Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD
- Genentech, Inc, United States
Figures
Figure 1
Immunization schematics and α-LptD mAb library characteristics.
(A) Cartoon of LptD immunization campaigns with purified E. coli LptDE protein, LptD cyclic peptides, and linear peptides. 10 rounds of protein or peptide injections were performed. Clones were …
Figure 2 with 2 supplements
Whole cell FACS binding and epitope binning of α-LptD mAbs.
(A) LptD mAb campaign summary of ELISA+ LptDE mAbs tested for surface binding on E. coli ΔwaaD and E. coli K-12 by FACS assay. Antibodies were scored FACS+ if the MFI was 2x above an isotype …
Figure 2—figure supplement 1
α-LptD mAb binding to E. coli K-12 and E. coli ΔwaaD strains by FACS analysis.
(A) Representative FACS traces for three ELISA+ α-LptD mAbs, 27C2 (purple), 3D10 (blue), and 27C8 (red), with E. coli ΔwaaD (top) and E. coli K-12 (bottom) cells. A non-binding isotype control mAb …
Figure 2—figure supplement 2
LptD ECL conservation among different Enterobacteriaceae species.
(A) Amino acid sequence alignment of LptD β-barrel region (amino acids 181–778) for E. coli (Ecoli), E. cloacae (Ecloacae), and K. pneumoniae (Kpneumo). Extent of ECLs based on available structures …
Figure 3 with 1 supplement
LptD ECLs are dispensable for E. coli growth.
(A) The ECL boundaries of LptD are highlighted as spheres and color-coded as indicated. Sequence boundaries are indicated Figure 2—figure supplement 2A. Structure based on S. flexneri LptDE with …
Figure 3—figure supplement 1
Expression of lptD loop mutants does not affect growth of E. coli expressing a wild-type copy of lptD.
Representative Western blots of (A) log-phase E. coli K-12 wild-type liquid cultures and (B) E. coli ΔwaaD cultures from overnight agar plates expressing each lptD loop mutant. Strains were grown in …
Figure 4 with 2 supplements
Mapping α-LptD mAb binding using LptD loop variants.
(A) 52 FACS+ LptD mAbs were screened for FACS binding to E. coli ΔwaaD expressing wild-type lptD. The mean fluorescent intensities (MFIs) are plotted. The blue bars highlight the eight …
Figure 4—figure supplement 1
Comprehensive FACS analysis on all lptD loop mutants.
52 FACS +LptD mAbs were screened for FACS binding to E. coli ΔwaaD expressing each lptD loop mutant (except L10, see text). The mean fluorescent intensities (MFIs) above background are normalized to …
Figure 4—figure supplement 2
Functional analysis of α-LptD antibodies.
Bacterial growth was measured (OD600) for (A) E. coli ΔwaaD and (B) E. coli K-12 after treatment with one representative plate of α-LptDE mAbs at 10 µg/mL for 4 hr. Black dashed line indicates media …
Figure 5 with 1 supplement
Antibody accessible ECLs of LptD.
Side view of LptD structure rendered in PyMol from E. coli LptDE with LptE removed (PDB 4RHB [Malojcic et al., 2015]). LptD was placed in a standard phosphatidylethanolamine membrane context (shown …
Figure 5—figure supplement 1
Diversity of the ECLs of LptD.
(A) Amino acid sequence alignments of each LptD ECL comparing E. coli (Ecoli), Pseudomonas aeruginosa (Paeruginosa), Acinetobacter baumannii (Abaumannii), K. pneumoniae (Kpneumoniae), and E. cloacae …
Tables
Table 1
Removal of extracellular LptD loops sensitizes E. coli to OM-excluded antibiotics.
https://doi.org/10.7554/eLife.46258.009| MIC (µg/ml)* | ||||
|---|---|---|---|---|
| Vancomycin | Rifampicin | |||
| LptD† | WT‡ | ΔwaaD§ | WT | ΔwaaD |
| WT | 128 | 64 | 8 | 0.125 |
| ΔL1 | 128 | 64 | 4 | 0.125 |
| ΔL2 | 8 | 4 | 0.25 | 0.0625 |
| ΔL3 | 128 | 16 | 4 | 0.125 |
| ΔL4 | 16 | 1 | 0.25 | 0.0156 |
| ΔL5 | 128 | 64 | 8 | 0.125 |
| ΔL6 | 128 | 64 | 2 | 0.0625 |
| ΔL7 | 128 | 64 | 4 | 0.125 |
| ΔL8 | 16 | 4 | 0.125 | 0.0625 |
| ΔL9 | 128 | 128 | 4 | 0.125 |
| ΔL10 | 64 | NG | 2 | NG |
| ΔL11 | 128 | 64 | 8 | 0.125 |
| ΔL12 | 128 | 64 | 8 | 0.125 |
| ΔL13 | 128 | 64 | 8 | 0.125 |
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*Minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic that completely inhibits bacterial growth.
†Conditional lptD E. coli strains carry an arabinose-inducible wild-type lptD and a plasmid-encoded copy of lptD with the indicated loop deletions (as indicated in Figure 3A). Only the plasmid copy of lptD is expressed in the absence of arabinose.
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‡The wild-type (WT) strain is a conditional E. coli K-12 with a chromosomal arabinose-inducible lptD.
§The ΔwaaD strain is a conditional E. coli ΔwaaD mutant with a chromosomal arabinose-inducible lptD.
Table 2
mAbs to accessible ECLs do not inhibit essential function of LptD.
https://doi.org/10.7554/eLife.46258.013| Growth inhibitory α-LptD mAbs* | |||||
|---|---|---|---|---|---|
| Antigen | Host | Clones | K-12† | ΔwaaD‡ | ΔwaaD + Rif.§ |
| Linear and cyclic LptD peptides | SD Rats | 576 | 0 | 0 | 0 |
| Purified LptDE | Mice | 3360 | 0 | 0 | 0 |
| Purified LptDE | SD Rats | 2400 | 0 | 0 | 0 |
| Cells and purified LptDE | SD Rats | 1494 | 0 | 0 | 0 |
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*For each antibody campaign, bacterial growth was measured (OD600) for E. coli ΔwaaD and E. coli K-12 after treatment with each antibody at 10 µg/mL for 4 hr. Growth inhibition was calculated as a percentage of growth compared to an untreated control. 50% growth inhibition was considered positive.
†WT (wild-type) is E. coli K-12.
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‡ΔwaaD is an E. coli ΔwaaD.
§ΔwaaD + Rif. is E. coli ΔwaaD grown in the presence sub-inhibitory rifampicin
Additional files
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Supplementary file 1
Statistical analysis of the initial growth rates for conditional lptD deletion strains complemented with lptD loop mutants.
Bacterial growth curves in Figure 3 and Figure 3—figure supplement 1 were analyzed by determining the doubling time (dt) during exponential growth phase and compared via the unpaired Student’s t test. The Bonferroni correction was applied to control for multiple comparisons.
- https://doi.org/10.7554/eLife.46258.016
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Supplementary file 2
Strains, plasmids, and primers used in this study.
Names, descriptions, and references for all key resources (bacterial strains, plasmid constructs, and primers) as described in the text.
- https://doi.org/10.7554/eLife.46258.017
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Supplementary file 3
Sequences of linear and cyclic peptides used for immunizations.
LptD peptides used for immunizations (Figure 1) were designed based on sequence conservation, surface exposure and loop location.
- https://doi.org/10.7554/eLife.46258.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.46258.019
