Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates

Immune Responses to Plague Infection in Wild Rattus rattus, in Madagascar: A Role in Foci Persistence?

Plague is endemic within the central highlands of Madagascar, where its main reservoir is the black rat, Rattus rattus. Typically this species is considered susceptible to plague, rapidly dying after infection inducing the spread of infected fleas and, therefore, dissemination of the disease to humans. However, persistence of transmission foci in the same area from year to year, supposes mechanisms of maintenance among which rat immune responses could play a major role. Immunity against plague and subsequent rat survival could play an important role in the stabilization of the foci. In this study, we aimed to investigate serological responses to plague in wild black rats from endemic areas of Madagascar. In addition, we evaluate the use of a recently developed rapid serological diagnostic test to investigate the immune response of potential reservoir hosts in plague foci.We experimentally infected wild rats with Yersinia pestis to investigate short and long-term antibody responses. Anti-F1 IgM and IgG were detected to evaluate this antibody response. High levels of anti-F1 IgM and IgG were found in rats one and three weeks respectively after challenge, with responses greatly differing between villages. Plateau in anti-F1 IgM and IgG responses were reached for as few as 500 and 1500 colony forming units (cfu) inoculated respectively. More than 10% of rats were able to maintain anti-F1 responses for more than one year. This anti-F1 response was conveniently followed using dipsticks.Inoculation of very few bacteria is sufficient to induce high immune response in wild rats, allowing their survival after infection. A great heterogeneity of rat immune responses was found within and between villages which could heavily impact on plague epidemiology. In addition, results indicate that, in the field, anti-F1 dipsticks are efficient to investigate plague outbreaks several months after transmission

Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium

Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases.

Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale

Transcriptional Regulation of opaR, qrr2–4 and aphA by the Master Quorum-Sensing Regulator OpaR in Vibrio parahaemolyticus

Background: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2–4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood. Methodology/Principal Findings: The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2–4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promote

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V

Within-subject comparison of two- versus three-implant-assisted mandibular overdenture : patient-based outcomes

RÉSUMÉ Objectifs: Cette étude cherche à évaluer l’influence de l’ajout d’un implant additionnel dans la région médiane mandibulaire sur la perception des patients porteurs d’une prothèse préexistante assistée par deux implants. Méthodes: Cette étude fait partie d’un essai clinique qui a été mené à l'Université de Montréal. Dix-sept personnes édentées (âge moyen de 61,9 ± 6,6 ans) ont reçu trois implants dans la région mandibulaire interforaminale. Deux implants ont été place près des trous mentonniers et le troisième au niveau de la ligne médiane. Au début de l’essai, les sujets ont été appareillés d’une prothèse de recouvrement mandibulaire assistée par les deux implants distaux. Ces implants étaient coiffés par des attaches individuelles Locator®. Le troisième implant n’a pas été mis en charge initialement et il est resté sans attache pour deux ans. Après cette période, une attache Locator a été installée sur le troisième implant et la prothèse de recouvrement a été modifiée pour accommoder ce mécanisme de rétention additionnel. Les mouvements antéropostérieurs de la prothèse tel que perçus par les patients ainsi que ceux évalués en cliniques ont été mesurés avant et après cette modification. La satisfaction des patients, leurs perceptions et leurs attentes vis-à-vis les prothèses mandibulaires ainsi que la volonté de payer ont été évaluées. La collecte de données a été effectuée à l'aide de questionnaires auto administré validés, à la suite de la modification et après six semaines d’utilisation. Des données sociodémographiques ont également été recueillies. Des statistiques descriptives et les essais non paramétriques ont été employés pour l'analyse statistique. Résultats: Les résultats ont indiqué une diminution statistiquement significative dans le mouvement antéropostérieur de la prothèse mandibulaire (p = 0,005) tel qu’évalué en clinique. Les patients ont rapporté une amélioration au niveau de la stabilité de la prothèse mandibulaire (p = 0,005), de même qu’au niveau de leur capacité à parler (p = 0,011) et à mastiquer les aliments durs (p = 0,012). L'ajout d'un troisième implant a répondu aux attentes des patients en ce qui concerne la stabilité (pour 94 % des patients), la rétention (100 %) et le confort (82,4%) de la prothèse mandibulaire. Sur une période de six semaines, la prothèse de recouvrement mandibulaire assistée par trois implants a contribué à l'augmentation de la satisfaction générale des patients, mais cette amélioration n'était pas statistiquement significative. Environ 80 % des patients recommanderaient ce type de prothèse à leurs pairs, mais seulement 47 % d'entre eux accepteraient de payer l’augmentation du coût de traitement associée à la pose d’un troisième implant. Conclusions: L'ajout d'un troisième implant dans la région médiane d’une prothèse préexistante assistée par deux implants a permis d'obtenir de meilleurs résultats au niveau de l’expérience du patient. Cependant, le coût supplémentaire du traitement peut influencer les choix du patient.ABSTRACT Objectives: This study aims to assess the impact of an additional midline implant to support an existing mandibular two-implant overdenture, on patient-based outcomes (patients’ satisfaction and expectations). Methods: This study was nested within a previous clinical trial conducted at the Université de Montréal. Seventeen edentulous individuals (mean age: 61.9 ± 6.6 years) received three threaded implants in the interforaminal mandibular area and a mandibular overdenture using two Locator® attachments. The midline implant was left unloaded over a two-year period. At the two-year follow-up, using a standard protocol, the third implant received a Locator® attachment and the overdenture was converted to a three-implant-assisted overdenture. The clinical and perceived anterior–posterior movements of mandibular prostheses were measured before and after the conversion. Patients’ expectation and satisfaction in regard to mandibular prosthesis as well as their willingness to pay the cost for the conversion were evaluated by using validated self-administered questionnaires. Data collection was conducted at baseline and after six weeks of wearing the converted mandibular prosthesis. Socio-demographic data were also collected. Descriptive statistics and non-paramteric tests were used for statistical analysis. Results: Data analysis revealed a statistically significant decrease in the anterior–posterior movement (p = 0.005) of overdenture as evaluated by clinicians. Study participants reported an increase in perceived stability of the overdenture (p = 0.005), and in their ability to speak (p = 0.011) and to chew hard food (p = 0.012). The addition of a third implant met the expectations of 94% of patients in regard to lower denture stability, 100% for retention, and 82.4% for comfort. The 3-implant-assisted mandibular overdenture increased patients’ general satisfaction over a short period of time, but this improvement was not statistically significant. About 80% of patients would recommend this type of prosthesis to their peers but only 47% of them would agree to pay a large increase in the cost of treatment compared to 2-implant overdenture. Conclusions: The addition of a midline third implant to an existing 2-mandibular-implant overdenture will lead to better patient-based outcomes. However, the additional cost of the treatment may influence patient preferences

