Centre de Recherche des Cordeliers, Sorbonne Université, INSERM, Université Paris Cité, Paris, France.
Service de Microbiologie, Hôpital Européen Georges Pompidou, AP-HP Assistance Publique-Hôpitaux de Paris, Paris, France.
Microbiol Spectr. 2023 Aug 17;11(4):e0521722. doi: 10.1128/spectrum.05217-22. Epub 2023 May 31.
Peptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family and by l,d-transpeptidases (LDTs), which are dispensable for growth in most bacterial species and whose physiological functions remain elusive. In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (Ldt), the anchoring of lipoprotein OprI to the peptidoglycan (Ldt), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (Ldt). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by Ldt and a greater extent of OprI anchoring to peptidoglycan, which is controlled by Ldt and Ldt. Consistently, deletion of each of the genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm. Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections.
肽聚糖是细菌细胞包膜的重要组成部分，它维持细胞质的渗透压，决定细胞的形状，并作为包膜聚合物（如脂蛋白）锚定的支架。肽聚糖聚合的最后交联步骤是由属于青霉素结合蛋白（PBP）家族的经典 d,d-转肽酶和 l,d-转肽酶（LDTs）完成的，LDTs对于大多数细菌物种的生长不是必需的，其生理功能仍不清楚。在这项研究中，我们研究了 LDTs 在浮游和生物膜条件下生长的铜绿假单胞菌中对细胞包膜合成的贡献。我们首先通过基因敲除、异源基因表达（在大肠杆菌中）和其中一种酶的直接酶活性测定，为铜绿假单胞菌的三个 LDT 分配了一个功能。我们发现，三个铜绿假单胞菌 LDT 催化肽聚糖交联（Ldt）、脂蛋白 OprI 与肽聚糖的锚定（Ldt）以及由此产生的肽聚糖-OprI 酰胺键的水解（Ldt）。系统发育树的构建表明，在不同物种中执行这三种功能的 LDT 不能分配到不同的进化谱系，这与 PBP 观察到的情况相反。我们表明，生物膜，但不是浮游细菌，显示出由 Ldt 形成的肽聚糖交联的比例增加，以及 OprI 与肽聚糖的锚定程度增加，这由 Ldt 和 Ldt 控制。一致地，每个基因的缺失都损害了生物膜的形成并增强了 EDTA 的杀菌活性。这些结果表明，LDTs 有助于稳定细菌细胞包膜，并使肽聚糖代谢适应生物膜中的生长。活性位点半胱氨酸 LDT 形成了一个功能上异源的酶家族，通过形成肽聚糖交联和动态锚定脂蛋白到肽聚糖，有助于细菌细胞包膜的生物发生。在这里，我们报告了以前未被表征的三个铜绿假单胞菌 LDT 的作用。我们表明，这些酶有助于抵抗 EDTA 的杀菌活性，并使细胞包膜聚合物适应生物膜中存在的条件。这些结果表明，LDTs 应该被认为是开发用于控制生物膜相关感染的药物-EDTA 联合的潜在靶标。
