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Monoclonal antibody targeting the ?-barrel assembly machine of Escherichia coli is bactericidal.

ABSTRACT: The folding and insertion of integral ?-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ?-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ?-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ?-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ?-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

SUBMITTER: Storek KM

PROVIDER: S-EPMC5889671 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Monoclonal antibody targeting the β-barrel assembly machine of <i>Escherichia coli</i> is bactericidal.

Storek Kelly M KM Auerbach Marcy R MR Shi Handuo H Garcia Natalie K NK Sun Dawei D Nickerson Nicholas N NN Vij Rajesh R Lin Zhonghua Z Chiang Nancy N Schneider Kellen K Wecksler Aaron T AT Skippington Elizabeth E Nakamura Gerald G Seshasayee Dhaya D Koerber James T JT Payandeh Jian J Smith Peter A PA Rutherford Steven T ST

Proceedings of the National Academy of Sciences of the United States of America 20180319 14

The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the <i>Escherichia coli</i> β-barrel assembly machine, BamA. In the a ...[more]

PMID: 29555747

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Project description:Integral ?-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the ?-barrel assembly machine (BAM). This essential complex, composed of a ?-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of ?-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the ?E stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of ?-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the ?-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which ?-barrel folding occurs.

Dataset Information

Monoclonal antibody targeting the ?-barrel assembly machine of Escherichia coli is bactericidal.

Publications

Monoclonal antibody targeting the β-barrel assembly machine of <i>Escherichia coli</i> is bactericidal.

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