Abstract
Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors two separate T6SSs on chromosomes 1 and 2, i.e., T6SS1 (VP1386-1420) and T6SS2 (VPA1025-1046). T6SS1 contains at least 7 putative operons: VP1386-1387, VP1388-1390, VP1392-1391, VP1393-1406, VP1400-1406, VP1409-1407, and VP1410-1420. V. parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) system that are highly expressed at low cell density and high cell density, respectively. ToxR is a membrane-bound virulence regulatory protein conserved across the Vibrio family. In the present work, we show that ToxR coordinates with AphA and OpaR to repress T6SS1 expression in V. parahaemolyticus. OpaR binds to the promoters of VP1388-1390, VP1400-1406, and VP1409-1407 to repress their transcription, but it appears to negatively regulate VP1393-1406 transcription in an indirect manner. By contrast, AphA negatively regulated the above four T6SS1 operons in an indirect manner. In addition, ToxR binds to the promoters of VP1400-1406 and VP1409-1407 to inhibit their transcription, but it presents an indirect interaction with VP1388-1390 and VP1393-1406 promoters. Notably, the expression of ToxR also manifested in a QS-dependent manner and the highest expression occurred at LCD. Meanwhile, the highest expression of T6SS1 occurred at an OD600 value of 0.6 to 0.8 due to the tight regulation of ToxR and QS, suggesting T6SS1 functions only during the mid-logarithmic growth phase. These observations provide significant insight into the molecular mechanism of T6SS1 gene regulation by QS and ToxR in V. parahaemolyticus.
Highlights
Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is mostly disseminated in marine and estuarine surroundings (Broberg et al, 2011; Lovell, 2017)
When the weight score of six was taken as the cutoff value, the OpaR box-like sequences were found for VP13881390, VP1400-1406 and VP1409-1407, while the ToxR boxlike sequences were found for VP1386-1387 and VP1409-1407 (Table 2)
At low cell density (LCD) (OD600 = 0.05–0.2), AphA negatively regulated the above four T6SS1 operons in an indirect manner; while ToxR binds to the promoters of VP1400-1406 and VP14091407 to inhibit their transcription, but it presents an indirect interaction with VP1388-1390 and VP1393-1406 promoters
Summary
Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is mostly disseminated in marine and estuarine surroundings (Broberg et al, 2011; Lovell, 2017). V. parahaemolyticus strains expresses a number of different virulence factors including thermostable direct hemolysin (tdh), TDH related hemolysin (trh), two type VI secretion systems (T6SS1 and T6SS2) as well as two type III secretion systems (T3SS1 and T3SS2) (Broberg et al, 2011). These factors play important roles in the strains surviving in the environment and causing human disease. The T6SS1 would enhance environmental fitness of V. parahaemolyticus in sea water, while the T6SS2 functions when the bacterium is inside a marine animal (Salomon et al, 2013)
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