Abstract
Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio strains that harbor a pVA1-like plasmid containing the pirA and pirB genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the pirA/pirB genes have not previously been investigated. In this study, we show that in the AHPND-causing Vibrio parahaemolyticus 3HP strain, the pirAvp and pirBvp genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirAvp and PirBvp proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, luxO and/or opaR (luxOD47E, ΔopaR, ΔluxO, and ΔopaRΔluxO), our results suggested that expression of the pirAvp and pirBvp genes was related to the QS system, with luxO acting as a negative regulator of pirAvp and pirBvp without any mediation by opaRvp. In the promoter region of the pirAvp/pirBvp operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that aphBvp was negatively controlled by LuxOvp, and that its expression paralleled the expression patterns of pirAvp and pirBvp. An electrophoretic mobility shift assay (EMSA) showed that AphBvp could bind to this predicted region, even though another QS transcriptional regulator, AphAvp, could not. Taken together, these findings suggest that the QS system may regulate pirAvp/pirBvp expression through AphBvp.
Highlights
Vibrio parahaemolyticus is an opportunistic marine pathogen often found in the ocean and estuary environment [1]
Several strains of V. parahaemolyticus were identified as causative agents of the newly emergent acute hepatopancreatic necrosis disease (AHPND) in shrimp [7]
AHPND-causing strains harbor a 70-kbp plasmid that produces the “Photorhabdus insect-related” (Pir)-like binary toxins, PirAvp and PirBvp [12]. These two AHPND-associated toxins form a complex that is structurally homologous to the insecticidal Cry toxin [12–14], and they have been confirmed as the key factors that drive AHPND pathogenesis [12]
Summary
Vibrio parahaemolyticus is an opportunistic marine pathogen often found in the ocean and estuary environment [1]. AHPND-causing strains harbor a 70-kbp plasmid (pVA1) that produces the “Photorhabdus insect-related” (Pir)-like binary toxins, PirAvp and PirBvp [12]. These two AHPND-associated toxins form a complex that is structurally homologous to the insecticidal Cry toxin [12–14], and they have been confirmed as the key factors that drive AHPND pathogenesis [12]. We show how LuxOvp, which is an important regulator of QS in Vibrio spp., affects the gene expression of the key AHPND pathogenic factors pirAvp and pirBvp. First, by monitoring the gene/protein expression of PirAvp and PirBvp during the growth of V. parahaemolyticus, we found the appearance of these two toxins is cell density-dependent. This is the first report to investigate the influence of the bacterial physiological system oTnhitshies tphireAfivrp/sptireBpvporgtetnoeisn.vOesutirgaftiendthineginsflwueilnl cbeeofhtehlepfbualctfeorrialthpehydseiovleolgoipcmalesnytsteomf AoPnHthNeDpiprArevvpe/nptiiroBnvpagnedn/oers.cOonutrrofilnsdtrinatgesgwiesillinbethheefluptfurlef.or the development of APHND prevention and/or control strategies in the future
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