Optimisation du protocole de recherche des Escherichia coli producteurs de Shiga-toxines (STEC) dans les aliments

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des agents pathogènes importants émergents en santé publique. Ces bactéries sont à l origine des épidémies de colites hémorragiques et de syndrome hémolytique et urémique (SHU). La grande majorité des cas est liée à la consommation d aliments contaminés par un type particulier de STEC appelés E. coli entérohémorragiques (EHEC) parmi lesquels on trouve les sérogroupes définis comme pathogènes par l AFSSA : O26, O103, O111, O145 et O157.Les bovins ont depuis longtemps été identifiés comme un important réservoir de STEC. Bien que la transmission des STEC à l'homme soit fréquemment associée à la consommation de viande, les produits laitiers ont également été impliqués dans les cas humains. Le développement de méthodes rapides pour la détection des STEC les plus impliqués en pathologie humaine est donc essentiel pour assurer la sécurité des produits alimentaires. Cependant, la détection des STEC dans les aliments est problématique principalement en raison de la diversité des sérogroupes de STEC et de l'absence de caractéristiques biochimiques communes permettant de les distinguer des autres E. coli.L'objectif de cette thèse était d optimiser le protocole de détection des STEC dans les aliments de manière à pouvoir proposer aux industriels des protocoles leur permettant une réelle maîtrise du danger STEC dans leur filière.Pour se faire, nous sommes intervenus à différentes étapes du protocole. Nous avons notamment sélectionné un milieu d enrichissement permettant la détection des E. coli O26 dans les fromages au lait cru : EPT+ acriflavine + CT. Nous avons également évalué les performances d une méthode de détection des E. coli O157 :H7 (VIDAS ECPT UP) dans la viande de boeuf. Cette nouvelle technique est basée sur l utilisation de protéines recombinante de phage qui ciblent spécifiquement les E. coli O157. A travers cette étude, nous avons démontré la possibilité d avoir des temps d enrichissement minimes pour l analyse d échantillon de 25g (6h) et la faisabilité d analyser des échantillons de 375g avec un ratio de dilution de et un enrichissement de 24h. Enfin nous nous sommes intéressés à l étape de confirmation en mettant au point un test d immuno-concentration automatisé (utilisant des protéines de phages spécifiques de nos bactéries cibles) permettant d immuno-concentrer les 5 sérogroupes les plus connus de STEC (O26, O103, O111, O145 et O157) en une seule foisEnterohemorrhagic Escherichia coli (EHEC) are an important subset of Shiga toxin producing E. coli (STEC) associated with foodborne infections. These bacteria can cause hemorrhagic diarrhoeal disease and Haemolytic and Uremic Syndrome which are associated with some predominant serotypes defined by AFSSA institute: O26, O103, O111, O145 and O157.Cattle are the primary reservoir of STEC. Although STEC transmission to humans is frequently associated with consumption of meat, dairy products have also been implicated in human cases. Sensitive and rapid detection methods for STEC are essential for the food industry to ensure a safe food supply. However, the detection of STEC in foods is problematic because it s an heterogeneous group which displays a broad range of both genotypic and phenotypic differences.The aim of this thesis was to optimize the research protocol of STEC strains in food in order to provide some protocols to the industries for improving risk management protocol.So, we involved in different steps of the protocol: the enrichment of E. coli O26 allowing their detection in raw milk cheeses after 24 h enrichment in EPT+ acriflavine + cefixime-tellurite. Then, we evaluated the VIDAS ECPT UP performances for the detection of E. coli O157: H7 in raw ground beef. Through this study, we showed the ability to reduce enrichment time for sample analysis of 25g (6h) and the feasibility of analyzing samples of 375g with a sample to broth ratio of after 24h enrichment. Thereafter we looked at the confirmation step. We developed and optimized automated VIDAS immuno-concentration of E. coli O157, O26, O103, O111 and O145 (VIDAS ESPT) with the use of recombinant phage proteins for captureDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 g/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 g/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl cells (ARP-8129; contributed by Dr. John C. Kappes and Dr. Xiaoyun Wu); anti–HIV-1 gp160 monoclonal antibody (N6/ PGDM1400x10E8v4) (ARP-13390; contributed by Drs. Ling Xu and Gary Nabel); HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) (ARP-3418; contributed by Dr. Nathaniel Landau and Aaron Diamond); plasmids pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11017, ARP-11018, ARP-11024, and ARP-11022; contributed by Drs. David Montefiori, Feng Gao, and Ming Li); plasmid pcDNA3.1(+)-expressing HIV-1 Env/Rev (ARP-11037; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11308; contributed by Drs. D. Montefiori, F. Gao, C. Wil- liamson, and S. Abdool Karim); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11309; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Sala- zar-Gonzalez); HIV-1 BG505 Env expression vector (BG505.W6M.ENV.C2) (ARP- 11518; contributed by Dr. Julie Overbaugh); HIV-1 Env expression vector (CRF02_AG clone 257) (ARP-11599; contributed by Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 CNE8 Env (ARP-12653; contributed by Drs. Linqi Zhang, Hong Shang, David Montefiori, Tsinghua University (Beijing, China), China Medical University (Bei- jing, China), and Duke University (Durham, NC); HIV-1 SF162 gp160 expression vector (ARP-10463; contributed by Drs. Leonidas Stamatatos and Cecilia Cheng- Mayer); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11034; contributed by Drs. B. H. Hahn, X. Wei, and G. M. Shaw); plasmid pcDNA3.1/V5- His TOPO-expressing HIV Env/Rev (ARP-11038; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11310; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); HIV-1 Env expression vector (p16845 env) (ARP-11503; contributed by Drs. R. Paranjape, S. Kulkarni, and D. Montefiori); HIV-1 1054 Env expression vector (p1054.TC4.1499) (ARP-11561) and 6244 Env expression vector (p6244_13.B5.4576) (ARP-11566; contributed by Drs. Beatrice H. Hahn, Brandon F. Keele, and George M. Shaw); HIV-1 ZM246F Env expression vector (pZM246F_C1G) (ARP-11830; contributed by Dr. Beatrice Hahn); HIV-1 Env expression vector (CRF02_AG clone 278) (ARP-11605; contributed by Drs. Michael Thomson, Ana Revilla, Elena Delgado, David Montefiori, Sonia P erez Castro, Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Majada- honda, Madrid, Spain), Complejo Hospitalario Santa Mar ıa Madre (Orense, Spain), Duke University (Durham, NC), and the CAVD; and NL4-3 Env expression vector (pDOLHIVenv) (from Dr. Eric Freed and Dr. Rex Risser). The following reagents were kindly provided by CAVD: X2988, ZM106.9, and 3817. We thank S. Tabruyn and F. Arbogast for their assistance with in vivo studies. We thank the SickKids-University Health Network Flow Cytometry Facility. This work wassupported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4- 169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fel- lowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the CanadaResearch Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Sci- ence, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fun

