Small RNA CjNC110 regulates the activated methyl cycle to enable optimal chicken colonization by Campylobacter jejuni

Abstract

Post-transcriptional gene regulation by non-coding small RNAs (sRNAs) is critical for colonization and survival of enteric pathogens, including the zoonotic pathogen Campylobacter jejuni. In this study, we utilized C. jejuni IA3902 (a representative isolate of the sheep abortion clone) and C. jejuni W7 (a highly motile variant of NCTC 11168, a human gastroenteritis strain) to further investigate regulation by sRNA CjNC110. Both motility and autoagglutination ability were confirmed to be phenotypes of conserved regulation by CjNC110. However, we demonstrated that W7∆CjNC110 does not change chicken colonization levels compared to W7 wild type, directly contrasting IA3902∆CjNC110, which had decreased colonization ability. Subsequently, we determined strain-specific phenotype variation between W7∆CjNC110 and IA3902∆CjNC110 when examining intracellular L-methionine (L-met) levels controlled by the activated methyl cycle (AMC). We hypothesized that the presence of a secondary system for L-met production conferred by MetAB in W7 but not IA3902 might explain the difference in both chicken colonization and L-met availability. Insertion of metAB within IA3902∆CjNC110 (naturally absent) restored intracellular L-met levels in IA3902∆CjNC110::metAB and overcame the colonization defect that resulted from mutagenesis of CjNC110 in IA3902. Deletion of metAB in W7∆CjNC110 (naturally present) led to a decrease in L-met in W7∆CjNC110∆metAB and a colonization defect which was otherwise masked in W7∆CjNC110. Our results indicate that regulation of the AMC leading to altered L-met availability is a conserved regulatory function of CjNC110 in C. jejuni and confirm that L-met generation via the AMC as activated by CjNC110 is critical for optimal host colonization.IMPORTANCEDuring this study, the regulatory action and conservation of function of CjNC110 between two different zoonotically important Campylobacter jejuni strains were examined. Critically, this work for the first time reveals regulation of L-methionine (L-met) production within the activated methyl cycle (AMC) by small RNA (sRNA) CjNC110 as a key factor driving C. jejuni optimal chicken colonization. As a growing body of evidence suggests that maintenance of L-met homeostasis appears to be critical for C. jejuni colonization, interventions targeting the AMC could provide a critical control point for therapeutic drug options to combat this zoonotic pathogen. Our results also indicate that even for conserved sRNAs such as CjNC110, strain-specific differences in phenotypes regulated by sRNAs may exist, independent of conserved regulatory action. Depending on the strain examined and accessory genomic content present, conserved regulatory actions might be masked, thus investigation in multiple strains may be warranted.

Keywords: Campylobacter; L-methionine; activated methyl cycle; chicken colonization; small RNAs.

Fig 1

W7 CjNC110 and luxS background mutants colonize at comparable levels to W7 WT at DPI 5, 12, and 19 (mean ± SEM). The ability of W7∆CjNC110 to colonize at comparable levels to WT directly contrasts IA3902∆CjNC110, which had hindered colonization at each DPI previously (23). Each dot represents an individual bird, and each color indicates the strain utilized displayed on the right (black box). Each bar represents the average CFU per g (log₁₀), with a minimum of six birds per strain each week. One-way ANOVA demonstrated no statistical difference in colonization at each DPI (P > 0.05).

Fig 2

W7∆CjNC110 increases motility and decreases autoagglutination, matching the biological trends of IA3902 isogenic mutants (23) (mean ± SEM). Colored bars indicate the average (A) motility in millimeter or (B) autoagglutination determined by A₆₀₀ at 24 h, using a minimum of at least three technical replicates from three independent studies. An increase in A₆₀₀ correlates to decreased autoagglutination ability, and a decrease in A₆₀₀ correlates to increased autoagglutination ability. For statistical analysis, one-way or two-way ANOVA with Tukey’s multiple comparison test was performed for each assay when appropriate. Significance (P < 0.05) is denoted by “*” and black lines when comparing W7 WT to all other strains.

Fig 3

(A) W7ΔCjNC110 had comparable hydrogen peroxide (H₂O₂) sensitivity and (B) intracellular L-met levels relative to W7 WT, contrasting the increased sensitivity to H₂O₂ and decreased L-met production in IA3902ΔCjNC110 demonstrated previously (23). Strains utilized are indicated at the bottom (x-axis). Colored bars indicate the average sensitivity to (A) H₂O₂ or (B) L-met metabolite concentration of each strain tested using three technical replicates from three independent studies per assay (mean ± SEM). For statistical analysis, each assay was analyzed independently, and each strain was compared to the other. One-way ANOVA tests demonstrated no significant difference per assay (P > 0.05).

Fig 4

IA3902∆CjNC110::metAB restores L-met and SAM production in IA3902∆CjNC110, while W7∆CjNC110∆metAB reduces L-met and SAM concentration to comparable levels observed for IA3902∆CjNC110 in vitro. (A) Intracellular L-met and (B) SAM were extracted from cell cultures at the early stationary phase of growth (12 h). The strains utilized are indicated at the bottom (x-axis). Each bar represents the average intracellular metabolite concentration of each strain tested using three technical replicates from three independent studies (mean ± SEM). For statistical analysis, each strain was compared to all the other strains using one-way ANOVA with Tukey’s multiple comparison test for each metabolite. Overlap in letters above each bar indicates no significance (P >  0.05) detected.

Fig 5

The reacquired MetAB system bolsters chicken colonization levels of IA3902ΔCjNC110 to comparable levels to W7ΔCjNC110 and IA3902 WT, and mutagenesis of metAB hinders W7ΔCjNC110 colonization at DPI 12 and 19. Each dot represents an individual bird, and each color indicates the strain utilized displayed at the top (black box). Each bar represents the average CFU per g (log₁₀), with a minimum of six birds per strain each week (mean ± SEM). For statistical analysis, each strain was compared to all the other strains using one-way ANOVA with Tukey’s multiple comparison test at each DPI. Overlap in letters above each bar indicates no significance (P >  0.05) was detected.

Fig 6

A predicted model for the role of sRNAs CjNC110 (activator) and CjNC140 (repressor) in regulation of intracellular L-met levels in C. jejuni. Model is based on computationally predicted binding sites with high-binding energy scores within the 5′ UTR of the mRNA to a stem-loop region of the sRNA. IA3902 does not encode metAB (illustrated by the red box with dashed lines) and is not expected to utilize this pathway. Each box represents an enzyme, and the primary substrate metabolite for each enzyme is provided as text. Enzyme box color indicates computationally predicted direct regulatory effect by CjNC110 (top half) or CjNC140 (bottom half): (i) basal expression is indicated by gray color fill, (ii) activation by red color fill, and (iii) repression by blue color fill.