Abstract
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.
Highlights
The outer membrane proteins of Gram-negative bacteria are synthesized in the cytoplasm and have to cross the inner membrane before being assembled into a correctly folded state in the outer membrane
Previous mutational and biochemical studies have shown that D227A or D229A single substitutions of the residue or the simultaneous L173S, L175S, R176A substitutions of the residues in BamB, produce a defective interaction with BamA in E. coli
When co-immunoprecipitation experiments were performed with the BamB variants harboring the single substitution L173S, R176A or D227A, BamA was detected at a level similar to that observed for the strain expressing the wild-type BamB protein
Summary
The outer membrane proteins of Gram-negative bacteria are synthesized in the cytoplasm and have to cross the inner membrane before being assembled into a correctly folded state in the outer membrane. BamA is conserved among bacteria, and homologues of the BAM complex are found in chloroplasts and mitochondria where they assemble b-barrel proteins [6,7,8]. The BAM complex has been shown to be involved in the assembly of integral outer membrane proteins (OMP) such as OmpA, LamB and the fimbrial usher protein FimD [10,11]. This complex is required for autotransporter biogenesis in several organisms [12,13,14,15]. Mutants unable to synthesize one or several Bam proteins have been shown to be more susceptible to different antibiotics including vancomycin, suggesting increased outer membrane permeability due to a defect in outer membrane integrity [11,16]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call