Tổng hợp và biểu hiện gen caf1 mã hóa kháng nguyên F1 của vi khuẩn Yersinia pestis

Yersinia pestis is the etiologic agent of plague, one of the most deadly infectious diseases described in the history of humanity. It was responsible for millions of deaths all over the world. Yersinia pestis also can be used as a highly lethal biological potential weapon. For plague diagnosis in humans as well as to detect Y. pestis in the environment, fraction 1 capsular antigen (F1) of the bacteria was usually used as a good marker. The aim of this study is to produce Y. pestis F1 antigen to serve as a material for development of immunochromatographic test strips for rapid detection of Y. pestis. Because of the difficulty in Y. pestis culture for DNA extraction as well as F1 antigen production, we artificially synthesized the target caf1 coding for F1 antigen for expression in Escherichia coli. After the codon optimization step, caf1 was synthesized by “gapless” PCR using 22 overlaping oligonucleotides cover the complete sequence of this gene. The sequencing result showed that we successfully synthesized the target gene. In total 6 clones sequence, there are 2 clones sequence which were 100% identity with reference sequence. The target sequence was then introduced into pET-52b(+) vector and expressed in E. coli BL21 (DE3) in the form of (His)10 affinity tag fusion. As the result of SDS-PAGE, the recombinant protein Caf1 of 18 kDa was highly expressed in E. coli as inclusion body form and was purified by His-tag affinity chromatography. The recombinant Caf1 was then confirmed by Western blot with His-tag antibody.Vi khuẩn Yersinia pestis là tác nhân gây bệnh dịch hạch, một trong những loại bệnh truyền nhiễm nguy hiểm nhất được biết cho đến nay đã gây ra hàng triệu ca tử vong trên thế giới và có thể được sử dụng như một vũ khí sinh học có tính hủy diệt cao. Để chẩn đoán bệnh dịch hạch ở người cũng như phát hiện Y. pestis trong môi trường, người ta thường dựa trên việc phát hiện kháng nguyên nang F1 của loại vi khuẩn này. Mục tiêu của nghiên cứu này là nhằm tạo kháng nguyên F1 của Y. pestis để làm nguyên liệu cho que thử sắc ký miễn dịch phát hiện nhanh Y. pestis. Do việc nuôi cấy Y. pestis để thu nhận kháng nguyên F1 hoặc DNA của vi khuẩn này rất khó khăn nên chúng tôi đã tiến hành tổng hợp nhân tạo gen mục tiêu caf1, mã hóa kháng nguyên F1, nhằm biểu hiện trong tế bào Escherichia coli. Sau khi tối ưu hóa mã bộ ba mã hóa amino acid (codon) cho E. coli, gen caf1 đã được tổng hợp bằng phương pháp “gapless” PCR. Kết quả giải trình tự cho thấy phương pháp này cho phép tổng hợp được trình tự gen mục tiêu với độ chính xác là 2/6 dòng plasmid. Tiếp đó, trình tự gen này đã được đưa vào vector pET-52b(+) và biểu hiện trong tế bào E. coli BL21 (DE3) ở dạng dung hợp với đuôi ái lực (His)10. Các kết quả điện di protein SDS-PAGE, tinh sạch protein bằng sắc ký ái lực Ni và Western blot cho thấy kháng nguyên tái tổ hợp F1 đã được tạo ra thành công với hiệu suất lớn ở dạng thể vùi trong tế bào E. coli

Beauty photoproduction measured using decays into muons in dijet events in ep collisions at s\sqrt{s}=318 GeV

The photoproduction of beauty quarks in events with two jets and a muon has been measured with the ZEUS detector at HERA using an integrated luminosity of 110 pb$^{- 1}$. The fraction of jets containing b quarks was extracted from the transverse momentum distribution of the muon relative to the closest jet. Differential cross sections for beauty production as a function of the transverse momentum and pseudorapidity of the muon, of the associated jet and of $x_{\gamma}^{jets}$, the fraction of the photon's momentum participating in the hard process, are compared with MC models and QCD predictions made at next-to-leading order. The latter give a good description of the data.Comment: 32 pages, 6 tables, 7 figures Table 6 and Figure 7 revised September 200