The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems

Abstract

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis--a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact-dependent growth inhibition (CDI) systems of γ-proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA-CT/cdiI coding regions, which are predicted to encode CdiA-CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA-CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA-CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA-CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.

Figure 1. Genomic organization of B. pseudomallei cdi gene clusters

The genomic neighborhoods of representative Bp CDI systems encoding the ten different CdiA-CT/CdiI sequence types (CDI types I through X) are presented. The Bp strain and encoding chromosome (if known) for each representative CDI system is given in parentheses. Bp 1106a carries two predicted CDI systems on chromosome I (types VII and X); these loci are labeled i and ii to distinguish the two systems. The cdiA and cdiB genes are shown in olive and yellow (respectively); and the cdiA-CT regions (corresponding to the 3′-end of cdiA genes) and cdiI genes are depicted in various colors. Genes related to transposases are shown in orange and all other coding sequences, including rhs (rearrangement hotspot) genes are white. Some systems contain additional small coding sequences between the cdiI and cdiB genes (white triangles). The asterisk indicates a putative immunity gene that is related to cdiI from Enterobacter aerogenes KCTC 2190.

Figure 2. B. pseudomallei CdiA-CTs inhibit cell growth

The CdiA-CT/CdiI toxin/immunity network was examined in E. coli cells using a two-plasmid strategy. CdiA-CT_II^1026b and the CdiA-CT^E478/CdiI^E478- DAS and CdiA-CT^E479/CdiI^E479-DAS complexes were produced from plasmid pCH450 upon induction with L-arabinose (indicated by the downward arrows). Each culture was also induced at 0 min with IPTG to produce Bp CdiI immunity protein (indicated in the inset legend) from a compatible pTrc plasmid. A) CdiA-CT_II^1026b production was induced at 30 min, and growth monitored by measuring the optical density at 600 nm (OD₆₀₀). B) CdiA-CT^E478/CdiI^E478-DAS complex production was induced at 30 min and growth monitored by OD₆₀₀. The CdiA-CT^E478 is liberated in these cells through degradation of ssrA(DAS)-tagged CdiI^E478 immunity protein. C) CdiA-CT^E479/CdiI^E479-DAS complex production was induced at 30 min and growth monitored by OD₆₀₀. The CdiA-CT^E479 is liberated in these cells through degradation of ssrA(DAS)-tagged CdiI^E479 immunity protein. In each panel, the black curve represents the growth of control cells carrying empty pCH450 and pTrc plasmid vectors.

Figure 3. CdiA-CT^E478 has tRNase activity similar to colicin E5

The CdiA-CT^E478/CdiI^E478-DAS complex was produced in E. coli ΔsspB cells and degradation of the ssrA(DAS)-tagged CdiI^E478 was initiated at 0 hr by induction of the SspB adaptor protein. RNA samples were taken at the indicated time points for northern blot analysis. Cells expressing SspB(Δ47), which does not efficiently deliver CdiI^E478-DAS to the ClpXP protease, were used as a control. Each gel lane contains 10 μg of total RNA, which was hybridized against oligonucleotide probes specific for E. coli tRNA₁^Tyr, tRNA^Asn, tRNA^His and tRNA₂^Arg. The gel migration positions for full-length and cleaved tRNA are indicated.

Figure 4. CdiA-CT^E479 has tRNase activity

CdiA-CT^E479/CdiI^E479 production was induced by IPTG in E. coli cells, and RNA samples taken for gel and northern blot analyses. RNA from cells expressing CdiA-CT^E479 with the Asp285Ala mutation was also analyzed. RNA was hybridized against oligonucleotide probes specific for E. coli tRNA₂^Arg and tRNA₃^Gly. The gel migration positions for ribosomal RNA (rRNA) and tRNA are indicated.

Figure 5. B. pseudomallei CdiI immunity proteins bind specifically to cognate CdiA-CT

Purified CdiA-CTs and His₆-tagged CdiI immunity proteins were incubated together, then subjected to Ni²⁺- affinity chromatography to bind CdiI-His₆ and any associated CdiA-CT. All fractions were run on SDS- polyacrylamide gels and stained with Coomassie blue. The lanes labeled input show the unfractionated CdiA-CT/CdiI-His₆ mixtures, the lanes labeled free show proteins that did not bind Ni²⁺ resin, and the lanes labeled bound show proteins that bound to the resin.

Figure 6. CdiI_II¹⁰²⁶ immunity protein specifically blocks CdiA-CT_II^1026b tRNase activity in vitro

Burkholderia thailandensis tRNA was incubated with purified CdiA-CT_II^1026b and analyzed by gel electrophoresis and northern blot hybridization. Different CdiI immunity proteins were included in reactions where indicated, and CdiA-CT_II^1026b containing the Asp214Ala mutation was also assayed for tRNase activity. RNA was hybridized against oligonucleotide probes specific for B. thailandensis tRNA_ICG^Arg and tRNA_UGC^Ala. The gel migration positions of full-length tRNA are indicated.

Figure 7. Growth inhibition mediated by the CDI_II^1026b system is contact-dependent

A) Bt E264 inhibitor cells carrying the Bp CDI_II^1026b system or empty cosmid vector (mock inhibitors) were co-cultured with Bt E264 target cells as described in Experimental Procedures. Cells were collected at the indicated time points, and viable counts for both inhibitor and target cells were determined as colony forming units per mL (CFU/mL). B) A filter competition assay was carried out as described in Experimental Procedures. Inhibitor cells expressing the CDI_II^1026b system, or mock inhibitor cells lacking the CDI_II^1026b system were separated from Bt6 target cells by filters with the indicated pore sizes, and viable target cell counts were determined after 24 h incubation. CFU/mL values were determined from three technical replicates and are expressed as the mean ± SEM.

Figure 8. B. pseudomallei CDI systems are modular

Bt E264 target cells were co-cultured with inhibitor cells expressing the Bp CDI_II^1026b system or chimeric CDI systems that deploy the CdiA-CTs from Bp K96243or Bp E479. Target cells were provided with Bp cdiI immunity genes as indicated. The competitive index was calculated as the inhibitor cell CFU/target cell CFU ratio at t = 0 h divided by the inhibitor cell CFU/target cell CFU ratio at t = 24 h. Competitive indices were determined from three technical replicates and are expressed as the mean ± SEM.

Figure 9. CDI-mediated growth inhibition requires CdiB and tRNase activity

Bt24 cells (B. thailandensis E264 ΔcdiAIB) carrying a plasmid-borne copy of the CDI_II^1026b system or its mutant derivatives that either lack the cdiB gene or encode the Asp3039Ala variant of CdiA_II^1026b (corresponding to the catalytically inactive Asp214Ala change in CdiA-CT_II^1026b) were incubated with Bt6 target cells. Bt7 target cells (expressing the cdiI_II^1026b immunity gene) were used as a negative control. Growth competition analysis was carried out as described in Experimental Procedures. CFU/mL values were determined from three technical replicates and are expressed as the mean ± SEM