QsvR represses the transcription of polar flagellum genes in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces dual flagellar systems, i.e., the sheathed polar flagellum (Pof) and numerous lateral flagella (Laf), both of which are strictly regulated by numerous factors. QsvR is an AraC-type regulator that controls biofilm formation and virulence of V. parahaemolyticus. In the present study, we showed that deletion of qsvR significantly enhanced swimming motility of V. parahaemolyticus, while the swarming motility was not affected by QsvR. QsvR bound to the regulatory DNA regions of flgAMN and flgMN within the Pof gene loci to repress their transcription, whereas it negatively controls the transcription of flgBCDEFGHIJ and flgKL-flaC in an indirect manner. However, over-produced QsvR was also likely to possess the binding activity to the regulatory DNA regions of flgBCDEFGHIJ and flgKL-flaC in a heterologous host. In summary, this work demonstrated that QsvR negatively regulated the swimming motility of V. parahaemolyticus via directly action on the transcription of Pof genes.

The phase variation between wrinkly and smooth colony phenotype affects the virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, undergoes wrinkly and smooth colony switching on the plate. The wrinkly spreader grew faster, had stronger motility and biofilm capacity when compared with the smooth one. However, whether the two phenotypes differ in their virulence still needs to be further investigated. In this study, the data showed that the smooth spreader had stronger virulence phenotypes, including the cytotoxicity against HeLa cells, antibacterial activity against E. coli, adhesive capacity toward HeLa cells, and lethality in zebrafish, relative to the wrinkly one. However, the colony morphology variation had no influence on the haemolytic activity. The mRNA levels of major virulence genes including T3SS1, T6SS1, and T6SS2 were significantly enhanced in the smooth colonies relative to those in the wrinkly colonies. Taken together, the presented work highlighted the different virulence profiles of the wrinkly and smooth colony phenotypes.

Atroposelective Synthesis of Axially Chiral Styrenes by Platinum- Catalyzed Stereoselective Hydrosilylation of Internal Alkynes.

Hydrofunctionalization of alkynes is one of the most efficient ways to access axially chiral styrenes with open-chained olefins. While great advances have been achieved for 1-alkynylnaphthalen-2-ols and analogues, atroposelective hydrofunctionalization of unactivated internal alkynes lags. Herein we reported a platinum-catalyzed atroposelective hydrosilylation of unactivated internal alkynes for the first time. With monodentate TADDOL-derived phosphonite L1 used as a chiral ligand, various axially chiral styrenes were achieved in excellent enantioselectivities with high E-selectivities. Control experiments showed that the NH-arylamide groups have significant effects on both the yields and enantioselectivities and could act as directing groups. The potential utilities of the products were shown by the transformations of the amide motifs of the products.

The Effect of Salinity on Biofilm Formation and c-di-GMP Production in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a moderately halophilic, salt-requiring organism that exhibits optimal growth at approximately 3% salt. Thus, salinity stress is one of the most important stimuli during its lifecycle. The bacterium possesses a strong ability to form biofilms on surfaces, which are thought to be involved in protecting it from adverse environmental conditions. In the present study, salinity-dependent biofilm formation by V. parahaemolyticus was investigated by combining crystal violet staining, colony morphology, intracellular c-di-GMP quantification and quantitative PCR. The results showed that biofilm formation by V. parahaemolyticus was significantly enhanced in low salinity growth conditions and was affected by incubation time. In addition, low salinity reduced intracellular c-di-GMP degradation in V. parahaemolyticus. Transcription of genes encoding ScrABC and ScrG proteins, which are involved in intracellular c-di-GMP metabolism, was inhibited by low salinity growth conditions. Thus, reduced intracellular c-di-GMP degradation in V. parahaemolyticus in low salinity growth conditions may be mediated by repression of scrG and scrABC transcription. Taken together, these results demonstrated for the first time that salinity regulates biofilm formation and c-di-GMP production in V. parahaemolyticus.

2-Amino-1,3,4-thiadiazoles in the 7-hydroxy-N-neopentyl spiropiperidine indolinyl series as potent P2Y1 receptor antagonists.

Blockade of the P2Y1 receptor is important to the treatment of thrombosis with potentially improved safety margins compared with P2Y12 receptor antagonists. Investigation of a series of urea surrogates of the diaryl urea lead 3 led to the discovery of 2-amino-1,3,4-thiadiazoles in the 7-hydroxy-N-neopentyl spiropiperidine indolinyl series as potent P2Y1 receptor antagonists, among which compound 5a was the most potent and the first non-urea analog with platelet aggregation (PA) IC50 less than 0.5 µM with 10 µM ADP. Several 2-amino-1,3,4-thiadiazole analogs such as 5b and 5f had a more favorable pharmacokinetic profile, such as higher Ctrough, lower Cl, smaller Vdss, and similar bioavailability compared with 3.

