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性调节通过一种似乎独特的途径发生。在大多数情况下，依赖于ATP的蛋白酶ClpP在被伴侣ClpX或ClpA识别后识别并降解SsrA标记的蛋白质。相反，金黄色葡萄球菌使用伴侣ClpC和适配器TrfA来帮助ClpP降解SsrA。这一新的机制进一步得到加强，因为观察到在缺乏TrfA的菌株中，SsrA在硫醇或氧化应激过程中继续发生破坏，这表明在ClpPC/TrfA之外还有一层蛋白分解调节。为了充分表征金黄色葡萄球菌中SsrA标记蛋白水平的调节，我们的第一个目标是发现金黄色葡萄球菌中SsrA降解基序的其他调节因子和适配器，并确定它们与已知的SsrA调节因子TrfA的相互作用。一个序列定义的金黄色葡萄球菌USA300 LAC JE2转座子突变体文库将被转化成一个能够表达快速成熟的带有SsrA标记的金星蛋白的质粒。为了在这次搜索中撒下最广泛的网，暴露在各种压力条件(如联胺、饥饿等)下的菌株的荧光模式。将会被获得。这些种群中的大多数菌株由于对SsrA标记的金星的持续降解而表现出低水平的荧光。然而，一小部分将具有影响SsrA降解的突变，当与缺乏clpC或trfA的菌株的模式相比时，将允许初步归类为各种控制途径。 随后将产生针对每个候选基因和trfA的双重干扰菌株，以进一步指导这一过程，以及与每个已识别基因的下游互补，以磨练金黄色葡萄球菌中SsrA调控的层次结构。在金黄色葡萄球菌中使用ClpPC/TrfA作为SsrA蛋白降解的介体，表明其他微生物使用的SsrA中已建立的基序可能对金黄色葡萄球菌无效。因此，金黄色葡萄球菌的SsrA序列可能包含新的降解信号，我们的第二个目标将是识别SsrA标签中的新基序，并评估各种残基在这些基序中的重要性。附加在金星上的SsrA的11个氨基酸的个体和组将被改变，以确定蛋白质降解调节的关键区域。由此产生的变化将通过监测荧光随时间的推移和暴露在各种压力下进行评估。最终，揭示金黄色葡萄球菌如何调节SsrA标记的蛋白质为更好地了解这种细菌如何调整其蛋白质组以适应具有挑战性的环境提供了机会，并可以为治疗感染提供金黄色葡萄球菌特有的靶点。

 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.