Deciphering the Roles of BamB and Its Interaction with BamA in Outer Membrane Biogenesis, T3SS Expression and Virulence in Salmonella

The folding and insertion of β-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) β-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella.

革兰氏阴性菌外膜中β桶形蛋白（β-barrel proteins）的折叠与插入过程由BAM复合物（BAM complex）介导，该复合物由外膜蛋白BamA以及BamB至BamE四种脂蛋白组成。在大肠杆菌（Escherichia coli）和/或沙门氏菌（Salmonella）中，脂蛋白BamB参与以下过程：(i) 外膜中的β桶形蛋白组装；(ii) 外膜对抗生素的通透性；(iii) 作为主要毒力因子的三型分泌系统（T3SS）的表达调控；以及(iv) 沙门氏菌的毒力。在大肠杆菌中，已有研究表明该蛋白可与BamA直接相互作用。本研究围绕BamB的结构-功能关系展开探究，以评估BamB在上述表型中的作用是否相互关联，以及这些作用是否依赖于BamB与BamA的相互作用。为此，我们将携带bamB基因点突变的重组质粒导入沙门氏菌bamB缺失突变株（ΔSalmonella bamB mutant）中。本研究证实，氨基酸残基L173、L175与R176对BamB的所有功能及其与BamA的相互作用均至关重要。此外，针对无法免疫沉淀BamA的D229A BamB变体所获得的实验结果表明，BamB与BamA的相互作用并非BamB在外膜蛋白组装、T3SS表达以及毒力相关功能中所绝对必需的条件。最后，本研究证实ΔbamB突变株的毒力缺陷与其抗菌剂敏感性升高并无关联，因为D227A BamB变体可完全恢复该突变株的毒力，但其抗生素敏感性与ΔbamB菌株并无显著差异。综上，本研究表明BamB的不同功能并非全部相互关联，且氨基酸残基L173、L175与R176可作为设计BamB抑制剂的优选位点，这类抑制剂至少可作为对抗沙门氏菌的抗生素替代疗法。