Abstract
As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.
Highlights
Cholera toxin (CT) production is induced during anaerobic respiration with trimethylamine N-oxide (TMAO) in Vibrio cholerae
We revealed that accumulation of ppGpp was induced in N16961 grown by anaerobic TMAO respiration, a condition leading to robust cholera toxin (CT) production
Identification of a V. cholerae Mutant Strain That Produced a Higher Level of CT during Anaerobic TMAO Respiration—Our previous studies demonstrated that CT production is and dramatically induced, whereas V. cholerae O1 serotype strains were grown anaerobically in the presence of TMAO [11]
Summary
Cholera toxin (CT) production is induced during anaerobic respiration with trimethylamine N-oxide (TMAO) in Vibrio cholerae. Other studies demonstrated that expression of virulence genes was substantially induced under anaerobic conditions through a transcriptional regulation that involved dimerization of AphB, a LysR-type transcriptional activator [12, 13] Together, these divergent results suggest that V. cholerae strains have. Stringent Response and CT Production in V. cholerae developed mechanisms that allow active responses to the oxygen-deprived condition, and expression of virulence-associated genes is differentially regulated in an anaerobic environment, a condition that probably mimics host intestine. This result suggests that TMAO is probably present in mammalian intestines, and V. cholerae may take advantage of its availability to support anaerobic respiratory growth in human intestine This finding led us to postulate the role of gut microbiotaderived metabolite as a signaling molecule to stimulate V. cholerae virulence. This report provides a novel insight into the signaling pathway that may contribute to the activation of CT production during adaptation to host-specific environments
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