生，进一步增强了这种新的机制，这表明除了ClpPC/TrfA之外还有另外一层蛋白水解调节。 为了充分表征SsrA标签蛋白在S.金黄色葡萄球菌，我们的第一个目标是发现更多的监管机构和衔接的SsrA降解基序在S。金黄色葡萄球菌，并确定它们与已知的SsrA调节剂TrfA的相互作用。构建了一个序列确定的S.金黄色葡萄球菌USA300 LAC JE2将用能够表达C-末端用SsrA标记的快速成熟Venus蛋白的质粒转化。为了在该搜索中撒下最广的网，来自暴露于各种应激条件（例如，二酰胺、饥饿等）的菌株的荧光模式被用于检测细胞的荧光。将获得。这些群体中的大多数菌株由于它们对SsrA标记的Venus的持续降解而将表现出低水平的荧光。然而，少数将具有影响SsrA降解的突变，当与缺乏clpC或trfA的菌株的模式相比时，将允许初始分类为各种控制途径。 随后将创建针对每个候选基因和trfA双重破坏的菌株，以进一步指导这一过程，以及与每个鉴定的基因的下游互补，以磨练S.金黄色。 ClpPC/TrfA在S.金黄色葡萄球菌作为SsrA蛋白水解的介质表明，其他微生物使用的SsrA中已建立的基序可能对S.金黄色。因此，S.金黄色葡萄球菌SsrA序列可能包含新的降解信号，我们的第二个目标将是确定新的基序中的SsrA标签，并评估在这些基序中的各种残基的重要性。将改变附加到Venus的SsrA的11个氨基酸的个体和组，以确定蛋白水解调节的关键区域。将通过监测荧光随时间的变化和暴露于各种应力期间的变化来评价所产生的变化。 最终，解开S。金黄色葡萄球菌调节SsrA标记的蛋白质提供了一个机会，以更好地了解这种细菌如何调整其蛋白质组，以挑战性的环境，并可以提供S。金黄色葡萄球菌-治疗感染的特异性靶点。

 DESCRIPTION (provided by applicant): Proteolytic regulation of the SsrA degradation motif in the human pathogen Staphylococcus aureus occurs through a seemingly unique pathway as compared to other prokaryotes. In most cases, the ATP- dependent protease ClpP recognizes and degrades SsrA-tagged proteins following recognition by either the chaperone ClpX or ClpA. S. aureus instead uses the chaperone ClpC and the adaptor TrfA to assist ClpP in SsrA degradation. This novel mechanism is further enhanced by the observation that SsrA breakdown continues to occur during thiol or oxidative stress in strains lacking TrfA, suggesting an additional layer of proteolytic regulation beyond ClpPC/TrfA. To fully characterize the regulation of SsrA-tagged protein levels in S. aureus, our first aim is to uncover additional regulators and adaptors of the SsrA degradation motif in S. aureus and determine their interplay with the known SsrA regulator TrfA. A sequence-defined transposon mutant library of S. aureus USA300 LAC JE2 will be transformed with a plasmid capable of expressing the rapidly maturing Venus protein that is C-terminally tagged with SsrA. To cast the widest net in this search, fluorescence patterns from strains exposed to a variety of stress conditions (e.g. diamide, starvation, etc.) will be obtained. Most strains in these populations will demonstrate low levels o fluorescence due to their persistent degradation of SsrA- tagged Venus. However, a small number will have mutations that affect SsrA degradation, which when compared to patterns from strains lacking clpC or trfA, will allow initial categorization into various control pathways. Subsequent strains doubly disrupted for each candidate gene and trfA will be created to further guide this process, as well as downstream complementation with each identified gene to hone a hierarchy of SsrA regulation in S. aureus. The use of ClpPC/TrfA in S. aureus as mediators of SsrA proteolysis suggests that the established motifs in SsrA used by other microbes may not be valid for S. aureus. Therefore, the S. aureus SsrA sequence may contain novel degradation signals, and our second aim will be to identify novel motifs in the SsrA tag, and to evaluate the importance of various residues in these motifs. Individual and groups of the eleven amino acids of SsrA appended to Venus will be altered to identify critical regions for proteolytic regulation. The resulting changes will be evaluated by monitoring fluorescence over time and during exposure to various stresses. Ultimately, unraveling how S. aureus regulates SsrA-tagged proteins provides an opportunity to better understand how this bacteria adjusts its proteome to challenging environments, and can provide S. aureus- specific targets for treating infections.