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

Abstract

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 Escherichia coli β-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 β-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 β-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.

Keywords: BamA; Gram-negative bacteria; LPS; membrane protein folding; β-barrel protein.

Fig. 1.

α-BamA mAb MAB1 kills E. coli ΔwaaD. (A) CFUs were quantified at indicated times after the addition of 10 nM MAB1, MAB2, or no antibody to the E. coli ΔwaaD strain. (B) Growth inhibition was measured by E. coli ΔwaaD density (OD₆₀₀) in the presence of MAB1 IgG, MAB2, or MAB1 Fab after 4 h. (C) Representative FACS traces of MAB1 surface-binding to E. coli ΔwaaD strains producing chimeric BamA proteins. The shaded trace is a control with no primary mAb. Mean fluorescent intensities (MFIs) for biological triplicate experiments are plotted in SI Appendix, Fig. S2B. (D) MAB1 dose–response inhibition of E. coli ΔwaaD strains producing chimeric BamA measured by OD₆₀₀ after 4 h of treatment. For all experiments, means and SDs of biological triplicates are plotted. Unpaired t tests were used to compare values to untreated controls or IC₅₀ values. IC₅₀ values are in SI Appendix, Table S3. ***P < 0.001.

Fig. 2.

MAB1 inhibits BamA OMP folding activity. (A) Representative Western blots of OMPs and controls from E. coli ΔwaaD in the presence or absence of 10 nM MAB1 or MAB2 after 1.5 h of treatment. (B) Induction of σ^E periplasmic stress response (rpoH P3-lacZ) in E. coli ΔwaaD by 10 nM MAB1 or MAB2. Data are a ratio of mAb to no mAb at times after mAb addition. (C) Influx of EtBr (ex515 nm, em600 nm, normalized to OD₆₀₀) into E. coli ΔwaaD after MAB1 or MAB2 treatment. (D) Fluorescence time-lapse microscopy of E. coli ΔwaaD cells expressing GPF (cytoplasm) and mCherry (periplasm) pretreated with 13 nM MAB1 or MAB2 for 1.5 h and imaged for 3 h. A representative image is shown. Means and SDs of biological triplicates are plotted in B and C. Unpaired t tests were used to compare values at each time point or antibody concentration tested. **P < 0.01, ***P < 0.001.

Fig. 3.

MAB1 binds to BamA extracellular loop 4 (L4). E. coli ΔwaaD producing BamA with site-directed substitutions in L4 were quantified and compared for FACS whole cell binding by MAB1 (A) and growth inhibition by MAB1 by bacterial density (OD₆₀₀) (B). BamA variants with reduced MAB1 binding and sensitivity shown in color; substitutions with no or subtle effects on activity of MAB1 are gray. Means and SDs of biological triplicates are plotted. The dotted line is the background control with no mAb. IC₅₀ values were calculated and compared with BamA – WT (0.018 ± 0.005 nM) by unpaired t test: E554Q (38.6 ± 7.2 nM, P < 0.01), H555Y (>50 nM, P < 0.001), E554Q/H555Y (>50 nM, P < 0.001), Y550N (0.030 ± 0.005 nM), D560S (0.038 ± 0.008 nM), Q561D (0.014 ± 0.002 nM), D562N (0.017 ± 0.003 nM), T566S (0.013 ± 0.005 nM), and T567A (0.011 ± 0.002 nM). (C) BAM rendered in PyMol from 5EKQ coordinates (16). BamA (gray), BamB (red), BamC (cyan), BamD (blue), and BamE (violet) are shown. Residues 554 and 555 are pink spheres. The β1-β16 lateral gate is indicated in green. The membrane space is approximated. Left and Right are rotated 90 °C relative to each other (BamBCDE are hidden in top view). Unpaired t tests were used to compare MFIs to WT or IC₅₀ values for each strain tested. ***P < 0.001.

Fig. 4.

An E. coli ΔwaaD, ΔlpxM mutant is resistant to MAB1. (A) Cartoon of LPS Kdo₂-lipid A with the acyl chain added by LpxM in red. (B) MAB1 growth inhibition of E. coli ΔwaaD; E. coli ΔwaaD, ΔlpxM; and E. coli ΔwaaD, ΔlpxM, plpxM complemented strains by cell density (OD₆₀₀). IC₅₀ values were calculated and compared with ΔwaaD (0.068 ± 0.0029 nM) by unpaired t tests: ΔwaaD, ΔlpxM (>13.3 nM, P < 0.001), and ΔwaaD, ΔlpxM, plpxM (0.017 ± 0.0013 nM, P < 0.001). (C) FACS whole-cell binding by MFI of MAB1 to the E. coli ΔwaaD and ΔwaaD, ΔlpxM strains. The dotted line is the background control with no mAb. (D) EtBr uptake into E. coli ΔwaaD and ΔwaaD, ΔlpxM strains measured in the absence of mAb. (E) Membrane fluidity of E. coli ΔwaaD and ΔwaaD, ΔlpxM strains measured using a fluorescent lipophilic probe, pyrenedecanoic acid (PDA), in the absence of mAb. The ratio of emission at 470 nm to emission at 405 nm normalized to the E. coli ΔwaaD strain is shown. For all experiments, means and SDs of biological triplicates are plotted. Unpaired t tests were used to compare values to ΔwaaD or IC₅₀ values for each strain tested. *P < 0.05, **P < 0.01.

Fig. 5.

LPS structure, NaCl concentration, and growth temperature change membrane fluidity and sensitivity to MAB1. (A, E, and I) EtBr uptake in E. coli ΔwaaD cells compared with E. coli ΔwaaG (A), grown at increasing NaCl concentrations (E), and grown at 37 °C, 30 °C, and 42 °C (I). Membrane fluidity of E. coli ΔwaaD strain (B, F, and J), MAB1 whole-cell binding by FACS (C, G, and K), and growth inhibition by MAB1 of E. coli ΔwaaD were compared with indicated strains and growth temperatures (D, H, and L). Membrane fluidity data are normalized to E. coli ΔwaaD strain grown at 37 °C. High temperatures caused unequal fluidity probe integration (SI Appendix, Fig. S9). The dotted line is the control with no antibody. For all experiments, means and SDs of biological triplicates are plotted. Unpaired t tests were used to compare values to ΔwaaD or ΔwaaD 37 °C. IC₅₀ values are in SI Appendix, Table S3. *P < 0.05, **P < 0.01, ***P < 0.001.