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. 2020 May 26;117(21):11715-11726.
doi: 10.1073/pnas.1922719117. Epub 2020 May 12.

Campylobacter jejuni BumSR directs a response to butyrate via sensor phosphatase activity to impact transcription and colonization

Kyle N Goodman  1 , Matthew J Powers  2 , Alexander A Crofts  2   3 , M Stephen Trent  2   4   5 , David R Hendrixson  6
Affiliations

Campylobacter jejuni BumSR directs a response to butyrate via sensor phosphatase activity to impact transcription and colonization

Kyle N Goodman et al. Proc Natl Acad Sci U S A. .

Abstract

Campylobacter jejuni monitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered that C. jejuni has the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified the C. jejuni butyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital for C. jejuni colonization of multiple hosts.

Keywords: BumS; Campylobacter jejuni; butyrate; short-chain fatty acids; two-component signal transduction system.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
The BumSR TCS is required for butyrate-modulated expression of peb3. (A) Expression of the peb3::astA transcriptional reporter in WT C. jejuni ΔastA and isogenic bumS and bumR mutants. The bumS and bumR mutants examined include a representative bumS::Tn and bumR::Tn mutant isolated in our genetic screen to identify Tn mutants defective for butyrate-dependent repression of peb3::astA expression and previously constructed C. jejuni ΔbumS and ΔbumR mutants with in-frame deletion of the coding sequence of each gene. C. jejuni strains were grown for 16 h in CDM alone (solid blue bars) or CDM supplemented with 12.5 mM butyrate (hatched blue bars) as indicated. The level of peb3::astA expression in each mutant is relative to the level of expression in WT C. jejuni grown without butyrate, which was set to 100 units. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate SDs of the average level of expression from three samples. Statistical significance was calculated in GraphPad Prism by ANOVA with Tukey’s test: * indicates the mutant grown in CDM alone had significantly increased or decreased expression relative to the WT strain grown in CDM alone; ** indicates the mutant grown with butyrate had a significantly increased or decreased expression relative to the WT strain grown with butyrate; *** indicates a strain showed a significantly different level of expression when grown in the presence of butyrate compared to growth in CDM alone (P < 0.05). (B) Production of BumR in WT and isogenic bumS and bumR mutants. Immunoblot analysis of the level of BumR in whole-cell lysates of WT and mutant strains after growth in CDM alone or CDM supplemented with 12.5 mM butyrate. RpoA served as a control for protein loading. Immunoblots were performed in triplicate, and values underneath the immunoblots represent quantified BumR mean signals relative to RpoA and normalized to WT, which was set to 100.
Fig. 2.
Fig. 2.
Effect of BumR receiver domain mutations on butyrate-modulated transcription. (A) Production of BumR in WT C. jejuni and bumR mutants. Immunoblot analysis of the level of BumR in whole-cell lysates of WT and mutant strains after growth in Mueller–Hinton media. RpoA served as a control for protein loading. (B) qRT-PCR analysis of peb3 transcription in WT C. jejuni and bumR mutants grown in CDM alone or CDM supplemented with 12.5 mM butyrate. The expression of peb3 in CDM alone (solid blue bars) and CDM with butyrate (hatched blue bars) in WT C. jejuni and mutant strains are shown. The level of peb3 transcription in WT grown in CDM alone as measured by qRT-PCR was set to 1. Expression of peb3 in WT grown in CDM with butyrate or mutants grown with or without butyrate is shown relative to the WT strain grown in CDM alone. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate SDs of the average level of expression from three samples. Statistical significance of ΔCT values relative to secD reference gene was calculated in GraphPad Prism by ANOVA with Tukey’s test: * indicates the mutant grown in CDM alone had significantly increased or decreased expression relative to the WT strain grown in CDM alone; ** indicates the mutant grown with butyrate had a significantly increased or decreased expression relative to the WT strain grown with butyrate; *** indicates a strain showed a significantly different level of expression when grown in the presence of butyrate compared to growth in CDM alone (P < 0.05).
