George Osei-Adjei

Regulatory actions of ToxR and CalR on their own genes and type III secretion system 1 in Vibrio parahaemolyticus

Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis. Type III secretion system 1 (T3SS1) is one of the virulence determinants of this bacteria. T3SS1 expression is regulated by ToxR and CalR. ToxR represses the transcription of T3SS1 genes via activation of CalR, which acts as a transcriptional repressor of T3SS1 genes. However, the transcriptional regulation mechanisms have not been elucidated. As showing in the present work, ToxR binds to the promoter DNA region of calR to activate its transcription. CalR occupies the promoter-proximal regions of each detected target operons in T3SS1 loci to repress their transcription, and thereby inhibiting T3SS1-dependent cytotoxicity. Moreover, a feedback CalR inhibits toxR and its own gene in a direct manner. Collectively, this work reported an interesting gene regulatory network involving the reciprocal regulation of ToxR and CalR, and their regulation on T3SS1 genes transcription in V. parahaemolyticus.Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis. Type III secretion system 1 (T3SS1) is one of the virulence determinants of this bacteria. T3SS1 expression is regulated by ToxR and CalR. ToxR represses the transcription of T3SS1 genes via activation of CalR, which acts as a transcriptional repressor of T3SS1 genes. However, the transcriptional regulation mechanisms have not been elucidated. As showing in the present work, ToxR binds to the promoter DNA region of calR to activate its transcription. CalR occupies the promoter-proximal regions of each detected target operons in T3SS1 loci to repress their transcription, and thereby inhibiting T3SS1-dependent cytotoxicity. Moreover, a feedback CalR inhibits toxR and its own gene in a direct manner. Collectively, this work reported an interesting gene regulatory network involving the reciprocal regulation of ToxR and CalR, and their regulation on T3SS1 genes transcription in V. parahaemolyticus.

Transcriptional regulation of cpsQ-mfpABC and mfpABC by CalR in Vibrio parahaemolyticus

The cpsQ‐mfpABC locus is transcribed as two operons, i.e., cpsQ‐mfpABC and mfpABC, in Vibrio parahaemolyticus, and both of them are all required for biofilm formation. CalR belongs to the LysR‐type transcriptional regulator family, and was originally identified as a repressor of the swarming motility and T3SS1 genes expression in V. parahaemolyticus. In the present work, a combination of qRT‐PCR, primer extension, LacZ fusion expression, electrophoretic mobility shift assay, and DNase I footprinting assays were employed to elucidate the regulatory mechanisms of cpsQ‐mfpABC and mfpABC by CalR. One transcription start site for each operon was detected and their activities were activated by CalR. His‐CalR protected two DNA regions upstream of mfpABC against DNase I digestion, but no binding sites were detected in the promoter region of cpsQ‐mfpABC, suggesting a direct and an indirect regulatory manner for mfpABC and cpsQ‐mfpABC transcription by CalR, respectively. Collectively, the results presented here confirmed a new physiological role for CalR that acts as an activator for cpsQ‐mfpABC and mfpABC transcription.

Transcriptional Regulation of the Type VI Secretion System 1 Genes by Quorum Sensing and ToxR in Vibrio parahaemolyticus

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.

Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios

Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

Reciprocal Regulation of OmpR and Hfq and Their Regulatory Actions on the Vi Polysaccharide Capsular Antigen in Salmonella enterica Serovar Typhi

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of human typhoid fever. S. Typhi expresses a major virulence determinant called Vi polysaccharide capsular antigen, which is encoded by the viaB locus containing 10 consecutive genes including tviA and tviB. Expression of Vi antigen is regulated by the two-component regulatory system EnvZ/OmpR and the global RNA-binding factor Hfq. In this study, we show that OmpR coordinates with Hfq to regulate the transcription of Vi antigen genes under osmotic stress conditions. OmpR binds to the promoters of tviA and its own genes to activate their transcription; however, it positively regulates tviB and negatively regulates hfq in an indirect manner. Moreover, Hfq reversely inhibits ompR, tviA and tviB, and positively regulates its own gene expression. Thus, we report of a complex gene regulatory network involving the reciprocal regulation and autoregulation of OmpR and Hfq, and their regulatory actions on Vi polysaccharide capsular antigen genes in S. Typhi.

CalR is required for the expression of T6SS2 and the adhesion of Vibrio parahaemolyticus to HeLa cells

Vibrio parahaemolyticus expresses one major virulence determinant T6SS2, which is constituted into three putative operons, i.e., VPA1027-1024, VPA1043-1028, and VPA1044-1046. CalR, a LysR-type transcriptional regulator, was originally identified as a repressor of the swarming motility and T3SS1 gene expression. As shown in this study, CalR binds to the promoter-proximal region of each of the three operons to activate their transcription, and moreover, CalR activates the adhesion of V. parahaemolyticus to HeLa cells. In addition, competitive EMSAs demonstrated that CalR acts as an antagonist of H–NS in V. parahaemolyticus. Collectively, these studies confirmed a new physiological role for CalR in V. parahaemolyticus.

The extracellular proteases produced by Vibrio parahaemolyticus

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.

OxyR positively and directly regulates Vi polysaccharide capsular antigen in Salmonella enterica serovar Typhi

Salmonella enterica serovar Typhi (S. Typhi) is a human enteropathogen that can overcome oxidative stress and survive in macrophages. OxyR is an important part of the antioxidant defense system. S. Typhi expresses a virulence (Vi) polysaccharide capsular antigen, which provides the bacterium with the ability to avoid host defenses and suppress detection by the innate immune system. This study investigated the effect of OxyR on Vi antigen in S. Typhi. In the oxyR mutant strain, microarray analysis, quantitative real time PCR and β-galactosidase assay confirmed that the viaB operon was positively regulated by OxyR. The Vi enzyme-linked immunosorbent assay and flow cytometry results showed that Vi capsule level was decreased in the oxyR mutant strain. Also, the EMSA revealed that OxyR directly binds to the promoter region of tviA. Thus, we propose that S. Typhi OxyR positively regulates expression of Vi capsule antigen in a direct manner.

Autoregulation of ToxR and Its Regulatory Actions on Major Virulence Gene Loci in Vibrio parahaemolyticus

Vibrio parahaemolyticus, the leading causative agent of seafood-associated gastroenteritis, harbors two major virulence gene loci T3SS1 and Vp-PAI (T3SS2 and tdh2). ToxR is a virulence regulator of vibrios. Cell density-dependent transcriptional pattern of toxR and its regulatory actions on T3SS1 and Vp-PAI have been previously reported, but the detailed regulatory mechanisms are still obscure. In the present work, we showed that the highest transcription level of toxR occurs at an OD600 = 0.2–0.4, which may be due to the subtle repression of ToxR and the quorum-sensing (QS) master regulator AphA. We also showed that ToxR is involved in regulating the mouse lethality, enterotoxicity, cytotoxicity, and hemolytic activity of V. parahaemolyticus. ToxR binds to the multiple promoter-proximal DNA regions within the T3SS1 locus to repress their transcription. In addition, ToxR occupies the multiple promoter-proximal DNA regions of Vp-PAI locus to activate their transcription. Thus, ToxR regulates the multiple virulence phenotypes via directly acting on the T3SS1 and Vp-PAI genes. Data presented here provide a deeper understanding of the regulatory patterns of ToxR in V. parahaemolyticus.