The Phenotypic Analysis of Lactobacillus plantarum shsp Mutants Reveals a Potential Role for hsp1 in Cryotolerance

Small heat shock proteins (sHSPs) are ubiquitous, low molecular weight (MW) proteins that share a conserved alpha-crystallin domain. sHSPs oligomers exhibit chaperon-like activities by interacting with unfolded substrates, thereby preventing their aggregation and precipitation. Unlike most lactobacilli, which have single shsp genes, three different sHSP-encoding genes, i.e., hsp1, hsp2, and hsp3, were previously identified in the probiotic Lactobacillus plantarum WCFS1. Early studies, including the characterization of the knock out (KO) mutant for hsp2, indicated a different organization and transcriptional regulation of these genes and suggested that the three L. plantarum sHSPs might accomplish different tasks in stress response. To unravel the role of sHSPs, KO mutants of hsp1 and hsp3 were generated using a Cre-lox based system. Mutation of either genes resulted in impaired growth capacity under normal conditions, heat-stress and stresses typically found during host interactions and food technological process. However, survival to heat shock and the level of thermal stabilization of cytoplasmic proteins were similar between mutants and parental strain. Transcriptional analysis revealed that in the mutant genetic backgrounds there is an upregulated basal expression of the un-mutated mate hsps and other stress-related genes, which may compensate for the loss of HSP function, hence possibly accounting for the lack of a remarkable susceptibility to heat challenge. HSP3 seemed relevant for the induction of thermotolerance, while HSP1 was required for improved cryotolerance. Cell surface properties and plasma membrane fluidity were investigated to ascertain the possible membrane association of sHSP. Intriguingly, the loss of hsp1 was associated to a lower level of maximal membrane fluidity upon heat stress. A role for HSP1 in controlling and improving membrane fluidity is suggested which may pertains its cryoprotective function