New azole antagonists with high affinity for the P2Y(1) receptor.

Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of the P2Y1 receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of the more potent thiadiazole ring system which leads to thiadiazole 4c as a new antiplatelet agent.

2-Aminothiazole based P2Y(1) antagonists as novel antiplatelet agents.

ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2µM) and acceptable PK in rats.

Potent P2Y1 urea antagonists bearing various cyclic amine scaffolds.

A number of new amine scaffolds with good inhibitory activity in the ADP-induced platelet aggregation assay have been found to be potent antagonists of the P2Y1 receptor. SAR optimization led to the identification of isoindoline 3c and piperidine 4a which showed good in vitro binding and functional activities, as well as improved aqueous solubility. Among them, the piperidine 4a showed the best overall profile with favorable PK parameters.

Discovery of diarylurea P2Y(1) antagonists with improved aqueous solubility.

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

CD40-targeting magnetic nanoparticles for MRI/optical dual-modality molecular imaging of vulnerable atherosclerotic plaques.

BACKGROUND AND AIMS: Acute coronary syndrome caused by vulnerable plaque rupture or erosion is a leading cause of death worldwide. CD40 has been reported to be highly expressed in atherosclerotic plaques and closely related to plaque stability. Therefore, CD40 is expected to be a potential target for the molecular imaging of vulnerable plaques in atherosclerosis. We aimed to design a CD40-targeted magnetic resonance imaging (MRI)/optical multimodal molecular imaging probe and explore its ability to detect and target vulnerable atherosclerotic plaques. METHODS: CD40-Cy5.5 superparamagnetic iron oxide nanoparticles (CD40-Cy5.5-SPIONs), which comprise a CD40-targeting multimodal imaging contrast agent, were constructed by conjugating CD40 antibody and Cy5.5-N-hydroxysuccinimide ester with SPIONs. During this in vitro study, we observed the binding ability of CD40-Cy5.5-SPIONs with RAW 264.7 cells and mouse aortic vascular smooth muscle cells (MOVAS) after different treatments, using confocal fluorescence microscopy and Prussian blue staining. An in vivo study involving ApoE-/- mice fed a high-fat diet for 24-28 weeks was performed. 24 h after intravenous injection of CD40-Cy5.5-SPIONs, fluorescence imaging and MRI were performed. RESULTS: CD40-Cy5.5-SPIONs bind specifically to tumor necrosis factor (TNF)-α-treated macrophages and smooth muscle cells. Fluorescence imaging results showed that, compared with the control group and the atherosclerosis group injected with non-specific bovine serum albumin (BSA)-Cy5.5-SPIONs, the atherosclerotic group injected with CD40-Cy5.5-SPIONs had a stronger fluorescence signal. T2-weighted images showed that the carotid arteries of atherosclerotic mice injected with CD40-Cy5.5-SPIONs had a significant substantial T2 contrast enhancement effect. CONCLUSIONS: CD40-Cy5.5-SPIONs could potentially serve as an effective MRI/optical probe for vulnerable atherosclerotic plaques during non-invasive detection.

Vibrio vulnificus septicemia in a hospitalized patient with hepatitis B virus-associated cirrhosis: A case report.

Vibrio vulnificus is usually transmitted by consumption of raw or undercooked seafood or exposure to seawater and can causes gastroenteritis, wound infection, and even sepsis. However, atypical or unclear sources of V. vulnificus infection have been reported. Here, we report a case of V. vulnificus infection presenting as septicemia in a 53-year-old man with hepatitis B virus-associated cirrhosis. The source of infection remained unclear as the patient reported no consumption of seafood or contact with seawater. Treatment with antibiotics was initiated prior to confirmation of V. vulnificus infection. This report provides an important reference for the diagnosis and treatment of V. vulnificus infection.

Palladium-catalyzed intramolecular asymmetric hydrocyclopropanylation of alkynes: synthesis of cyclopropane-fused γ-lactams.

A palladium-catalyzed intramolecular asymmetric hydrocyclopropanylation of alkynes via C(sp3)-H activation has been developed for the synthesis of cyclopropane-fused γ-lactams. The presented strategy proceeds in a selective and 100% atom-economical manner. A range of cyclopropane-fused γ-lactams were prepared from readily available substrates in good yields and enantioselectivities with a chiral phosphoramidite ligand.

