RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae

Abstract

Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

Fig. 1.

The toxT 5′ UTR modulates temperature-dependent translation of LacZ. (A) Schematic of the predicted structure of the toxT 5′ UTR. The secondary structure was derived from Mfold (16). Shown are the relevant nucleotides within the putative fourU thermometer and the SD sequence; the complete nucleotide sequence and structure is shown in Fig. S1. The start codon and the U5 and U7 residues are indicated. (B) Effect of temperature on LacZ translation from toxT 5′ UTR. The toxT 5′UTR-lacZ (WT) construct, and the U5C, U7C, and U5C U7C (U5, 7C) mutant toxT 5′ UTR-lacZ constructs were expressed from the pBAD promoter in plasmids pBO1296, pBO1826, pBO1829, and pKEK1807, respectively. Plasmids were transformed into V. cholerae O1 strains O395 (Classical), C6706 (El Tor), and E. coli strain DH5α, and strains assayed for β-galactosidase activity during midlog growth (OD₆₀₀ of 0.4–0.5) at the temperatures indicated in the presence of 0.1% arabinose. Translation from the WT UTR was significantly greater at 37 °C than at 25 °C (P < 0.01), and translation from WT UTR was significantly greater than from U5C and U5,7C UTRs at 37 °C (P < 0.001); Student’s two-tailed t test.

Fig. 2.

The toxT 5′ UTR fourU RNA thermometer facilitates temperature-dependent access to the SD sequence. Enzymatic cleavage of 5′ ³²P-end-labeled toxT RNA was carried out at 20 °C, 30 °C, and 37 °C. RNA fragments were separated on 15% polyacrylamide. (A) RNase T1 (cuts 3′ of single-stranded guanines) was used at the indicated concentrations [0.01 U (+) and 0.1 U (++)]. (B) RNase V1 (cuts double-stranded, stacked regions) was used at 0.1 U. Lane L is the alkaline ladder, lane N contains no RNase, and RNA was cleaved with 0.1 U T1 at 42 °C in lane T. WT indicates the native toxT 5′ UTR, and U5, 7C indicates the U5C U7C toxT 5′ UTR. The SD sequence is labeled, and select nucleotides are noted.

Fig. 3.

The toxT 5′ UTR fourU RNA thermometer facilitates temperature-dependent translation of ToxT. (A) Schematic representation of the 5′-toxT UTR-toxT-FLAG transcript of pKEK1733. E. coli DH5α (E. coli), V. cholerae O1 ΔtoxT strains KKV2426 (Vc El Tor) and KKV2288 (Vc Classical) carrying plasmid pKEK1733 were grown at the indicated temperatures in the presence of 0.1% arabinose. (B) Schematic representation of the 5′-araBAD-UTR-toxT-FLAG transcript of pKEK1789. E. coli DH5α (E. coli), V. cholerae O1 strain C6706 (Vc El Tor), and V. cholerae O1 strain O395 (Vc Classical) carrying plasmid pKEK1789 (5′-araBAD-UTR) were grown at the indicated temperatures in the presence of 0.1% arabinose. Western immunoblot using anti-FLAG monoclonal antibody was performed in A and B with whole-cell lysates (OD₆₀₀ = 0.6). (C) Mutations in the fourU thermometer disrupt temperature-dependent translation of ToxT. E. coli DH5α carrying plasmids pKEK1733 (5′-toxT UTR), pKEK1734 (5′-U5C toxT UTR), pKEK1735 (5′-U7C toxT UTR), or pKEK1736 (5′-U5C U7C toxT UTR) were grown at 37 °C in the presence of 0.1% arabinose. Western immunoblot using anti-FLAG monoclonal antibody was performed with whole-cell lysates (OD₆₀₀ = 0.6).

Fig. 4.

The toxT 5′ UTR fourU RNA thermometer facilitates temperature-dependent expression of CT. V. cholerae O1 ΔtoxT strains KKV2288 (Classical) and KKV2426 (El Tor) carrying plasmids pKEK1682 (5′-toxT UTR-toxT), pKEK1683 (5′-U5C toxT UTR-toxT), pKEK1685 (5′-U7C toxT UTR- toxT), or pKEK1686 (5′-U5C U7C toxT UTR- toxT) were grown at 25 °C and 37 °C in the presence of 0.1% arabinose and harvested at identical cell densities (OD₆₀₀ = 0.6). mRNA abundance for (A) toxT and (B) ctxB were determined by quantitative RT-PCR (Materials and Methods). (C) Supernatants were assayed for CT.

Fig. 5.

The toxT 5′ UTR fourU RNA thermometer controls virulence of V. cholerae. (A) V. cholerae O1 classical strains KKV598 (WT), KKV2333 (U5C 5′toxT UTR), and KKV2368 (U7C 5′toxT UTR) were coinoculated with the wild-type strain O395 perorally into infant mice. **P < 0.01, Student t test; n.s., not significant. (B) V. cholerae O1 classical strains KKV598 (WT) and KKV2425 (U5C, U7C 5′toxT UTR), and V. cholerae O1 El Tor strain KKV2382 (U5C, U7C 5′toxT UTR) were coinoculated with the wild-type strains O395 or C6706 perorally into infant mice. No U5C U7C mutant bacteria were recovered from the intestine, detection limit (d.l.) is 5 × 10⁻⁵. ***P < 0.001, Student t test. Inocula in A and B contained a ratio of ∼1:1 mutant/wild-type; intestinal homogenates were recovered at 24 h after inoculation and cfu of wild-type and mutant strains determined. The competitive index is given as the output ratio of mutant/wild-type divided by the input ratio of mutant/wild-type; each value shown is from an individual mouse.