The zCOSMOS 10k-Bright Spectroscopic Sample

We present spectroscopic redshifts of a large sample of galaxies with I_(AB) < 22.5 in the COSMOS field, measured from spectra of 10,644 objects that have been obtained in the first two years of observations in the zCOSMOS-bright redshift survey. These include a statistically complete subset of 10,109 objects. The average accuracy of individual redshifts is 110 km s^(–1), independent of redshift. The reliability of individual redshifts is described by a Confidence Class that has been empirically calibrated through repeat spectroscopic observations of over 600 galaxies. There is very good agreement between spectroscopic and photometric redshifts for the most secure Confidence Classes. For the less secure Confidence Classes, there is a good correspondence between the fraction of objects with a consistent photometric redshift and the spectroscopic repeatability, suggesting that the photometric redshifts can be used to indicate which of the less secure spectroscopic redshifts are likely right and which are probably wrong, and to give an indication of the nature of objects for which we failed to determine a redshift. Using this approach, we can construct a spectroscopic sample that is 99% reliable and which is 88% complete in the sample as a whole, and 95% complete in the redshift range 0.5 < z < 0.8. The luminosity and mass completeness levels of the zCOSMOS-bright sample of galaxies is also discussed

The Atacama Cosmology Telescope: Physical Properties and Purity of a Galaxy Cluster Sample Selected via the Sunyaev-Zel'dovich Effect

We present optical and X-ray properties for the first confirmed galaxy cluster sample selected by the Sunyaev-Zel'dovich Effect from 148 GHz maps over 455 square degrees of sky made with the Atacama Cosmology Telescope. These maps, coupled with multi-band imaging on 4-meter-class optical telescopes, have yielded a sample of 23 galaxy clusters with redshifts between 0.118 and 1.066. Of these 23 clusters, 10 are newly discovered. The selection of this sample is approximately mass limited and essentially independent of redshift. We provide optical positions, images, redshifts and X-ray fluxes and luminosities for the full sample, and X-ray temperatures of an important subset. The mass limit of the full sample is around 8e14 Msun, with a number distribution that peaks around a redshift of 0.4. For the 10 highest significance SZE-selected cluster candidates, all of which are optically confirmed, the mass threshold is 1e15 Msun and the redshift range is 0.167 to 1.066. Archival observations from Chandra, XMM-Newton, and ROSAT provide X-ray luminosities and temperatures that are broadly consistent with this mass threshold. Our optical follow-up procedure also allowed us to assess the purity of the ACT cluster sample. Eighty (one hundred) percent of the 148 GHz candidates with signal-to-noise ratios greater than 5.1 (5.7) are confirmed as massive clusters. The reported sample represents one of the largest SZE-selected sample of massive clusters over all redshifts within a cosmologically-significant survey volume, which will enable cosmological studies as well as future studies on the evolution, morphology, and stellar populations in the most massive clusters in the Universe.Comment: 20 pages, 15 figures, 6 tables. Accepted for publication in ApJ. Higher resolution figures available at: http://peumo.rutgers.edu/~felipe/e-prints

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states

Photobiomodulation reduces the cytokine storm syndrome associated with Covid-19 in the zebrafish model

Although the exact mechanism of the pathogenesis of COVID-19 is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red PBM as an attractive therapy to downregulate the cytokine storm caused by COVID-19 from a zebrafish model. RT-PCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that rSpike was responsible for generating systemic inflammatory processes with significantly increased pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a, coa1) mRNA markers, with a pattern like those observed in COVID-19 cases in humans. On the other hand, PBM treatment decreased the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most impacted metabolic pathways between PBM and the rSpike-treated groups were related to steroid metabolism, immune system, and lipids metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19, and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials.publishedVersio

Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity