The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is a marine bacterium that is globally recognized as the leading cause of seafood-borne gastroenteritis. V. parahaemolyticus uses various toxins and two type 3 secretion systems (T3SS-1 and T3SS-2) to subvert host cells during infection. We previously determined that V. parahaemolyticus T3SS-1 activity is upregulated by increasing the expression level of the master regulator ExsA under specific growth conditions. In this study, we set out to identify V. parahaemolyticus genes responsible for linking environmental and growth signals to exsA gene expression. Using transposon mutagenesis in combination with a sensitive and quantitative luminescence screen, we identify HlyU and H-NS as two antagonistic regulatory proteins controlling the expression of exsA and, hence, T3SS-1 in V. parahaemolyticus Disruption of hns leads to constitutive unregulated exsA gene expression, consistent with its known role in repressing exsA transcription. In contrast, genetic disruption of hlyU completely abrogated exsA expression and T3SS-1 activity. A V. parahaemolyticushlyU null mutant was significantly deficient for T3SS-1-mediated host cell death during in vitro infection. DNA footprinting studies with purified HlyU revealed a 56-bp protected DNA region within the exsA promoter that contains an inverted repeat sequence. Genetic evidence suggests that HlyU acts as a derepressor, likely by displacing H-NS from the exsA promoter, leading to exsA gene expression and appropriately regulated T3SS-1 activity. Overall, the data implicate HlyU as a critical positive regulator of V. parahaemolyticus T3SS-1-mediated pathogenesis.IMPORTANCE Many Vibrio species are zoonotic pathogens, infecting both animals and humans, resulting in significant morbidity and, in extreme cases, mortality. While many Vibrio species virulence genes are known, their associated regulation is often modestly understood. We set out to identify genetic factors of V. parahaemolyticus that are involved in activating exsA gene expression, a process linked to a type III secretion system involved in host cytotoxicity. We discover that V. parahaemolyticus employs a genetic regulatory switch involving H-NS and HlyU to control exsA promoter activity. While HlyU is a well-known positive regulator of Vibrio species virulence genes, this is the first report linking it to a transcriptional master regulator and type III secretion system paradigm.

Keywords: HlyU; Vibrio; enteric pathogens; gene regulation; transposons; type III secretion.

FIG 1

Integration of an exsA promoter-luxCDABE fusion into the tdhS locus (tdhS::exsA-lux) results in bioluminescence and a normal T3SS-1 secreted protein profile. (A) Schematic representation of the constructed exsA promoter-luxCDABE fusion inserted within the tdhS chromosomal locus. (B) Comparison of bioluminescence emitted by the indicated V. parahaemolyticus strains. Bacteria were cultured under T3SS-1-inducing conditions, sampled after 3 h of growth, and then assessed for light emission. cps/OD, counts per second divided by optical density (600 nm) of the cell suspension at time of collection. (C) Total secreted protein profiles of V. parahaemolyticus strains as determined by SDS-PAGE. Bacteria were grown in LB medium or LB supplemented with MgSO₄ and EGTA (induced [Ind.] for T3SS-1 activity). Labeled protein species have been previously identified by mass spectrometry analyses in wild-type V. parahaemolyticus (35). M, protein standard.

FIG 2

Bioluminescence scatterplot of Vp-lux Tn5 insertional mutants. Transposon insertion mutants were assayed for bioluminescence emission. Each dot represents an individual mutant, and two representative controls (Vp-lux strain) are indicated as red asterisks. The vertical and horizontal bars represent boundaries used to categorize mutants based on light emission (exsA promoter activity) and cell growth, respectively. The box encompasses high-light-emitting mutants selected for further study. CPS, counts per second.

FIG 3

Schematic diagram of transposon insertion sites within selected genes of interest identified by luminescence screening of the Vp-lux Tn5 mutant library. The triangles represent approximate transposon insertion sites with the approximate DNA base location in the V. parahaemolyticus genome (NBCI accession numbers NC_004603 and NC_004605; I, chromosome I).

FIG 4

Protein secretion and exsA promoter activity assays with the Vp-lux (tdhS::exsA-lux) strain and specific transposon insertion mutants. (A) Total secreted proteins were collected from culture supernatants of the indicated Tn5 insertion mutants and subjected to SDS-PAGE, followed by Coomassie blue staining. A protein ladder was included (M), and previously identified proteins are labeled. (B) Luciferase assays were conducted with the indicated Tn5 insertion mutants and the hns::Tn5 ΔhlyU double mutant (strain Vp-EL) (n = 2). The error bars indicate standard deviations from the mean values. A two-tailed t test was conducted to compare data sets. *, P < 0.05; **, P < 0.005. (C) Complementation of the hlyU::Tn5 mutant with pVSV105 encoding hlyU was performed, followed by a luciferase assay. A two-tailed t test was used to compare data sets. **, P < 0.005; ****, P < 0.0001.

FIG 5

V. parahaemolyticus ΔhlyU mutant (strain hlyU1) is deficient for secretion of T3SS-1 proteins and exhibits reduced host cell cytotoxicity during infection. (A) Total secreted proteins were collected and subjected to SDS-PAGE, followed by Coomassie staining. A protein standard was included (M), and previously identified protein species are labeled. (B) Percent cytotoxicity was calculated after infection of HeLa cells with various strains by measuring released lactate dehydrogenase levels. All strains contained pVSV105 or phlyU-FLAG as indicated. Statistical bars indicate standard deviations from the means, and statistical significance was determined by a paired two-tailed t test compared to the V. parahaemolyticus control. ***, P < 0.001. ns, not significant.

FIG 6

Electrophoresis mobility shift assay (EMSA) and DNA footprinting assays indicate HlyU binding to a region within the exsA promoter. (A) Six percent TBE-polyacrylamide gels were loaded with reaction mixtures containing exsA promoter DNA and increasing amounts of purified HlyU-His or bovine serum albumin (BSA). An exsA promoter DNA-only control was included as indicated. The white box indicates slow-migrating, concentrated DNA species. (B) A 6% TBE-polyacrylamide gel was loaded with reaction mixtures containing nleH1 DNA and increasing amounts of purified HlyU-His. Following electrophoresis, gels were stained with SYBR green to specifically stain DNA. (C) A DNase I footprinting assay using purified HlyU-His was used to identify a DNA binding region for HlyU-His within the exsA promoter. The approximate base pair numbers of the HlyU-His footprint region are indicated (259 to 315 bp) and are based on capillary electrophoresis internal size standards. The DNA sequence associated with the identified HlyU protected region is displayed below the chromatogram. A 7-bp inverted repeat (labeled in blue) and separated by 14 bp is centrally located within the HlyU-His protected region.

FIG 7

Schematic diagram of the intergenic region involved in exsA gene regulation. The ExsA binding motif and H-NS binding site have been previously identified. A putative HlyU binding site, as identified in this study, is shown. The ExsA binding motif has three box elements, each composed of conserved DNA sequences: 1, GC box; 2, A box; 3, TTAGN4TT. Protein-DNA interactions at specific sites within the exsA promoter region are shown. Plus and minus signs indicate positive and negative regulatory roles, respectively, on exsA promoter activity.