Characterization of a new mechanism of cell wall synthesis regulation in the cholera pathogen

肽聚糖（PG）是细菌细胞壁的重要组成部分，细菌依靠这种结构来维持细胞的形状和完整性。PG的合成途径已被广泛研究，并确定了许多参与合成的酶。然而，由于许多这些蛋白质似乎有重叠的活动，大多数因素的具体作用尚未精确确定。此外，人们对PG的合成是如何受到时空调控的知之甚少。在霍乱病原霍乱弧菌中，两个同源蛋白PBP1a和PBP1b在PG合成中起关键作用。我们对突变体的分析表明，PBP1a是主要因素，因为缺乏PBP1a的细胞（mrcA突变体）产生较少的PG，高度容易受到渗透挑战，并且在非规范d -氨基酸存在下无法生长或维持其棒状，这些非规范d -氨基酸在固定期释放。相反，缺乏PBP1b的细胞缺乏显著的表型。此外，我们发现mrcA突变体在缺乏lpoA或vc1887的菌株中大量表型。在大肠杆菌中，LpoA的作用是激活PBP1a；然而，没有发现VC1887的功能，VC1887是一种在弧菌科外缺乏可识别基序或同源物的周质蛋白。在这里，我提出的实验，以更好地表征在霍乱弧菌PG合成过程。特别是，我将评估PBP1a， PBP1b和VC1887的作用，这是一个新的PG生产贡献者。最后，我将进行高通量筛选，以确定PBP1a/VC1887/LpoA的特异性抑制剂，以确定特异性抑制弧菌生长的药物。总的来说，这些研究将产生关于控制重要病原体PG合成的新的基础知识，并可能产生治疗霍乱的新药物。

Peptidoglycan (PG) is an essential component of the bacterial cell wall, a structure that bacteria rely on to maintain cell shape and integrity. PG synthetic pathways have been extensively studied, and numerous enzymes that contribute to it have been identified. However, as many of these proteins appear to have overlapping activities, the specific roles of most factors have not been precisely determined. Moreover, there is little knowledge of how PG synthesis is spatio-temporally regulated. In Vibrio cholerae, the etiologic agent of cholera, two homologous proteins, PBP1a and PBP1b play key roles in PG synthesis. Our analyses of mutants suggest that PBP1a is the dominant factor, as cells lacking PBP1a (mrcA mutants) produce less PG, are highly susceptible to osmotic challenge, and fail to grow or maintain their rod shape in the presence of non-canonical D-amino acids, which are released in stationary phase. In contrast, cells lacking PBP1b lack a dramatic phenotype. Additionally, we have found that mrcA mutants are largely phenocopied by strains lacking either lpoA or vc1887. In E. coli, the role of LpoA is to activate PBP1a; however, no function has been identified for VC1887, a periplasmic protein that lacks recognizable motifs or homologs outside of the Vibrionaceae. Here, I present experiments to better characterize the process of PG synthesis in V. cholerae. In particular, I will assess the roles of PBP1a, PBP1b and VC1887, which is a novel contributor to PG production. Finally, I will conduct a high throughput screen to identify specific inhibitors of PBP1a/VC1887/LpoA, in order to identify agents that specifically inhibit the growth of Vibrios. In aggregate, these studies will yield new fundamental knowledge regarding the control of PG synthesis in an important pathogen and may yield new agents to treat cholera.