Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species

Abstract

Bacteria that express contact-dependent growth inhibition (CDI) systems outcompete siblings that lack immunity, suggesting that CDI mediates intercellular competition. To further explore the role of CDI in competition, we determined the target cell range of the CDIEC93 system from Escherichia coli EC93. The CdiAEC93 effector protein recognizes the widely conserved BamA protein as a receptor, yet E. coli EC93 does not inhibit other enterobacterial species. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Consistent with this hypothesis, other enterobacteria are sensitized to CDIEC93 upon the expression of E. coli bamA and E. coli cells become CDIEC93 resistant when bamA is replaced with alleles from other species. Our data indicate that BamA loops 6 and 7 form the CdiAEC93-binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAEcoli and CdiAEC93 play a role in self-nonself discrimination.

Importance: Contact-dependent growth inhibition (CDI) systems are widespread among Gram-negative bacteria, enabling them to bind to neighboring bacterial cells and deliver protein toxins that inhibit cell growth. In this study, we tested the role of CDI in interspecies competition using intestinal isolate Escherichia coli EC93 as an inhibitor cell model. Although E. coli EC93 inhibits different E. coli strains, other bacterial species from the intestine are completely resistant to CDI. We show that resistance is due to small variations in the CDI receptor that prevent other species from being recognized as target cells. CDI receptor interactions thus provide a mechanism by which bacteria can distinguish siblings and other close relatives (self) from more distant relatives or other species of bacteria (nonself). Our results provide a possible means by which antimicrobials could be directed to one or only a few related bacterial pathogens by using a specific receptor "zip code."

FIG 1

Enterobacteria are resistant to CDI^EC93. Wild-type E. coli EC93 (cdiA⁺) or E. coli EC93 ∆cdiA mutant cells were mixed with the indicated target species at a 10:1 ratio, and the suspension were incubated on LB agar for 20 h. Cells were harvested, washed, and replated on LB agar supplemented with streptomycin to enumerate viable target cells as CFU. All competitions were conducted at least twice, and the reported values are the mean ± the standard error of the mean.

FIG 2

Expression of bamA^Ecoli sensitizes enterobacteria to CDI^EC93. (A) Competitions between E. coli EC93 and S. Typhimurium target cells carrying plasmid pZS21 or plasmids that express E. coli bamA or acrAB. (B) Competitions between E. coli EC93 and C. freundii cells carrying plasmid pZS21 or pZS21-bamA⁺. Plasmid pcdiI^EC93 corresponds to pDAL741 and constitutively expresses the cdiI immunity gene from E. coli EC93. (C) Competitions between E. coli EC93 and E. aerogenes cells carrying plasmid pZS21, pZS21-bamA⁺, and/or pcdiI^EC93, as indicated. For all competitions, wild-type E. coli EC93 (cdiA⁺) or E. coli EC93 ∆cdiA mutant cells were cocultured with target bacteria at a 10:1 ratio on LB agar for 20 h. Cells were harvested, washed, and replated on LB agar supplemented with streptomycin for enumeration of viable target cells as CFU. All competitions were conducted at least twice, and the reported values are the mean ± the standard error of the mean.

FIG 3

Predicted membrane topology and conservation of the BamA β-barrel domain. (A) Predicted topology of the BamA β-barrel based on the crystal structure of B. pertussis FhaC (Protein Data Bank code 2QDZ). Residues are numbered according to E. coli BamA. The percent identity for each residue is indicated as a heat map. Predicted extracellular loops 4, 6, and 7 are indicated. (B) Description of the BamA^Ecoli loop variants used in this study. The mutated residues are indicated, and the HA peptide epitope sequence is Val-Asp-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala.

