Contact-Dependent Growth Inhibition (CDI) and CdiB/CdiA Two-Partner Secretion Proteins

Abstract

Bacteria have developed several strategies to communicate and compete with one another in complex environments. One important mechanism of inter-bacterial competition is contact-dependent growth inhibition (CDI), in which Gram-negative bacteria use CdiB/CdiA two-partner secretion proteins to suppress the growth of neighboring target cells. CdiB is an Omp85 outer-membrane protein that exports and assembles CdiA exoproteins onto the inhibitor cell surface. CdiA binds to receptors on susceptible bacteria and subsequently delivers its C-terminal toxin domain (CdiA-CT) into the target cell. CDI systems also encode CdiI immunity proteins, which specifically bind to the CdiA-CT and neutralize its toxin activity, thereby protecting CDI(+) cells from auto-inhibition. Remarkably, CdiA-CT sequences are highly variable between bacteria, as are the corresponding CdiI immunity proteins. Variations in CDI toxin/immunity proteins suggest that these systems function in bacterial self/non-self recognition and thereby play an important role in microbial communities. In this review, we discuss recent advances in the biochemistry, structural biology and physiology of CDI.

Keywords: biofilms; self/non-self recognition; toxin/immunity proteins; type V secretion.

Fig. 1. Contact-dependent growth inhibition (CDI)

a) CDI⁺ bacteria carry cdiBAI gene clusters that encode CdiB-CdiA two-partner secretion proteins and CdiI immunity proteins. b) Model for the CdiB/CdiA complex. CdiB is represented by the crystal structure of B. pertussis FhaC (PDB: 3NJT), and CdiA is modeled as concatenated β-helices from E. coli Ag43 (PDB: 4KH3). c) CdiA binds to receptors on neighboring bacteria and delivers its C-terminal toxin domain (red star) into the target cell. If target cells lack immunity (left pathway), then their growth is inhibited. In contrast, CdiI neutralizes the toxin in isogenic CDI⁺ bacteria, preventing growth inhibition (right pathway).

Fig. 2. Domain structure of CdiA

a) Architectures of putative CdiA proteins. Predicted domain structures are presented for CdiA proteins from E. coli EC93 (Uniprot: Q3YL96), M. catarrhalis O35E (A5JFL2), B. pseudomallei 1026b (I1WVY3) and P. syringae DC3000 (Q880E1). The domain structure of B. pertussis FhaB is also presented for comparison. The extended signal peptide region (ESPR) and TPS transport domain are required for TpsA/CdiA secretion. FHA-1 (PF05594) and FHA-2 (PF13332) peptide repeats were first identified in FhaB and are predicted to form β-helical structures^; . In many species, the pretoxin-VENN domain (PF04829) demarcates the variable C-terminal (CT) region, which contains the CDI toxin activity. DUF637 (PF04830) is a domain of unknown function found in a subset of CdiA proteins. HINT indicates the pretoxin-HINT domain (hedgehog intein; PF07591), and HNH indicates a predicted colicin DNase domain (PF12639). The restriction endonuclease (REase) domain at the C-terminus of CdiA^Bp1026b was determined though X-ray crystallography. b) Domain structures of CdiA-CT regions from different E. coli CdiA proteins. CdiA-CT^EC93 and CdiA-CT^KTE214 are formed from single domains, but many other CdiA-CTs are composed of two domains. The extreme C-terminal domain usually contains nuclease activity, whereas the function of the variable N-terminal domain is not known. E. coli strains are indicated as superscripts and domains are color-coded to indicate sequence variation. The position of the common VENN peptide motif is indicated.

Fig. 3. Crystal structures of selected CdiA-CT/CdiI complexes

Toxin/immunity protein complexes from B. pseudomallei 1026 (PDB: 4G6V), E. cloacae ATCC 13047 (PDB: 4NTQ) and E. coli TA271 (PDB: 4G6U) are presented^; . All CdiA-CT structures contain only the C-terminal nuclease domain, with the exception of CdiA-CT^TA271, for which a portion of the N-terminal domain (shown in red) has been resolved.

Fig. 4. Genomic organization and horizontal gene transfer

a) The cdi gene cluster from E. coli EC93 is depicted with the cdiA-CT/cdiI_o1 orphan gene pair. The genes rendered in gray encode a predicted IS3-family transposase. The cdiA-CT_o1 toxin coding sequence is outlined in red and lacks an initiating methionine codon. b) E. coli cdi genes are found on genomic islands. A family of cdi gene containing islands is inserted at several different tRNA genes in E. coli strains.

Fig. 5. Burkholderia pseudomallei cdi loci

Organization of the 10 cdi sequence types found in strains of B. pseudomallei. The cdiA-CT to cdiB regions are shown from representative strains, with cdiA genes identified by their ordered locus tags. Immunity genes are shown in green, bcpO in brown and predicted orphan immunity genes in light blue. The direct repeats surrounding the orphan cdiI immunity gene within the type locus are shown as arrows.

Fig. 6. E. coli EC93 ΔcdiA mutants are defective for biofilm formation

Biofilms of wild-type (cdiA⁺) and ΔcdiA strains of E. coli EC93 after 24 h incubation in a flow-cell. Inset scale bars equal 40 µm. The left panels show three-dimensional reconstructions of biofilm structures. Images courtesy of Loni Townsley and Fitnat Yildiz (UC Santa Cruz).