CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria

Abstract

Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiA^EC93 from Escherichia coli EC93 and OmpC-specific CdiA^EC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiA^EC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiA^EC93 that carries the corresponding sequence from CdiA^EC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads.IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads.

Keywords: bacterial competition; cell-cell adhesion; self/nonself discrimination; toxin immunity proteins; type V secretion system.

FIG 1

Localization of the BamA^Eco-binding region within CdiA^EC93. (A) CdiA^EC93 domain architecture. Domains and peptide repeat regions are depicted according to InterPro. TPS indicates the two-partner secretion transport domain, and also shown are FHA-1 (PF05594), FHA-2 (PF13332), the pretoxin-VENN domain (PF04829), and the CdiA-CT region. His₆ epitope-tagged CdiA_EC93 fragments used for BamA interaction experiments are depicted. (B) Affinity copurification of BamA with His₆-tagged CdiA^EC93 fragments. Refolded BamA^Eco or BamA^ECL was incubated with His₆-tagged CdiA^EC93 and then subjected to Ni²⁺-affinity chromatography. The input fraction and final elution samples were analyzed by immunoblotting using polyclonal antisera to BamA^Eco.

FIG 2

CdiA^EC93-CdiA^EC536 chimeras. CdiA^EC93 and CdiA^EC536 sequences are shown schematically. Shared residues are indicated in white, and effector-specific residues are indicated in blue and yellow. The residues demarcating the receptor-binding region (RBR) and covarying region (CVR) are indicated, as is the VENN motif that defines the CdiA-CT toxin region.

FIG 3

Cell-cell adhesion. (A) Flow cytometric analysis of CDI-dependent cell-cell adhesion. GFP-labeled inhibitor cells were mixed at a 5:1 ratio with DsRed-labeled target bacteria, and the cell suspension was analyzed by flow cytometry for dual green/red fluorescent events. CdiA protein identity is indicated schematically in the left margin, and target cell genetic backgrounds are indicated along the top. (B to G) CdiA-dependent cell-cell adhesion was quantified for each effector protein: mock CDI⁻ (B), CdiA^EC93 (C), CdiA^EC536 (D), CdiA^EC93 with the receptor-binding region (RBR) from CdiA^EC536 (E), CdiA^EC93 with the covarying region (CVR) from CdiA^EC536 (F), and CdiA^EC93 with both RBR and CVR from CdiA^EC536 (G). The fraction of target bacteria bound to inhibitor cells was quantified for two independent experiments. Data are presented as averages ± standard errors. UPEC, uropathogenic E. coli.

FIG 4

Competition cocultures. Target bacteria with the indicated bamA and ompC alleles were cocultured with inhibitor strains that express the following CdiA effectors: mock CDI^– (A), CdiA^EC93 (B), CdiA^EC536 (C), CdiA^EC93 with the receptor-binding region (RBR) from CdiA^EC536 (D), CdiA^EC93 with the covarying region (CVR) from CdiA^EC536 (E), and CdiA^EC93 with both RBR and CVR from CdiA^EC536 (F). Viable inhibitor and target cells were enumerated as CFU milliliter⁻¹, and the competitive index was calculated as final inhibitor-to-target cell ratio divided by the initial ratio for each coculture. Competitive indices are reported as averages ± standard errors for two independent experiments. UPEC, uropathogenic E. coli.

FIG 5

Comparison of class I, II, and III CdiA effectors. The CdiA^EC93, CdiA^EC536, and CdiA^STECO31 proteins are presented schematically. Residues shared by all three proteins are shown in white, and residues shared by two CdiAs are shown in gray. Effector-specific residues are shown in blue (CdiA^EC93), green (CdiA^EC536), and orange (CdiA^STECO31).

FIG 6

CdiA^STECO31 uses Tsx as a receptor to bind target bacteria. (A) E. coli ∆tsx mutants are resistant to CdiA^STECO31-mediated growth inhibition. Eight independent transposon insertions within tsx were identified in selections for CDI-resistant mutants. Inhibitor cells (CDI^STECO31 or CDI⁻ mock) were cocultured with tsx⁺ or Δtsx target bacteria in shaking broth for 4 hours as described in Materials and Methods. Viable inhibitor and target cells were enumerated as CFU, and the competitive index was calculated as the final inhibitor-to-target cell ratio divided by the initial ratio. Competitive indices are presented as averages ± standard errors. (B) Tsx is required for CdiA^STECO31-dependent cell-cell adhesion. The fraction of red fluorescent target bacteria bound to green fluorescent inhibitor cells was quantified for two independent experiments. Data are presented as averages ± standard errors.

FIG 7

Topological model for CdiA. The constituent domains of cell surface CdiA are labeled and color coded as in Fig. 1. CdiB is represented as a barrel in the outer membrane (OM). The proposed model is based on unpublished protease protection data and the work of Noël et al. (48).