Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L , D -transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile . We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non– C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile , the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

中文翻译：

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肽聚糖转肽酶家族的鉴定表明，艰难梭菌需要非规范交联才能生存


大多数细菌都被含有肽聚糖 (PG) 的细胞壁包围，肽聚糖是一种由通过短肽交联连接在一起的聚糖链组成的大型聚合物。交联有两种主要类型，根据所涉及的氨基酸称为 4-3 和 3-3。 4-3 交联由青霉素结合蛋白产生，而 3-3 交联由 L , D -转肽酶 (LDT) 产生。在大多数细菌中，主要的交联模式是 4-3 交联，这些交联对于生存能力至关重要，而 3-3 交联仅占一小部分，并不是必需的。然而，在机会性肠道病原体艰难梭菌中，大约70%的交联是3-3。我们在此表明​​，3-3 个交联和 LDT 对于艰难梭菌的生存能力至关重要。我们还表明，艰难梭菌有五个 LDT，其中三个具有 YkuD 催化结构域，如所有先前已知的 LDT 一样，两个具有 VanW 催化结构域，其功能迄今为止尚不清楚。五个 LDT 表现出广泛的功能冗余。 VanW 结构域蛋白存在于许多革兰氏阳性细菌中，但在其他谱系中很少见。我们测试了 7 种非艰难梭菌 VanW 结构域蛋白，并在 3 例中证实了 LDT 活性。总之，我们的研究结果揭示了一个以前未被识别的 PG 交联酶家族，将催化功能分配给 VanW 结构域，并证明 3-3 交联对于艰难梭菌的生存能力至关重要，这是首次证明这一点在任何细菌种类中。 LDT 在艰难梭菌中的重要性使其成为选择性杀死艰难梭菌的抗生素的潜在目标。