Table_1_Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape.DOCX

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as “peptidoglycan-binding anchor” (Pba).

外膜蛋白A（outer membrane protein A, OmpA）家族包含一个进化保守结构域，可将革兰氏阴性菌的外膜与半刚性肽聚糖（peptidoglycan, PG）层相连。具有临床重要性的致病菌铜绿假单胞菌（Pseudomonas aeruginosa）携带多种OmpA家族蛋白（OprF、OprL、PA0833及PA1048），这些蛋白均共享PG结合结构域。这类蛋白对细菌细胞形态、膜稳定性、生物被膜及外膜囊泡（outer membrane vesicle, OMV）的形成具有关键作用。除其他OmpA家族蛋白外，计算机模拟分析（in silico analysis）显示，基因座为PA1041的假定外膜蛋白属于携带OmpA结构域的脂蛋白，因此是潜在的毒力因子。本研究旨在评估PA1041的PG结合蛋白功能，并阐明其对细菌表型的影响。研究人员通过蛋白质印迹法（Western blot）确认临床菌株中存在PA1041表达产生的脂蛋白，并通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）验证了其PG结合活性。利用琼脂糖凝胶珠ELISA，研究团队发现该脂蛋白可结合内消旋二氨基庚二酸（meso-diaminopimelic acid, mDAP）——一种存在于肽聚糖五肽侧链中的氨基酸。研究人员针对参考菌株PAO1及其缺失该脂蛋白的对应转座子突变株PW2884开展了表型变化检测。透射电子显微镜观察显示，突变株的细胞极区附近出现周质空间扩张的现象。此外，研究团队观察到OMV释放量升高，该结果可通过纳米颗粒追踪分析得到验证。尤为重要的是，缺失该脂蛋白的突变株可在流动池中形成厚实但结构松散紊乱的生物被膜。综上，基因座PA1041编码的脂蛋白可将外膜锚定至肽聚糖层；突变株仍可存活，但会出现包括生物被膜形成紊乱在内的多种严重表型改变。基于铜绿假单胞菌PW2884突变株的表型特征及该蛋白的功能，研究人员将基因座标签为PA1041的脂蛋白命名为‘肽聚糖结合锚定蛋白（peptidoglycan-binding anchor, Pba）’。