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首页> 外文期刊>PLoS Genetics >The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli
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The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli

机译：美西林抗性组揭示肽聚糖内肽酶在刺激大肠杆菌中细胞壁合成中的作用

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Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.
机译：细菌细胞通常被网状大分子包围，这种大分子称为由杂聚物肽聚糖（PG）构成的细胞壁。该基质的生物发生是青霉素和相关β-内酰胺的目标。这些药物会抑制称为青霉素结合蛋白（PBP）的PG合成酶的转肽酶活性，从而阻止新生壁物质交联到现有网络中。 β-内酰胺美西林在大肠杆菌的细胞延伸机制中特异性靶向PBP2酶。过去，对美西林的耐药性的低通量选择在确定参与细胞壁生物发生和β-内酰胺抗生素杀伤活性的机制方面一直很有用。在这里，我们使用转座子测序（Tn-Seq）作为高通量方法来鉴定大肠杆菌基因组中几乎所有的mecillinam耐药基因座，为揭示PG组装和耐药性的新机制提供了全面的资源。严格应答或Rcs包膜应力应答的诱导先前与美西林抗性有关。因此，除了野生型细胞外，我们还对这些应答缺陷的突变体进行了Tn-Seq分析。因此，通过确定电阻组中每个电阻位点的应力响应依赖性，大大提高了数据集的实用性。认为独立于应激反应的抗性基因座最有可能识别细胞壁生物发生的直接调节剂，因此我们将下游分析的重点放在了电阻组的这一子集中。这些等位基因之一的表征导致令人惊讶的发现，即通过刺激一部分PBP刺激PG的合成，内切肽酶在细胞壁上的交联酶的过度生产会促进美西林抗药性。我们对该激活机制的分析表明，与该领域的普遍观点相反，PG合酶和PG裂解酶不需要在多酶复合物中发挥作用来扩展细胞壁基质。

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