AphA directly activates the transcription of polysaccharide biosynthesis gene scvE in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a seafood-borne pathogen, is capable of forming biofilms on surfaces. Exopolysaccharide (EPS) plays crucial roles in holding bacterial cells together and keeping biofilm attached on the surface. The cpsA-K and scvA-O gene clusters are responsible for EPS synthesis in V. parahaemolyticus. AphA, the master quorum sensing (QS) regulator operating at low cell density (LCD), positively regulates transcription of cpsA-K and scvA-O, but lacks the detailed mechanisms. The present data showed that the aphA mutant produced smooth colonies, whereas the wild-type strain produced wrinkled colonies. AphA bound the regulatory DNA region of scvE to activate its transcription, whereas it positively regulated transcription of cpsA and scvA in an indirect manner. The transcriptional level of scvE gradually decreased with increasing cell density, which correlated with the expression level of aphA. Taken together, this work elucidated how AphA regulated the biofilm-associated colony morphology variation in V. parahaemolyticus through its regulatory actions on the expression of EPS genes.

Complete genome sequence and comparative analysis of a Vibrio vulnificus strain isolated from a clinical patient.

Vibrio vulnificus is an opportunistic, global pathogen that naturally inhabits sea water and is responsible for most vibriosis-related deaths. We investigated the genetic characteristics of V. vulnificus isolated from the clinical blood culture specimen of a patient with hepatitis B virus cirrhosis in 2018 (named as V. vulnificus VV2018) by whole genome sequencing (WGS). VV2018 belonged to a novel sequencing type 620 (ST620) and comprised two circular chromosomes, containing 4,389 potential coding sequences (CDSs) and 152 RNA genes. The phylogenetic tree of single nucleotide polymorphisms (SNPs) using 26 representative genomes revealed that VV2108 grouped with two other V. vulnificus strains isolated from humans. The pan-genome of V. vulnificus was constructed using 26 representative genomes to elucidate their genetic diversity, evolutionary characteristics, and virulence and antibiotic resistance profiles. The pan-genome analysis revealed that VV2018 shared a total of 3,016 core genes (≥99% presence), including 115 core virulence factors (VFs) and 5 core antibiotic resistance-related genes, and 309 soft core genes (≥95 and <99% presence) with 25 other V. vulnificus strains. The varG gene might account for the cefazolin resistance, and comparative analysis of the genetic context of varG revealed that two genes upstream and downstream of varG were conserved. The glycosylation (pgl) like genes were found in VV2018 compared with Pgl-related proteins in Neisseria that might affect the adherence of the strain in hosts. The comparative analysis of VV2018 would contribute to a better understanding of the virulence and antibiotic resistance profiles of V. vulnificus. Meanwhile much work remains to be done to better understand the function of pgl-like genes in V. vulnificus.

QsvR and OpaR coordinately repress biofilm formation by Vibrio parahaemolyticus.

Mature biofilm formation by Vibrio parahaemolyticus requires exopolysaccharide (EPS), type IV pili, and capsular polysaccharide (CPS). Production of each is strictly regulated by various control pathways including quorum sensing (QS) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). QsvR, an AraC-type regulator, integrates into the QS regulatory cascade via direct control of the transcription of the master QS regulators, AphA and OpaR. Deletion of qsvR in wild-type or opaR mutant backgrounds altered the biofilm formation by V. parahaemolyticus, suggesting that QsvR may coordinate with OpaR to control biofilm formation. Herein, we demonstrated both QsvR and OpaR repressed biofilm-associated phenotypes, c-di-GMP metabolism, and the formation of V. parahaemolyticus translucent (TR) colonies. QsvR restored the biofilm-associated phenotypic changes caused by opaR mutation, and vice versa. In addition, QsvR and OpaR worked coordinately to regulate the transcription of EPS-associated genes, type IV pili genes, CPS genes and c-di-GMP metabolism-related genes. These results demonstrated how QsvR works with the QS system to regulate biofilm formation by precisely controlling the transcription of multiple biofilm formation-associated genes in V. parahaemolyticus.

