The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence‐associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E‐cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence‐associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST‐OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST‐OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs‐associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs‐induced cleavage of E‐cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence‐associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.This work was funded by BBSRC awards BB/F020988/1 and BB/K004514/1. Moredun Research Institute receives funding via Rural and Environment Science and Analytical Services (RESAS) division of the Scottish Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms

The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes

Dietary reference values for vitamin K

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) derives dietary reference values (DRVs) for vitamin K. In this Opinion, the Panel considers vitamin K to comprise both phylloquinone and menaquinones. The Panel considers that none of the biomarkers of vitamin K intake or status is suitable by itself to derive DRVs for vitamin K. Several health outcomes possibly associated with vitamin K intake were also considered but data could not be used to establish DRVs. The Panel considers that average requirements and population reference intakes for vitamin K cannot be derived for adults, infants and children, and therefore sets adequate intakes (AIs). The Panel considers that available evidence on occurrence, absorption, function and content in the body or organs of menaquinones is insufficient, and, therefore, sets AIs for phylloquinone only. Having assessed additional evidence available since 1993 in particular related to biomarkers, intake data and the factorial approach, which all are associated with considerable uncertainties, the Panel maintains the reference value proposed by the Scientific Committee for Food (SCF) in 1993. An AI of 1 mu g phylloquinone/kg body weight per day is set for all age and sex population groups. Considering the respective reference body weights, AIs for phylloquinone are set at 70 mu g/day for all adults including pregnant and lactating women, at 10 mu g/day for infants aged 7-11 months, and between 12 mu g/day for children aged 1-3 years and 65 mu g/day for children aged 15-17 years. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

ニュースレター No.8

Kyoto University Global COE Program: Global Center for Education and Research on Human Security Engineering for Asian Megacities = 京都大学グローバルCOE プログラム「アジア・メガシティの人間安全保障工学拠点