RNA-based thermoregulation of a Campylobacter jejuni zinc resistance determinant

Abstract

RNA thermometers (RNATs) trigger bacterial virulence factor expression in response to the temperature shift on entering a warm-blooded host. At lower temperatures these secondary structures sequester ribosome-binding sites (RBSs) to prevent translation initiation, whereas at elevated temperatures they "melt" allowing translation. Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide yet little is known about how it interacts with the host including host induced gene regulation. Here we demonstrate that an RNAT regulates a C. jejuni gene, Cj1163c or czcD, encoding a member of the Cation Diffusion Facilitator family. The czcD upstream untranslated region contains a predicted stem loop within the mRNA that sequesters the RBS to inhibit translation at temperatures below 37°C. Mutations that disrupt or enhance predicted secondary structure have significant and predictable effects on temperature regulation. We also show that in an RNAT independent manner, CzcD expression is induced by Zn(II). Mutants lacking czcD are hypersensitive to Zn(II) and also over-accumulate Zn(II) relative to wild-type, all consistent with CzcD functioning as a Zn(II) exporter. Importantly, we demonstrate that C. jejuni Zn(II)-tolerance at 32°C, a temperature at which the RNAT limits CzcD production, is increased by RNAT disruption. Finally we show that czcD inactivation attenuates larval killing in a Galleria infection model and that at 32°C disrupting RNAT secondary structure to allow CzcD production can enhance killing. We hypothesise that CzcD regulation by metals and temperature provides a mechanism for C. jejuni to overcome innate immune system-mediated Zn(II) toxicity in warm-blooded animal hosts.

Fig 1. The Cj1164c/Cj1163c intergenic region mediates temperature dependent regulation of a downstream gene in E. coli.

a. Diagrammatic representation of the C. jejuni NCTC 11168 operon containing the Cj1163c/czcD gene with the shaded arrowhead indicating the associated σ⁷⁰ promoter identified by RNAseq analyses [36,37]. Located between Cj1164c and czcD is a 75 bp untranslated intergenic region (SLczcD) that was cloned between reporter genes malE (red) encoding maltose binding protein and gfp (green) on plasmid pMSLG downstream of the inducible tac promoter P_tac. b. Predicted SLczcD secondary structure from Mfold analysis with the czcD start codon underlined and ribosome binding site (RBS) indicated. Site directed mutations predicted to enhance (A41G and A36U) or disrupt (CC29,30GG and CC29,30UU) secondary structure are indicated, whilst bases in bold indicate the region removed in the pMSLG^ΔSL construct. Boxed base pairs indicate where disrupting and subsequent compensatory mutations were introduced as indicated. c. E. coli pMSLG-containing cells were grown at 25, 30 or 37°C and standardised whole cell lysates western blotted and probed with anti-MBP (red) and anti-GFP (green) antisera. All GFP signals were standardised to the corresponding internal loading control MBP signal and normalised to the 25°C value. Relative GFP quantifications are provided below the blots. These data are representative of at least three replicate experiments. d. Similar analyses as (c) with extracts from strains containing variants of pMSLG. The pMSLG^ΔSL plasmid had the bases in bold in (b) removed. The CC29,30GG; CC29,30UU; A41G and A36U derivatives are as indicated in (b). All GFP signals were standardised to the corresponding internal loading control MBP signal and normalised to the pMSLG value. Relative GFP quantifications are provided below the blots. These data are representative of at least three replicate experiments. e. Similar analyses as (c) with compensatory mutants (boxed in b) in the predicted stem region of SLczcD on pMSLG. The SLczcD G59:C38 predicted base pair was disrupted by introducing a G59C mutation and base pairing restored with a C38G mutation. In a similar manner the U34:A63 predicted base pair was disrupted with a U34A mutation and restored with an A63U mutation. All GFP signals were standardised to the corresponding internal loading control MBP signal and normalised to the pMSLG value. Relative GFP quantifications are provided below the blots. These data are representative of at least three replicate experiments.

Fig 2. The SLczcD mediates temperature-dependent post transcriptional regulation of CzcD in C. jejuni.

a. Western blot analysis of the C. jejuni NCTC11168 CjCzcDhis strain and derivatives with deletion of the SLczcD (CjΔSL) and the derepressing mutation CC29,30GG (CjCC29,30GG). Standardised whole cell lysates were probed with anti-RecA (green) as a loading control and anti-histidine (red) to detect CzcD. Cultures were grown for 24 hours in MH broth at the temperatures indicated. All CzcD signals were standardised to the corresponding internal RecA loading control signal and normalised. Relative CzcD quantifications are provided below the blots normalised to the corresponding 32°C CzcD value for each of the three strains to allow effect of temperature to be quantified (upper row) or to the CjCzcDhis value at each temperature allowing comparison of CjΔSL and CjCC29,30GG strains with CjCzcDhis (lower row). These data are representative of at least three replicate experiments. b. RT-qPCR analysis of czcD expression levels for strain CjCzcDhis grown for 24 hours in MH broth at the temperatures indicated. The CT values were normalised against the data at 32°C. Data are the mean of three biological replicates with standard error indicated. Comparison of ΔΔCt mean values by t test gave non-significant p values of 0.90 (37 vs 32°C) and 0.78 (42 vs 32°C).

