Instituto de Investigaciones Biotecnológicas Dr Rodolfo A Ugalde, Instituto Tecnológico de Chascomús, IIB-INTECH, CONICET, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
Infect Immun. 2013 Mar;81(3):956-64. doi: 10.1128/IAI.01158-12. Epub 2013 Jan 14.
Secretion of proteins in Gram-negative bacteria is a high-energy-consuming process that requires translocation across two membranes and a periplasmic space composed of a mesh-like layer, the peptidoglycan. To achieve this, bacteria have evolved complex secretion systems that cross these barriers, and in many cases there are specific peptidoglycanases that degrade the peptidoglycan to allow the proper assembly of the secretion machinery. We describe here the identification and characterization of a muramidase in Brucella abortus that participates in the intracellular multiplication in professional and nonprofessional phagocytes. We demonstrated that this protein has peptidoglycanase activity, that a strain with a clean deletion of the gene displayed a defect in the early stages of the intracellular multiplication curve, and that this is dependent on the lytic activity. While neither the attachment nor the invasion of the strain was affected, we demonstrated that it had a defect in excluding the lysosomal marker LAMP-1 but not in acquiring the reticulum endoplasmic marker calnexin, indicating that the gene participates in the early stages of the intracellular trafficking but not in the establishment of the replicative niche. Analysis of the assembly status and functionality of the VirB secretion apparatus indicated that the mutant has affected the proper function of this central virulence factor.
革兰氏阴性菌的蛋白质分泌是一个高能耗的过程，需要跨两个膜和一个由网格状层组成的周质空间进行易位，该层由肽聚糖组成。为了实现这一目标，细菌已经进化出了复杂的分泌系统，这些系统可以跨越这些障碍，并且在许多情况下，有特定的肽聚糖酶可以降解肽聚糖，以允许适当组装分泌机器。我们在这里描述了布鲁氏菌中一种参与专业和非专业吞噬细胞内增殖的粘肽酶的鉴定和特征。我们证明了这种蛋白质具有肽聚糖酶活性，一种基因缺失的菌株在细胞内增殖曲线的早期阶段表现出缺陷，并且这种缺陷依赖于溶菌活性。虽然菌株的附着和入侵不受影响，但我们证明它在排除溶酶体标记物 LAMP-1 方面存在缺陷，但在获取网质内质标记物 calnexin 方面没有缺陷，表明该基因参与细胞内运输的早期阶段，但不参与复制小生境的建立。对 VirB 分泌装置的组装状态和功能的分析表明，突变体影响了这种主要毒力因子的正常功能。