Quorum sensing and QsvR tightly control the transcription of vpa0607 encoding an active RNase II-type protein in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative, halophilic bacterium, is a leading cause of acute gastroenteritis in humans. AphA and OpaR are the master quorum sensing (QS) regulators operating at low cell density (LCD) and high cell density (HCD), respectively. QsvR is an AraC-type protein that integrates into the QS system to control gene expression by directly controlling the transcription of aphA and opaR. However, the regulation of QsvR itself remains unclear to date. In this study, we show that vpa0607 and qsvR are transcribed as an operon, vpa0607-qsvR. AphA indirectly activates the transcription of vpa0607 at LCD, whereas OpaR and QsvR directly repress vpa0607 transcription at HCD, leading to the highest expression levels of vpa0607 occurs at LCD. Moreover, VPA0607 acts as an active RNase II-type protein in V. parahaemolyticus and feedback inhibits the expression of QsvR at the post-transcriptional level. Taken together, this work deepens our understanding of the regulation of QsvR and enriches the integration mechanisms of QsvR with the QS system in V. parahaemolyticus.

Enantioselective Syntheses of 2-Azabicyclo[2.2.1]heptanes via Brønsted Acid Catalyzed Ring-Opening of meso-Epoxides.

A chiral phosphoric acid-catalyzed ring-opening of meso-epoxides was developed. A range of 2-azabicyclo[2.2.1]heptanes were obtained in high yields with excellent enantioselectivities. In addition, the hydroxyl and amide groups in the products provided handles for further derivatization.

[ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus].

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the ß-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.

Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018-2020.

Vibrio parahaemolyticus is the leading cause of acute seafood-associated gastroenteritis worldwide. The aim of this study was to investigate the presence of virulence genes, biofilm formation, motor capacities and antimicrobial resistance profile of V. parahaemolyticus isolates isolated from clinical samples in Nantong during 2018-2020. Sixty-six V. parahaemolyticus strains isolated from stool specimens of diarrheal patients were examined. The PCR results showed that there were two tdh+trh+ isolates, four tdh-trh- isolates and sixty tdh+trh- isolates, accounting for 3.0%, 6.1% and 90.9%, respectively. All the tdh carrying isolates manifested the positive reactions for the Kanagawa phenomenon (KP) test. Most of the isolates harbored at least one of the specific DNA markers of 'pandemic group' strains, suggesting that the dominant isolates of V. parahaemolyticus in Nantong might belong to the new O3: K6 or its serovariants. All tdh+ isolates possessed the Vp-PAI genes, but no tdh-trh- isolates carried the T3SS2 genes. All isolates were biofilm producers and had relatively strong motor capacities. In addition, the V. parahaemolyticus isolates were resistant to ampicillin (98.5%), cefuroxime (75.6%), cefepime (66.7%), piperacillin (59.1%) and ampicillin/sulbactam (50.0%), but sensitive to ciprofloxacin (100.0%), levofloxacin (100.0%), trimethoprim-sulfamethoxazole (98.5%), gentamicin (98.5%), amikacin (97%), meropenem (71.2%), and ceftazidime (56.1%). Multidrug-resistant isolates in clinical might be related to the inappropriate use of antimicrobials in aquaculture.

Transcriptomic Analysis of Vibrio parahaemolyticus Underlying the Wrinkly and Smooth Phenotypes.

Vibrio parahaemolyticus, a causative agent of seafood-associated gastroenteritis, undergoes opaque-translucent (OP-TR) colony switching associated with capsular polysaccharide (CPS) production. Here, we showed that V. parahaemolyticus was also able to naturally and reversibly switch between wrinkly and smooth phenotypes. More than 1,000 genes were significantly differentially expressed during colony morphology switching, including the major virulence gene loci and key biofilm-related genes. The genes responsible for type III secretion system 1 (T3SS1), type VI secretion systems (T6SS1 and T6SS2), and flagellar synthesis were downregulated in the wrinkly spreader phenotype, whereas genes located on the pathogenicity island Vp-PAI and those responsible for chitin-regulated pili (ChiRP) and Syp exopolysaccharide synthesis were upregulated. In addition, we showed that the wrinkly spreader grew faster, had greater motility and biofilm capacities, and produced more c-di-GMP than the smooth type. A dozen genes potentially associated with c-di-GMP metabolism were shown to be significantly differentially expressed, which may account for the differences in c-di-GMP levels between the two phenotypes. Most importantly, dozens of putative regulators were significantly differentially expressed, and hundreds of noncoding RNAs were detected during colony morphology switching, indicating that phenotype switching is strictly regulated by a complex molecular regulatory network in V. parahaemolyticus. Taken together, the presented work highlighted the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization. IMPORTANCE We showed that Vibrio parahaemolyticus was able to naturally and reversibly switch between wrinkly and smooth phenotypes and disclosed the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes between the two colony morphology phenotypes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization.