Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. Recent genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 identified two sets of genes for the type III secretion system (TTSS), TTSS1 and TTSS2. Here, we constructed a series of mutant strains from RIMD2210633 to determine whether the two putative TTSS apparatus are functional. The cytotoxic activity of mutant strains having a deletion in one of the TTSS1 genes was significantly decreased compared with that of the parent and TTSS2-related mutant strains. In an enterotoxicity assay with the rabbit ileal loop test, intestinal fluid accumulation was diminished by deletion of the TTSS2-related genes while TTSS1-related mutants caused a level of fluid accumulation similar to that of the parent. VopD, a protein encoded in the proximity of the TTSS1 region and a homologue of the Yersinia YopD, was secreted in a TTSS1-dependent manner. In contrast, VopP, which is encoded by a pathogenicity island on chromosome 2 and is homologous to the Yersinia YopP, was secreted via the TTSS2 pathway. These results provide evidence that V. parahaemolyticus TTSSs function as secretion systems and may have a role in the pathogenicity of the organism. This is the first report of functional TTSSs in Vibrio species. The presence of TTSS apparatus gene homologues was demonstrated in other vibrios, such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio tubiashii, suggesting that some other vibrios also contain TTSS and that the TTSS has a role in protein secretion in those organisms during interaction with eukaryotic cells.Vibrio parahaemolyticus, one of the human-pathogenic vibrios, is a gram-negative halophilic bacterium that naturally inhabits marine and estuarine environments. The organism causes three major types of clinical illness: gastroenteritis (the most common illness), wound infections, and septicemia (4,7,10,16). Almost all of the clinical V. parahaemolyticus isolates from diarrheal patients show ␤-type hemolysis on Wagatsuma agar (22), a specialized blood agar medium. This hemolysis has been called the Kanagawa phenomenon (KP), and it is considered to be a good marker of pathogenic strains. The thermostable direct hemolysin (TDH) is responsible for the KP (10). TDH is a protein toxin composed of 165 amino acid residues, and it displays several biological activities, i.e., hemolytic activity, enterotoxicity, cytotoxicity, and cardiotoxicity (10,17,23,25). Thus, TDH has been considered a major virulence factor of the organism. The overall mechanism of pathogenesis by V. parahaemolyticus, however, has not yet been elucidated.The type III secretion system (TTSS) is an apparatus used by several gram-negative pathogenic bacteria to secrete and translocate virulence factor proteins into the cytosol of eukaryotic cells (11). The TTSS apparatus is well conserved among these bacteria, whereas the specific properties of the effectors and, hence, the resulting symptomatic effects on the host organism vary ...

SummaryVibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADPribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.

ObjectiveTo evaluate the efficacy and safety of the oral Janus kinase (JAK) inhibitor peficitinib versus placebo in Japanese patients with rheumatoid arthritis (RA).MethodsIn this multicentre, double-blind, parallel-group, placebo-controlled phase III study, patients with RA and inadequate response to methotrexate (MTX) were randomised 1:1:1 to placebo, peficitinib 100 mg once daily or peficitinib 150 mg once daily with MTX for 52 weeks. Based on baseline randomisation, at week 12, non-responders receiving placebo were switched to peficitinib until the end of treatment; the remaining patients were switched to peficitinib at week 28. Primary efficacy variables were American College of Rheumatology (ACR)20 response rate at week 12/early termination (ET) and change from baseline in van der Heijde-modified total Sharp score (mTSS) at week 28/ET.Results519 patients were randomised and treated. Significantly more (p<0.001) peficitinib (58.6%, 100 mg; 64.4%, 150 mg) than placebo (21.8%) recipients achieved ACR20 response at week 12/ET. Significantly lower (p<0.001) mean changes from baseline in mTSS at week 28/ET occurred in peficitinib (1.62, 100 mg; 1.03, 150 mg) than placebo (3.37) recipients. Peficitinib was associated with haematological and biochemical parameter changes, and increased incidence of serious infections and herpes zoster-related disease. One death from suicide occurred in a patient in the placebo group after switching to peficitinib 100 mg.ConclusionsIn Japanese patients with RA and inadequate response to MTX, peficitinib demonstrated significant superiority versus placebo in reducing RA symptoms and suppressing joint destruction. Peficitinib had an acceptable safety and tolerability profile, with no new safety signals compared with other JAK inhibitors.Trial registration numberNCT02305849.

