H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

Abstract

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity.

Fig. 1.

Deletion of hns de-represses Hcp1 expression and secretion. V. parahaemolyticus POR1-derivative deletion strains containing endogenously C-terminal myc-tagged hcp1 were grown in MLB media for 5 h at 30 °C with or without 20 µM phenamil. Expression (cells) and secretion (medium) of Hcp1–myc were detected by immunoblot using anti-myc antibodies. The loading control (LC) is shown for total protein lysate.

Fig. 2.

VP1391 and VP1407 are required for T6SS1 anti-bacterial activity. Viability counts of E. coli (prey) before (0 h) and after (4 h) co-culture. E. coli were co-cultured with V. parahaemolyticus (attacker) POR1 or the following POR1 derivative strains, POR1Δhcp1 and POR1Δvp1391, or POR1Δvp1407, carrying an empty vector or a vector encoding the arabinose-inducible expression of vp1391 (pVP1391) or the expression of vp1407 from its endogenous promoter (pVP1407). Mixed cultures at a 4 : 1 OD₆₀₀ ratio (attacker : prey) were spotted on MLB plates containing 0.1 % (w/v) arabinose and incubated at 30 °C for 4 h.

Fig. 3.

H-NS, AphA, LafK and ToxRS are not required for T6SS1 anti-bacterial activity. Viability counts of E. coli (prey) and V. parahaemolyticus deletion strains (attacker) 4 h after co-culture. Mixed cultures at a 4 : 1 OD₆₀₀ ratio (attacker : prey) were spotted on MLB plates containing 0.1 % (w/v) arabinose and incubated at 30 °C for 4 h.

Fig. 4.

H-NS does not mediate repression of Hcp1 expression at 37 °C. Expression of endogenously C-terminal myc-tagged Hcp1 in V. parahaemolyticus POR1 and the Δhns (POR1 derivative) strains harbouring an empty vector or a vector for the expression of hns driven by its native promoter (pH-NS). Cultures were grown in different media (LB or MLB) and at different temperatures (30 °C or 37 °C) and in the absence or presence of phenamil as indicated. Expression of Hcp1–myc was detected by immunoblot using anti-myc antibodies. The loading control (LC) is shown for total protein lysate.

Fig. 5.

H-NS represses T6SS1 anti-bacterial activity under low salt conditions. (a) Viability counts of E. coli (prey) before (0 h) and after (4 h) co-culture. E. coli were co-cultured with V. parahaemolyticus (attacker) POR1 or the POR1 derivative strains, Δhcp1, or Δhns. Mixed cultures at a 4 : 1 OD₆₀₀ ratio (attacker : prey) were spotted on LB or MLB plates and incubated at 30 °C or 37 °C for 4 h. (b) Viability counts of E. coli (prey) 4 h after co-culture. E. coli were co-cultured with V. parahaemolyticus (attacker) POR1 or the POR1 derivative strains, Δhcp1, or Δhns, harbouring an empty vector or a vector for the expression of hns driven by its native promoter (pH-NS). Mixed cultures at a 4 : 1 OD₆₀₀ ratio (attacker : prey) were spotted on LB or MLB plates and incubated at 30 °C for 4 h.