A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

Abstract

Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA 'exoprotein' onto the cell surface. An additional small immunity protein (CdiI) protects CDI(+) cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI(+) cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.

Figure 1. Analysis of CdiA chimeras

a, Target cells expressing CdiI_EC93 or CdiI₅₃₆ were co-cultured with inhibitor cells expressing cdi genes from either E. coli EC93 or UPEC 536. After 3 h, the number of viable target cells was determined as colony forming units (CFU) per ml (mean ± s.d., n = 4 experiments). b, The C-terminal ∼200 residues of the indicated CdiAs diverge following a conserved VENN peptide motif (shown by different colors). The CdiI proteins from each system are also highly variable. c, Target cells expressing CdiI from E. coli UPEC 536, E. coli EC93, Y. pestis CO92 (accession Q7CGD9), and D. dadantii 3937 (CDI module 2, see Supplementary Table 1) were protected from CDI mediated by chimeric CdiA₅₃₆ proteins containing cognate, but not heterologous, CdiA-CT (mean ± s.d., n = 2 experiments).

Figure 2. CdiA-CT contains growth inhibitory activity

a, Growth of E. coli cells expressing CdiA-CT_EC93 from plasmid pDAL778. Co-expression of cognate CdiI_EC93, but not heterologous CdiI₅₃₆, protected cells from growth inhibition. The mean ± s.d. is shown, n = 2 experiments. b, The 268 residue CdiA-CT_EC93 peptide is depicted along with various truncation constructs indicating the number of residues deleted. Each CdiA-CT construct was tested for growth inhibitory activity when expressed in E. coli cells (“++” indicates that growth was blocked immediately after CdiA-CT_EC93 induction, “+” indicates that growth was blocked after a 2 - 3 hr delay, and “-” indicates no growth inhibition).

Figure 3. CdiI immunity protein binds specifically to cognate CdiA-CT and blocks activity

a, Purified CdiA-CT and CdiI-His₆ proteins from UPEC 536 and D. dadantii 3937 (CDI module 2, 3937-2) were mixed in vitro with Ni²⁺-NTA resin. Aliquots of resin bound and unbound fractions were analyzed by SDS-PAGE and Coomassie blue staining. b, A bacterial two-hybrid system (BACTH) based on adenylate cyclase activity was used to monitor CdiA-CT/CdiI binding in vivo. A β-galactosidase reporter was used to measure adenylate cyclase activity. Expression of two-hybrid T25-cdiA-CT/cdiI-T18 fusion constructs resulted in significant β-galactosidase activity. T25-gfpmut3/cdiI-T18 fusions were used to control for background β-galactosidase activity. Fluorescence microscopy of cells expressing each D. dadantii 3937 construct confirms GFP expression of the control. P values were obtained using an unpaired, two-tailed t-test (mean ± s.d., n = 2 experiments). c, Purified CdiA-CT_3937-2 was incubated with linear pUC19 DNA in the presence and absence of cognate and heterologous CdiI. Reactions were analyzed by native agarose gel electrophoresis and ethidium bromide staining. d, Purified CdiA-CT₅₃₆ was incubated with an S100 cell extract (100,000 x g supernatant), in the presence and absence of cognate and heterologous CdiI. Reactions were analyzed by denaturing gel electrophoresis. Top panel, ethidium bromide (EtBr) staining for total tRNA (arrow indicates tRNA; asterisk indicates degradation products); lower panels, Northern blot analyses of tRNA^His and tRNA_1B^Ala. Arrows indicate full-length tRNAs.

Figure 4. CDI systems function in intrastrain growth competition

a, Str^R CDI⁺ EC93 cells were mixed 1:1 with rif^R EC93 ΔcdiA-CTΔcdiI cells that either contained no plasmid (cdiI^-), pBR322 plasmid (vector), or CdiI_EC93-expressing plasmid (cdiI_EC93). After 3 hr of co-culture, cells were plated onto LB medium, and rif^R and str^R CFU/ml were quantified to calculate the competitive index [(rif^R CFU/str^R CFU)_{3 hr} / (rif^R CFU/str^R CFU) _{0 hr}]. The mean ± s.d. is shown (n = 2 experiments), P value = 0.002. b. Gent^R CDI⁺ D. dadantii 3937 cells were mixed 100:1 with nal^R D. dadantii 3937 ΔcdiA-CT_3937-1 ΔcdiI_3937-1 alone (cdiI^-), or complemented with heterologous cdiI from D. dadantii EC16 (cdiI_EC16), or cognate cdiI (cdiI_3937-1). Cell mixtures were inoculated onto chicory leaves (see Supplementary methods), incubated for the indicated times, and viable counts were determined. The competitive index (CFU nal^R /CFU gent^R) was calculated as described for panel ‘a’ above at each time point. The mean ± s.d. is shown (n = 2 experiments). P value at 24 h = 0.00004. P values were obtained using an unpaired, two-tailed t-test.