Search

International audience; Introduction and objectives: Epithelial cells are the first barrier to infection. Pathogens such as Salmonella and mastitis-causing S. aureus strains can penetrate these cells and survive in the intracellular milieu. Although the cross-presentation of bacterial antigens on MHC class I has been observed in dendritic cells, the role of epithelial cells in the process is still unclear. Basedon this, we aimed to verify the capacity of swine gut epithelial cells and bovine mammary epithelial cells to present bacterial antigens and stimulate cytotoxic T lymphocytes.Materials and methods: Bovine and porcine epithelial cells transiently transfected with murine MHC-I were exposed to strains of Salmonella carrying genes involved in the invasion and intracellular fate of Salmonella tagged with the peptide SIINFEKL (OVA 256-264). A murine CD8+ T lymphocyte clone (B3Z) was then brought into contact with infected epithelial cells and the release of IL2 was quantified by ELISA.Results, discussion and conclusion: We observed that swine and bovine epithelial cells have the ability to cross-present antigens derived from the degradation of bacterial proteins. The intensity of CD8+ T cells responses varied depending on the tagged bacterial factor and suggested a more efficient presentation of antigens derived of Salmonella factors involved in intracellular survival and late replication. These preliminary data indicate the role of epithelial cells in the induction of adaptive immune response and our experimental strategy might be applied to pathogens other than Salmonella. We believe that a better understanding of the mechanisms regulating the host cytotoxic response and the fate of pathogens in the intracellular niche might provide new avenues to fight persistent bacterial infections, meeting a major animal and public health concern.

Supplementary Materials; International audience; Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

International audience

Physiological and pathological conditions that affect the folding capacity of the endoplasmic reticulum (ER) provoke ER stress and trigger the unfolded protein response (UPR). The UPR aims to either restore the balance between newly synthesized and misfolded proteins or if the damage is severe, to trigger cell death. However, the molecular events underlying the switch between repair and cell death are not well understood. The ER-resident chaperone BiP governs the UPR by sensing misfolded proteins and thereby releasing and activating the three mediators of the UPR: PERK, IRE1 and ATF6. PERK promotes G2 cell cycle arrest and cellular repair by inducing the alternative translated p53 isoform p53ΔN40 (p53/47), which activates 14-3-3σ via suppression of p21(CDKN1A). Here we show that prolonged ER stress promotes apoptosis via a p53-dependent inhibition of BiP expression. This leads to the release of the pro-apoptotic BH3-only BIK from BiP and activation of apoptosis. Suppression of bip mRNA translation is mediated via the specific binding of p53 to the first 346-nt of the bip mRNA and via a p53 trans-suppression domain located within the first seven N-terminal amino acids of p53ΔN40. This work shows how p53 targets BiP to promote apoptosis during severe ER stress and further illustrates how regulation of mRNA translation has a key role in p53-mediated regulation of gene expression during the UPR.

International audience; The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome replication and maintenance but also highly antigenic. Hence, EBV evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA at a minimal level to ensure its essential function thereby, at the same time, minimizing immune recognition. Defining intervention points where to interfere with EBNA1 immune evasion is an important step to trigger an immune response against EBV-carrying cancers. Thanks to a yeast-based assay that recapitulates all the aspects of EBNA1 self-limitation of expression, a recent study by Lista et al. [Nature Communications (2017) 7, 435-444] has uncovered the role of the host cell nucleolin (NCL) in this process via a direct interaction of this protein with G-quadruplexes (G4) formed in GAr-encoding sequence of EBNA1 mRNA. In addition, the G4 ligand PhenDC3 prevents NCL binding on EBNA1 mRNA and reverses GAr-mediated repression of translation and antigen presentation. This shows that the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to unveil EBV-carrying cancers to the immune system and that the yeast model can be successfully used for uncovering drugs and host factors that interfere with EBV stealthiness.

International audience

International audience

International audience; In this study we employed an in vivo approach coupled to 2D-DIGE and microarrays analysis to explore the response of porcine mesenteric lymph nodes (MLN) to S. Typhimurium infection at genomic and proteomic levels. MLN samples were collected from four control and twelve infected pigs (at 1, 2 and 6 days post infection) for protein and RNA purification. Protein was screened by differential in gel analysis and changes in transcriptome were assessed by Bayesian Analysis of Time Series. Afterwards, bioinformatic tools were employed to analyse and integrate data from both strategies. Comparison of genomic and proteomic data uncovered unique enriched mechanisms from each methodology. However, common functional changes were also observed, highlighting “Antigen processing and presentation” as the most significantly altered pathway in both approaches. Since both methodologies confirmed the induction of this mechanism in porcine MLN upon S. Typhimurium infection, we stress molecules taking part in this route as candidates for studies focusing on the genetic improvement of Salmonella resistance in pigs.

