Functional Characterization of Pseudomonas Contact Dependent Growth Inhibition (CDI) Systems

Abstract

Contact-dependent inhibition (CDI) toxins, delivered into the cytoplasm of target bacterial cells, confer to host strain a significant competitive advantage. Upon cell contact, the toxic C-terminal region of surface-exposed CdiA protein (CdiA-CT) inhibits the growth of CDI- bacteria. CDI+ cells express a specific immunity protein, CdiI, which protects from autoinhibition by blocking the activity of cognate CdiA-CT. CdiA-CT are separated from the rest of the protein by conserved peptide motifs falling into two distinct classes, the "E. coli"- and "Burkholderia-type". CDI systems have been described in numerous species except in Pseudomonadaceae. In this study, we identified functional toxin/immunity genes linked to CDI systems in the Pseudomonas genus, which extend beyond the conventional CDI classes by the variability of the peptide motif that delimits the polymorphic CdiA-CT domain. Using P. aeruginosa PAO1 as a model, we identified the translational repressor RsmA as a negative regulator of CDI systems. Our data further suggest that under conditions of expression, P. aeruginosa CDI systems are implicated in adhesion and biofilm formation and provide an advantage in competition assays. All together our data imply that CDI systems could play an important role in niche adaptation of Pseudomonadaceae.

Fig 1. Analysis of cdiA_PA2462 and cdiA_PA0041 gene expression profile.

Expression of cdiA_PA2462 and cdiA_PA0041 in WT and ΔrsmA strains grown 7 h under agitation (planktonic) or without agitation (static) in LB medium. For each gene, expression was normalized to 16S expression and is shown relative to the WT planktonic condition level. Error bars represent the standard error of the mean from three independent experiments. Data were analyzed for significance using a two-tailed Student’s t-test. ** 0.025<p-value <0.01; *** p-value<0.01; ns: non-significant.

Fig 2. The growth of cdiBAI_PA2462 and cdiBAI_PA0041 mutants is inhibited by contact with a wild-type strain.

Gentamycin-resistant wild-type and mutant target bacteria were mixed with or without wild-type or ΔrsmA attackers and the number of viable target cells was calculated as the number of CFU/ml after contact (t_24h) divided by the number of CFU/ml before contact (t_0h). Error bars represent the standard error of the mean from three independent experiments.

Fig 3. Roles of CdiA_PA2462, CdiA_PA0041 in growth competition.

All experiments were performed in a ΔrsmA background. A) ΔBAI_PA2462 and B) ΔBAI_PA0041 target strains were mixed with different attacker cells. Error bars represent the standard error of the mean from three independent experiments.

Fig 4. Roles of adhesion and biofilm formation in competition assays.

(A). CLSM of adhesive structures formed by WT, cdiA_PA2462 and cdiA_PA0041 mutants (ΔA_PA2462 and ΔA_PA0041 respectively) at 8 h post-inoculation. All strains carry a gfp gene inserted at the glmS locus on the P. aeruginosa PAO1 chromosome. White scale bar = 10 μm. (B) and (C). After staining with crystal violet, biofilm was quantified over time by measuring the optical density at 550 nm (OD₅₅₀). Each strain was tested in triplicate experiment. Data were examined for significance using a two-tailed Student’s t-test. *** p-value<0.01; ns: non-significant. D. Competition experiments were performed in a ΔrsmA background. WT or ΔpilAΔfliC attacker strains were mixed with ΔBAI_PA2462 or BAI_PA0041 target bacteria. Error bars represent the standard error of the mean from three independent experiments.

Fig 5. CdiI_PA2462 and CdiI_PA0041 play a protective role.

All experiments were carried out in a ΔrsmA background. Wild-type (WT) attacker was mixed with different gentamycin-resistant mutant targets: (A) ΔBAI_PA2462 carrying or not the pJN107 plasmid encoding the CdiI_PA2462 protein or the BA_PA2462 mutant. No arabinose was added to produce CdiI_PA2462 from pJN107-I_PA2462; (B) BAI_PA0041 carrying or not the pJN107 plasmid encoding the CdiI_PA0041 protein or the BA_PA0041 mutant. As control, ΔBAI_PA0041 expressing cdiI_PA0041 was tested alone in inter-bacterial competition (grey bar). 0.5% arabinose was used to induce CdiI_PA0041.

Fig 6. Intracellular toxicity of CdiA-CT_PA2462 and CdiA-CT_PA0041 in E. coli cells and protection by their cognate CdiI immunity proteins.

Production of (A) CdiA-CT_PA2462 and (B) CdiA-CT_PA0041 were repressed with 0.5% glucose (grey bars) or induced with 1% arabinose (black bars). Error bars represent the standard error of the mean from three independent experiments. C. Purified CdiA-CT_PA0041 and CdiI-His₆ proteins were mixed in vitro with Ni²⁺-NTA resin. Fractions were analyzed by SDS-PAGE and Coomassie blue staining. Control experiments with purified proteins incubated alone are shown on the left panel while CdiA-CT/CdiI-His₆ mixtures are shown on the right panel. Marker lane (kDa) is shown on the left.

Fig 7. Representative CdiA proteins of each class of pre-toxin motif.

The signal peptide (SP) is a sequence potentially recognized by the sec machinery and cleaved during the export through the inner membrane. TPS domain found in N-terminal part of TpsA allows targeting to the TpsB transporter and subsequent translocation through the outer membrane. Haemagglutinin repeats are highly divergent repeats found in FHA-like proteins. DUF637 is a conserved region found in bacterial haemagglutinins or hemolysins. WVHN, VENN, LYVT, DAMV, NEALV are the pre-toxin motifs delimiting the conserved N-terminal and the variable CT domains.

Fig 8. CdiA-CT encoded by P. aeruginosa PA7, PA14 and P. syringae pv. tomato DC3000 inhibit E. coli cell growth.

Production of (A) CdiA-CT_{PSPA7_2777,} (B) CdiA-CT_{PA14_32790} and (C) CdiA-CT_{PSPTO_3229} was repressed with 0.5% glucose (grey bars) and induced with 1% arabinose (black bars). CdiI-His₆ proteins were produced from pTrc99a with 1 mM IPTG. The number of CFU/ml was calculated after 6 h of culture and the graphs show the mean of three independent experiments.