Fig. 3.
Fig. 3.
Colonization dynamics of WT C. jejuni and isogenic ΔbumS and ΔbumR mutants over time in the avian intestinal tract. Day of hatch chicks were orally infected with ∼100 colony-forming units (CFU) of WT C. jejuni (blue circles), isogenic ΔbumS (red triangles), and ΔbumR (gray diamonds) mutants. Chicks were killed at days 1, 4, 7, or 14 postinfection and the levels of each C. jejuni strain in A the proximal small intestine, (B) distal small intestines, (C) ceca, and (D) large intestines was determined (reported as CFU per gram of content). Each closed symbol represents the level of C. jejuni in a single chick. Open symbols represent chicks with C. jejuni levels below the limit of detection (<100 CFU per gram of content). Horizontal bars represent geometric mean for each group. Statistical analysis was performed using the Mann–Whitney U test (*P < 0.05).
Fig. 4.
Fig. 4.
Analysis of C. jejuni butyrate-modulated genes and the BumSR regulon. (A) qRT-PCR analysis of transcription of genes initially identified by RNAseq analysis of WT C. jejuni to be modulated in expression by 12.5 mM butyrate during growth in CDM. The expression of each gene in WT C. jejuni grown in CDM alone (solid blue bars) as measured by qRT-PCR was set to 1. Expression of each gene in WT C. jejuni grown in CDM supplemented with 12.5 mM butyrate (hatched blue bars) is shown relative to WT grown in CDM alone. (B) qRT-PCR analysis of transcription of genes initially identified by RNAseq analysis to be altered in C. jejuni ΔbumS and/or ΔbumR relative to WT C. jejuni. The expression of each gene in WT C. jejuni grown in CDM alone (solid blue bars) as measured by qRT-PCR was set to 1. Expression of each gene in ΔbumS (red bars) and ΔbumR (gray bars) is shown relative to WT. Dotted lines indicates divisions between gene sets (classes I to III) show similar effects in expression due to deletion of bumS and/or bumR. (C) Analysis of the ability of BumSR TCS regulon members to be modulated by butyrate for transcription. Select genes that were altered in expression in C. jejuni ΔbumS or ΔbumR were analyzed for butyrate-modulated expression in WT C. jejuni grown in CDM alone (solid blue bars) and in CDM supplemented with 12.5 mM butyrate (hatched blue bars) by qRT-PCR. The expression of each gene in WT C. jejuni grown in CDM alone was set to 1. Expression of each gene in WT C. jejuni grown in CDM supplemented with 12.5 mM butyrate is shown relative to WT grown in CDM alone. For (A–C), results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate SDs of the average level of expression from three samples. Statistically significant differences in gene expression between WT C. jejuni grown with or without butyrate is indicated by * in A and C (P < 0.05). For (B), different colored asterisks indicate statistically significant differences (P < 0.05) in gene expression between: WT C. jejuni and both ΔbumS and ΔbumR, black *; WT C. jejuni and ΔbumS only, red *; and WT C. jejuni and ΔbumR only, gray * (P < 0.05). Statistical analysis of ΔCT values relative to secD reference gene was calculated by the Student’s t test.
Fig. 5.
Fig. 5.
The BumSR TCS is not required for acetate- or lactate-modulated gene expression. (A) qRT-PCR analysis of transcription of peb3 and Cjj0438 in WT C. jejuni and ΔbumS or ΔbumR mutants grown in CDM alone or CDM supplemented with 100 mM acetate. The expression of peb3 and Cjj0438 in WT C. jejuni grown in CDM alone (sold blue bars) as measured by qRT-PCR was set to 1. Expression of genes in WT grown in CDM supplemented with acetate (hatched blue bars), ΔbumS without (solid red bars), or with acetate (hatched red bars), or ΔbumR without (solid gray bars) or with acetate (hatched gray bars) is shown relative to WT grown in CDM alone. (B) qRT-PCR analysis of transcription of Cjj0683 and ggt in WT C. jejuni and ΔbumS or ΔbumR mutants grown in CDM alone or CDM supplemented with 25 mM l-lactate. The expression of Cjj0683 and ggt in WT C. jejuni grown in CDM alone (sold blue bars) as measured by qRT-PCR was set to 1. Expression of genes in WT grown in CDM supplemented with l-lactate (hatched blue bars), ΔbumS without (solid red bars) or with l-lactate (hatched red bars), or ΔbumR without (solid gray bars) or with l-lactate (hatched gray bars) is shown relative to the WT strain grown in CDM alone. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate SDs of the average level of expression from three samples. Statistical significance of ΔCT values relative to recA reference gene (for acetate analysis in A) or secD reference gene (for lactate analysis in B) was calculated in GraphPad Prism by ANOVA with Sidak’s test: * indicates statistically significant differences in gene expression between a strain grown in CDM alone and in CDM supplemented with acetate or lactate (P < 0.05).
