Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium's ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host.IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

中文翻译：

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肽聚糖水解酶RipA和Ami1对于宿主中结核分枝杆菌的复制和持续存在至关重要。

细胞壁聚合物肽聚糖（PG）的合成和裂解是精心策划的过程，对于细菌的生长和存活至关重要。然而，尚需阐明许多作用于结核分枝杆菌中PG的酶的功能和重要性。我们证明了N-乙酰基村m-1-丙氨酸酰胺酶Ami1的活性对于结核分枝杆菌中的细胞分裂而言是可有可无的，但有助于细菌在小鼠慢性感染过程中持续存在。此外，d，1-内肽酶RipA，一种预测的必需酶，对于结核分枝杆菌的生存是必需的，但是对于在体外和体内有效的细胞分裂是必需的。RipA的消耗会使结核分枝杆菌对利福平和靶向细胞包膜的抗生素敏感。Ami1有助于在缺乏RipA的细胞中维持残留的细胞分裂，但是Ami1提供的部分冗余在感染过程中是不够的，因为RipA的耗尽阻止了结核分枝杆菌在巨噬细胞中复制并导致小鼠中细菌的急剧杀伤。值得注意的是，RipA对于在小鼠中持久存在结核分枝杆菌至关重要，这表明在慢性小鼠感染期间需要细胞分裂。尽管有多种酶作用于具有冗余功能的PG，但我们已经鉴定出两种PG水解酶，它们对于结核分枝杆菌的复制和在宿主中的持久性至关重要。重要结核病（TB）是全球主要的健康负担，有160万人屈服每年都会患这种疾病。寻找新药以改善当前的化疗方案对于减轻这种全球健康负担至关重要。细胞壁聚合物肽聚糖（PG）已成为细菌病原体中非常成功的药物靶标，因为许多当前使用的抗生素靶向这种大分子的合成。但是，编码具有明显冗余功能的PG合成酶和PG修饰酶的大量基因阻碍了结核分枝杆菌PG合成中新药靶标的鉴定。在这里，我们证明了两种PG裂解酶对M的毒力很重要。特别是d，l-肽链内切酶RipA代表了潜在的有吸引力的药物靶标，因为它的耗尽导致结核分枝杆菌从宿主中清除，并使细菌对利福平，一线TB药物和几个细胞壁高度敏感靶向抗生素。