CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans.

Fig. 1.

(A to E) csrAregulates tryptophanase activity to promote toxin-mediated killing of C. elegansby EPEC. (A and B) Young adult worms were exposed to confluent lawns of EPEC, csrAmutant, csrAmutant complemented with a functional csrAallele expressed from a multicopy plasmid [csrA(pCRA16)], and tnaAmutant and monitored for paralysis (A) and killing (B) on LB agar supplemented with tryptophan (LBW). Worms were considered paralyzed if they failed to traverse an entire body length on prodding. Worm mortality was assayed by transferring the pathogen-exposed worms onto NGM plates containing nonpathogenic E. coliOP50 and assaying for motility 24 h later. Error bars indicate standard deviations of results from at least three independent experiments, with each employing at least two biological replicates. A one-way analysis of variance (ANOVA) was used to assess statistical significance. A Pvalue cutoff of <0.05 was considered statistically significant. The calculated Pvalues for both the paralysis and killing assays were <0.02. (C and D) Paralysis (C) and killing (D) of the nematodes were assayed in the presence of EPEC, csrAmutant, csrAmutant overexpressing tnaA[csrA(pMD6)], tnaAmutant, and tnaAmutant overexpressing csrA[tnaA(pCRA16)] essentially as described above. A one-way ANOVA was used to assess statistical significance. The calculated Pvalues for both the paralysis and killing assays were <0.02. (E) Tryptophanase activity was measured from lysates of bacteria cultivated on agar plates. The rate of hydrolysis of SOPC, a chromogenic tryptophan analogue, to ONTP was measured as described previously (2). Error bars indicate standard deviations of results from at least two independent experiments, each with at least three replicates. The unpaired Student ttest was employed to assay for statistical significance between the indicated samples. A Pvalue cutoff of <0.01 was considered statistically significant. **, P< 0.01.

Fig. 2.

(A to D) TnaB but not AroP or Mtr imports tryptophan into EPEC to stimulate toxin-dependent killing of C. elegans. (A) Comparative analysis of the primary structure of the boxA-rutriboelement and the TnaC leader peptide of the tnaCABoperon in E. coliK-12 and EPEC/EHEC. The translation termination codon of tnaCand the translation initiation codon of tnaAare indicated by dashed underlines. The intersite distance between the terminal nucleotide of rutand the translational initiation nucleotide of tnaAis 197 bases. (B and C) Young adult worms were exposed to EPEC and its congenic mutant derivatives, the tnaB, mtr, and aroPmutants and the tnaBcomplemented strain [tnaB(ptnaB)], and assayed for paralysis (B) and killing (C). Error bars indicate the standard deviations of results from at least three independent experiments, with each using at least two replicates. A one-way ANOVA was used to assess statistical significance. A Pvalue cutoff of <0.05 was considered statistically significant. The calculated Pvalues for both the paralysis and killing assays were <0.02. (D) Specific tryptophanase activity was assayed in the tnaBmutants as described above. The unpaired Student ttest was employed to assay for statistical significance between the indicated samples. A Pvalue cutoff of <0.01 was considered statistically significant. **, P< 0.01; ns, no statistically significant difference.

Fig. 3.

Model for the role of CsrA and TnaB in the regulation of tnaAand toxin production. CsrA positively regulates tryptophanase activity independently of the tryptophan permease TnaB. TnaB is the primary importer of tryptophan when EPEC is cultivated on LB medium. Imported tryptophan stimulates the expression of tnaA, as is evident by elevated tryptophanase activity. In turn, tryptophanase catabolizes tryptophan to synthesize exotoxins, which paralyze and kill C. elegans. csrAand tnaAalso regulate the LEEin A/E pathogens. Activating and repressive circuits are depicted as thin lines with arrowheads and blunt ends, respectively. Arrows with dashed lines represent the LEE. Curved arrows indicate catalytic reactions.