Fig 3. CzcD is a Zn(II) exporter and Zn(II)/Co(II) resistance determinant.

a. C. jejuni NCTC 11168 (circles), CjczcD^- (squares) and the genetically complemented CjczcD⁺ strain (triangles) were grown for 24 hours at 37°C in MH broth from a starting OD₆₀₀ of 0.05 with varying concentrations of metals and the OD₆₀₀ measured. For the zinc experiment, data for all three strains are presented whilst for other metals only the wild type and CjczcD^- strain are included. Data points are the mean of three biological repeats, each with three technical repeats and error bars represent standard error of the biological repeats. b. Cellular Zn(II) content of C. jejuni NCTC 11168 (wt) and the CjczcD^- strain was measured by ICP-MS. Cells were grown for 24 hours in MH broth with and without 25 μM Zn(II) supplementation as indicated and the Zn(II) content of mid logarithmic phase cells measured. Data are the mean of three biological replicates with standard error of the mean indicated. The * symbol indicates a p value of <0.05 from a two way ANOVA test.

Fig 4. Zn(II) induction of CzcD is SLczcD independent.

(a) The C. jejuni NCTC 11168 czcDhis strain (CjCzcDhis) and the derivative lacking SLczcD (CjΔSL) were grown from an initial OD₆₀₀ of 0.05 for 24 hours at 37°C in MH broth with Zn(II), Co(II) and copper added at the indicated concentrations. Cells were harvested and following SDS-PAGE of soluble lysates, western blots were probed with anti-GroEL (green) as a loading control and anti-histidine to detect CzcD (red). Relative CzcD signal values are presented below the blots and were standardised to the GroEL internal control signal and then normalised to the no added metal condition. These data are representative of at least three replicate experiments. (b) qRT-PCR of the czcD transcript for the wild type strain grown with and without added Zn(II) as indicated. The data were obtained from three biological replicates and error bars represent the calculated standard error of the RQ values of the biological repeats. A one sample t-test applied to the mean of ΔΔCt values gave a significant p value of <0.05.

Fig 5. The SLczcD influences C. jejuni temperature dependent Zn(II) sensitivity.

Following eight hours of growth in MEM-α containing varying Zn(II) concentrations and at three incubation temperatures, OD₆₀₀ values of C. jejuni NCTC 11168 (a), a derivative lacking SLczcD (b) and the czcD^- mutant strain (c) were measured. All readings were normalised to the OD₆₀₀ value obtained from media lacking Zn(II) and expressed as a percentage. Each data point is the average of three biological replicates each performed in technical triplicate with error bars indicating the standard error of the biological repeats.

Fig 6. CzcD and associated temperature regulation are involved in Galleria mellonella larval killing.

a. Strains of C. jejuni were grown in MH broth, washed twice in PBS and ~10⁶ CFU injected into G. mellonella larvae that were incubated at 37°C and scored daily for survival. Strains were wild-type NCTC 11168, czcD insertionally inactivated mutant (CjczcD^-), the derived genetically complemented strain (CjczcD⁺), the derivative lacking SLczcD (Cj^wtΔSL) or with the derepressing mutation CC29,30GG (CjCC29,30GG). Data represent five biological replicates for all strains except CjczcD⁺ with four biological replicates. Each biological replicate consisted of injecting ten larvae (i.e. n = 50 or 40). Pairwise p values were calculated using a Log-rank (Mantel-Cox) test implemented in GraphPad Prism. Data for the CjczcD^- strain was statistically significantly different when compared to all four other strains with p values of <0.0001, <0.0001, 0.0001 and 0.0004 for the wild-type, CjczcD⁺, CjCC29,30GG and CjΔSL comparisons, respectively. No other pairwise comparisons were statistically significant. b. Similar experiment as for (a) except C. jejuni strains were grown and larvae incubated at 32°C, and larvae were injected with ~10⁷ CFU. Data represent three biological replicates with ten larvae per experiment per strain (n = 30). P values were calculated as in (a). The results obtained for pairwise comparison of the wild-type with CjCC29,30GG and CjΔSL strains were statistically significant with p values of 0.0009 and <0.0001, respectively. There was no statistically significant difference between the CjCC29,30GG and CjΔSL strains.