International audience

International audience

National audience

International audience

National audience

International audience; Intending to check the occurrence of the subclinical mastitis and to identify its causative agents, were collected 122 quarter samples of 31 cows from the herd of a small cheese industry placed in the city of Nossa Senhora do Livramento, MT. The samples were submitted to the Whiteside test and among these, the positive ones were analyzed in microbiological exams. 23 (74,2%) cows were carrying subclinical mastitis and the infected quarters percentage was 44,3% (54/122). The Staphylococcus were the most predominant pathogens (45,4%), and were identified as S. intermedius (41,4%), S. aureus (17,2%), S. warneri (6,9%), S. carnosus (3,4%). The strains that didn´t have their identification confirmed remained as Coagulase Negative Staphylococcus (27,6%) and Coagulase Positive Staphylococcus (3,4%). Also were isolated coliforms (15,6%), Candida kefyr (7,8%), Torulopsis glabrata (7,8%), Micrococcus sp. (4,7%), Sarcina sp.(3,1%), and other microorganisms (15,6%). The S. intermedius was the most predominant among all the etiological agents found, corresponding to 18,4% of the isolations. It was supposed that mastitis occurred in a high percentile because of the absence of prevention and control strategies.; Com o objetivo de verificar a ocorrência da mastite subclínica e identificar seus agentes etiológicos foram coletadas 122 amostras dos quartos mamários de 31 vacas do rebanho de uma queijaria em Nossa Senhora do Livramento, MT. Submeteram-se as amostras ao teste de Whiteside e destas as positivas foram analisadas por exames microbiológicos. 74,2% (23/31) das vacas demonstraram-se portadoras de mastite subclínica a um nível de infecção de 44,3% (54/122) dos quartos. Os Staphylococcus foram os patógenos mais incidentes (45,4%), sendo identificados como S. intermedius (41,4%), S. aureus (17,2%), S. warneri (6,9%), S. carnosus (3,4%). As cepas que não tiveram sua identificação confirmada permaneceram como Staphylococcus coagulase negativa (27,6%) e Staphylococcus coagulase positiva (3,4%). Isolou-se ainda coliformes (15,6%), Candida kefyr (7,8%), Torulopsis glabrata (7,8%), Micrococcus sp. (4,7%), Sarcina sp. (3,1%) e outros microrganismos (15,6%). A espécie S. intermedius foi a mais predominante dentre todos os agentes etiológicos encontrados, correspondendo a 18,4% dos isolamentos. Presumiu-se que a mastite ocorreu em um elevado percentual pela não adoção de medidas de controle e prevenção.

Setenta cepas de Staphylococcus coagulase positiva isoladas de leite de vacas com mastite subclínica e de queijo tipo Minas Frescal foram submetidas a testes bioquímicos para identificação das espécies e analisadas quanto a produção de algumas enzimas tais como: catalase, coagulase, linase, desoxirribonuclease (Dnase), hemolisinas, penicilinase, protease e termonuclease (TNase). Foram identificadas as espécies S. aureus, S. aureus subsp., anaerobius, S. delphini, S. hyicus, S. intermedius, S. schleiferi subsp. coagulans. As cepas que não tiveram sua identificação confirmada permaneceram com Staphylococcus coagulase positiva (SCP). A espécie predominante foi S. aureus (51,4 por cento) seguido dos SCP (22,8 por cento). Todas as cepas produziram pelo menos uma ou mais enzimas analisadas, sendo S. aureus a única espécie que apresentou resultados positivos na análise de todas as enzimas e obteve o maior percentual para a produção de TNase (50 por cento). Com relação à biotipagem, 93,3 por cento das cepas de S. aureus foram consideradas de origem bovina juntamente com 100 por cento das cepas SCP. A contaminação de alimentos por cepas patogênicas de Staphylococcus spp. pode resultar em problemas para a Saúde Pública, bem como em prejuízos para a indústria de alimentos.

International audience