H-NS is a repressor of major virulence gene loci in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a leading cause of seafood-associated diarrhea and gastroenteritis, harbors three major virulence gene loci T3SS1, Vp-PAI (T3SS1+tdh2) and T6SS2. As showing in this study, the nucleoid-associated DNA-binding regulator H-NS binds to multiple promoter-proximal regions in each of the above three loci to repress their transcription, and moreover H-NS inhibits the cytotoxicitiy, enterotoxicity, hemolytic activity, and mouse lethality of V. parahaemolyticus. H-NS appears to act as a major repressor of the virulence of this pathogen. Date presented here would promote us to gain a deeper understanding of H-NS-mediated silencing of horizontally acquired virulence loci in V. parahaemolyticus.

Transcription of exsD is repressed directly by H-NS in Vibrio parahaemolyticus

“…It has been previously shown that V. parahaemolyticus H-NS acts as a transcriptional repressor of the T3SS1 genes, exsA and exsB (VP1700), through direct protein-DNA association [13]. This study demonstrates that there is a similar mechanism for the repression of exsD by H-NS in V. parahaemolyticus.…”

“…The wild-type (WT) V. parahaemolyticus strain RIMD 2210633 and its hns nonpolar mutant (Dhns) have been described previously [13]. Briefly, the 540 and 618 bp DNA regions upstream and downstream of hns were amplified by PCR, purified, and used as templates to create an 1158 bp deletion construct that was subsequently inserted between the Pst I and Sph I sites of pDS132.…”

“…For complementation of the Dhns [13], a PCR-generated DNA fragment containing the hns coding region together with an upstream synthetic ribosome binding site (RBS) was cloned between the XbaI and HindIII sites of the pBAD33 vector harboring an arabinose P BAD promoter and a chloramphenicol resistance gene. The recombinant plasmid pBAD33-hns was verified by DNA sequencing, and subsequently transformed into Dhns, yielding the complemented mutant strain Dhns/pBAD33-hns.…”

“…For EMSA [13], the 5 0 ends of this DNA fragment were labeled using [g-32 P] ATP and T4 polynucleotide kinase. DNA binding was performed in a 10 ml reaction volume containing binding buffer [1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5) and 10 mg/ml salmon sperm DNA], radio-labeled DNA, and increasing amounts of His-H-NS.…”

“…V. parahaemolyticus strains transformed with recombinant or empty pHRP309 plasmids were grown as described previously. b-galactosidase activity was measured using the b-Galactosidase Enzyme Assay System (Promega) [13].…”