FIG 4

Heterologous bamA confers CDI^EC93 resistance on E. coli. (A) Competitions between E. coli EC93 and E. coli ∆bamA::cat mutant cells complemented with plasmid-borne copies of bamA from the indicated species. For all competitions, wild-type E. coli EC93 (cdiA⁺) or E. coli EC93 ∆cdiA mutant cells were cultured with target bacteria at a 1:1 ratio in LB broth for 4 h. Cultures were sampled at 0 and 4 h and plated onto LB agar supplemented with ampicillin to enumerate viable target cells as CFU per milliliter. (B) Adhesion between CDI^EC93 inhibitor and target cells. GFP-labeled E. coli MC4100 cells carrying cosmid pDAL660∆1-39 (CDI^EC93 +) or pWEB::TNC (CDI^EC93 −) were used as inhibitors and mixed in 5-fold excess with DsRed-labeled target bacteria expressing bamA genes from the indicated species. Cell suspensions were analyzed by flow cytometry, and the percentage of red-fluorescent target cells aggregated with green-fluorescent inhibitors was calculated. The reported values are the mean ± the standard error of the mean from two independent experiments. Typhim., Typhimurium.

FIG 5

BamA^Ecoli loops 6 and 7 are required for CDI^EC93. (A and B) Competitions between E. coli EC93 and E. coli ∆bamA::cat targets that express the indicated bamA^Ecoli alleles. For all competitions, wild-type E. coli EC93 (cdiA⁺) or E. coli EC93 ∆cdiA mutant cells were cultured with target bacteria at a 1:1 ratio in LB broth for 4 h. Cultures were sampled at 0 and 4 h and plated on LB agar supplemented with ampicillin to enumerate viable target cells as CFU per milliliter. (C) Adhesion between CDI^EC93 inhibitor and target cells. GFP-labeled E. coli MC4100 cells carrying cosmid pDAL660∆1-39 (CDI^EC93 +) or pWEB::TNC (CDI^EC93 −) were used as inhibitors and mixed in 5-fold excess with DsRed-labeled target bacteria expressing the indicated bamA^Ecoli alleles. Cell suspensions were analyzed by flow cytometry, and the percentage of red-fluorescent target cells aggregated with green-fluorescent inhibitors was calculated. The reported values are the mean ± the standard error of the mean from two independent experiments.

FIG 6

BamA^Ecoli loops 6 and 7 are sufficient for CDI^EC93. (A) Competitions between E. coli EC93 and E. coli ∆bamA::cat targets that express the indicated bamA alleles. For all competitions, wild-type E. coli EC93 (cdiA⁺) or E. coli EC93 ∆cdiA mutant cells were cultured with target bacteria at a 1:1 ratio in LB broth for 4 h. Cultures were sampled at 0 and 4 h and plated on LB agar supplemented with ampicillin to enumerate viable target cells as CFU per milliliter. (B) Adhesion between CDI^EC93 inhibitor and target cells. GFP-labeled E. coli MC4100 cells carrying cosmid pDAL660∆1-39 (CDI^EC93 +) or pWEB::TNC (CDI^EC93 −) were used as inhibitors and mixed in 5-fold excess with DsRed-labeled target bacteria expressing the indicated bamA alleles. Cell suspensions were analyzed by flow cytometry, and the percentage of red-fluorescent target cells aggregated with green-fluorescent inhibitors was calculated. The reported values are the mean ± the standard error of the mean from two independent experiments.

FIG 7

BamA_Ec6/7^Ecloacae allows delivery of another CdiA-CT. (A) Northern blot analysis of tRNA_ICG^Arg from E. coli EPI100 carrying cosmid pDAL879 (inhibitors) and E. coli CH9350 (targets). (B) Northern blot analysis of RNA isolated from competition cocultures. Inhibitors were mixed with target cells expressing the indicated bamA alleles at a 1:1 ratio and incubated for 1 h. RNA was isolated from the mixed cultures and analyzed by Northern hybridization using a radiolabeled probe to tRNA_ICG^Arg. Target cells from the sample on the far right also expressed the cognate cdiI_o1^EC93 immunity gene from plasmid pDAL867.