Fig. 6.
Fig. 6.
BumS phosphatase activity is specific for phospho-BumR. (A) Dephosphorylation of phospho-BumR by BumS. After autophosphorylation of BumR by Ac[32P], increasing concentrations of BumS were added as indicated by BumS:BumR molar ratios and then incubated for 10 min. (B) Dephosphorylation of phospho-BumR by BumS over time. After autophosphorylation of BumR by Ac[32P], mock treated or BumS at a 1:2 BumS:BumR molar ratio was added and incubated for up to 60 min. Reactions were stopped at various times indicated. (C) Dephosphorylation of phospho-FlgRΔCTD by BumS. After autophosphorylation of FlgRΔCTD by Ac[32P], BumS was added at 1:2 or 1:1 BumS:FlgRΔCTD molar ratio for 20 min. (D) Comparison of dephosphorylation of phospho-BumR by BumS and FlgS. After autophosphorylation of BumR by Ac[32P], BumS or FlgS were added at 1:2 molar ratio to BumR and the reactions were incubated for 10 min.
Fig. 7.
Fig. 7.
BumS phosphatase activity is dependent on specific H box residues. (A and B) In vitro phosphatase activities WT BumS, BumSQW196-7EL (an H box mutant), and BumSA338G (a D box mutant) over time. After autophosphorylation of BumR by Ac[32P], BumS proteins were added at 1:2 BumS:BumR molar ratios for up to 60 min. Reactions were stopped at various times indicated. (A) Representative phosphatase assay of WT BumS or BumS mutants for phospho-BumR. (B) Quantification of three independent phosphatase assays for WT BumS, BumSQW196-7EL, and BumSA338G. The level of phospho-BumR in each assay was normalized to phospho-BumR at 1 min of mock sample, which was set to 100%. No appreciable decrease in phospho-BumR in the absence of BumS was observed for the duration of the experiment (up to 60 min) as observed in A. Error bars indicate SDs of the relative level of phospho-BumR at indicated time after addition of BumS from three independent experiments. Statistical significance to compare WT to mutant proteins was calculated in GraphPad Prism by ANOVA with Dunnett’s test (*P < 0.05).
Fig. 8.
Fig. 8.
Electrophoretic mobility shift assays for analysis of DNA-binding activity of BumR. (A) Comparison of BumR binding to target promoter with or without phosphorylation. Recombinant BumR alone or after autophosphorylation with Li-AcP was added to peb3, Cjj0580, cetA, or recA promoter DNAs at concentrations ranging from 0 to 1.5 μM. (B) Binding of 1.5 μm BumR after autophosphorylation with Li-AcP to radiolabeled promoter DNAs for peb3, Cjj0580, or cetA in the presence of increasing concentrations of the same unlabeled specific competitor DNA or unlabeled noncompetitor DNA (recA promoter).
Fig. 9.
Fig. 9.
Model for butyrate-modulated transcription of C. jejuni genes via the BumSR TCS. BumR is hypothesized to be endogenously phosphorylated by other kinases or high-energy phosphodonors in the cytoplasm of C. jejuni. BumS is proposed to primarily function as a phosphatase to control the level of phosphorylation of BumR and its activity as a transcriptional regulator. BumR shows increased affinity for target promoter DNA in its phosphorylated form and can function as a repressor or activator depending on the promoter. Exogenous butyrate produced by the intestinal microbiota is either sensed directly by BumS after transport to the cytoplasm, sensed indirectly by its conversion to another metabolite that is sensed by BumS, or sensed indirectly by its influence on another pathway that alters the level of a factor directly sensed by BumS. In any case, increased levels of exogenous butyrate are expected to have a negative influence on BumS phosphatase activity, which results in increased phospho-BumR levels to influence transcriptional outcomes.
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