Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, USA.
Department of Microbiology, Cornell University, Ithaca, New York, USA.
mBio. 2021 Apr 6;12(2):e03596-20. doi: 10.1128/mBio.03596-20.
The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by "autolytic" enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from fully complemented growth and morphology of an EP-insufficient , highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in , aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion. Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in , one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.
细菌细胞壁主要由肽聚糖（PG）组成，PG 是维持细胞形状、生长和结构完整性所必需的多氨基糖。PG 由 A/B 类青霉素结合蛋白（a/bPBPs）和形状、伸长、分裂和孢子形成（SEDS）蛋白合成，如 RodA（作为 Rod 系统细胞伸长机制的一部分），并通过“自溶”酶降解以适应生长过程。人们认为，自溶酶（特别是内肽酶 [EP]）通过创建间隙来促进 PG 的合成和掺入，这些间隙由 PG 合成酶进行修补和铺设，但自溶酶与 PG 合成之间的确切关系仍不完全清楚。在这里，我们研究了 EP 耗竭对腹泻病原体 中 PG 合成的后果。我们发现，EP 耗竭导致严重的形态和分裂缺陷，但这些细胞继续增加质量并异常掺入新的细胞壁物质。在 Rod 系统抑制剂存在的情况下，质量增加会继续进行，但在 aPBPs 抑制时细胞会裂解，这表明在这些条件下 aPBPs 是结构完整性所必需的。即使在长时间的 EP 耗竭后，Rod 系统虽然不是观察到的质量增加所必需的，但仍然保持功能。最后，来自 的 EP 的异源表达完全弥补了 EP 不足的 的生长和形态，这突出表明 PG 合成酶不一定通过与 EPs 的直接相互作用发挥作用。总的来说，我们的研究结果表明，在 EP 不足的情况下，aPBPs 对于结构完整性变得至关重要，而 Rod 系统无法促进适当的细胞扩张。细菌细胞壁的合成和周转必须紧密协调，以避免结构完整性失效和细胞死亡。这种协调的细节知之甚少，特别是在细胞壁周转酶是否以及如何为不同的细胞壁合成机器（aPBPs 和 Rod 系统）的活性所必需的方面。我们的研究结果表明，在 中，一类周转酶，即内肽酶，是适当的细胞伸长和分裂所必需的。在 EP 不足的情况下，aPBPs 对于维持结构完整性变得至关重要，而 Rod 系统在这些条件下仍然活跃，但对细胞扩张的贡献很小。我们的研究结果表明，aPBPs 在识别由非主要内肽酶形成的自溶酶产生的细胞壁间隙方面比 Rod 系统更具多功能性，这增加了我们对自溶酶和细胞壁合成酶之间协调的理解。对内肽酶生物学的深入了解可能会促进针对这些必需周转过程的抗生素的开发。