ObjectivesTo investigate the efficacy and safety of peficitinib, an oral Janus kinase inhibitor, in patients with rheumatoid arthritis (RA).MethodsIn this double-blind phase III study, patients with RA and an inadequate response to prior disease-modifying anti-rheumatic drugs (DMARDs) were randomised to peficitinib 100 mg once daily, peficitinib 150 mg once daily, placebo or open-label etanercept for 52 weeks’ treatment; placebo-treated patients were switched at week 12 to peficitinib 100 or 150 mg once daily. The primary endpoint was American College of Rheumatology (ACR)20 response at week 12/early termination (ET). Secondary endpoints (assessed throughout) included ACR20, ACR50 and ACR70 response, changes from baseline in disease activity scores (DAS)28 and ACR core parameters, adverse events (AEs) and changes in clinical or laboratory measurements.ResultsIn total, 507 patients received treatment. ACR20 response rates at week 12/ET were significantly higher in the peficitinib 100 mg (57.7%) and 150 mg (74.5%) groups versus placebo (30.7%) (p<0.001). ACR50/70 response rates were also higher for both peficitinib doses versus placebo. Improvements in ACR response were maintained until week 52. Changes from baseline in DAS28-C-reactive protein/erythrocyte sedimentation rate and the ACR core set were significantly greater for both peficitinib doses versus placebo at week 12/ET (p<0.001). AE incidence was similar across treatment arms. Incidence of serious infection and herpes zoster-related disease was higher with peficitinib versus placebo, but with no clear dose-dependent increase.ConclusionsIn patients with RA and inadequate response to DMARDs, peficitinib 100 mg once daily or 150 mg once daily was efficacious in reducing RA symptoms and was well tolerated compared with placebo.Trial registration numberNCT02308163.

Vibrio parahaemolyticus, one of the human-pathogenic vibrios, is a Gram-negative halophilic bacterium that naturally inhabits marine and estuarine environments. The bacterium causes food-borne gastroenteritis, most frequently associated with the consumption of raw or undercooked seafood (1). Consumption of sufficiently high numbers of the organism can cause watery diarrhea, septicemia and even death (7,10). Almost all the clinical isolates of V. parahaemolyticus show β-hemolysis on Wagatsuma agar, a special blood agar medium (24). The hemolysis has been called the Kanagawa-phenomenon (KP), and is considered a good marker to differentiate human pathogenic V. parahaemolyticus from the non-pathogenic strains. To date, the thermostable direct hemolysin (TDH), has been shown to have a role in KP (6,27). In 1988, Honda et al. (9) discovered a new hemolysin in KP-negative clinical V. parahaemolyticus isolates. This hemolysin was referred to as TDH-related hemolysin (TRH). It is immunologically similar to TDH, and shares approximately 67% identity with TDH at the amino acid level (9). TDH and TRH show several common biological properties, such as hemolytic activity, enterotoxicity and cytotoxicity (5,6,8,10,16,17,23,25,29,31). V. parahaemolyticus strains isolated from patients with diarrhea produce TDH or TRH or both, whereas environmental isolates rarely produce these proteins (6, 27). Thus, TDH and TRH have been considered to be involved in the gastrointestinal disorders caused by V. parahaemolyticus (6).Although live V. parahaemolyticus shows cytotoxicity and enterotoxicity (2, 15, 25), the virulence factors responsible for these activities have not been fully elucidated. Recently, we completed the genome sequencing of a KP-positive (TDH-producing) V. parahaemolyticus RIMD2210633 strain (13). In this study, we constructed tdh-deletion mutants from the sequenced RIMD2210633 strain, and examined the hemolytic activity, cytotoxicity and enterotoxicity of the mutant strains Abstract: The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.