Search

Finding strategies for decolonizing gut carriers of multidrug-resistant Escherichia coli (MDR-Ec) is a public-health priority. In this context, novel approaches should be validated in preclinical in vivo gut colonization models before being translated to humans. However, the use of mice presents limitations. Here, we used for the first time Zophobas morio larvae to design a new model of intestinal colonization (28-days duration, T28). Three hyperepidemic MDREc producing extended-spectrum β-lactamases (ESBLs) or carbapenemases were administered via contaminated food to larvae for the first 7 days (T7): Ec-4901.28 (ST131, CTX-M-15), Ec-042 (ST410, OXA-181) and Ec-050 (ST167, NDM-5). Growth curve analyses showed that larvae became rapidly colonized with all strains (T7, ~106-7 CFU/mL), but bacterial load remained high after the removal of contaminated food only in Ec-4901.28 and Ec-042 (T28, ~103-4 CFU/mL). Moreover, larvae receiving a force-feeding treatment with INTESTI bacteriophage cocktail (on T7 and T10 via gauge needle) were decolonized by Ec-4901.28 (INTESTI-susceptible); however, Ec-042 and Ec-050 (INTESTIresistant) did not. Initial microbiota (before administering contaminated food) was very rich of bacterial genera (e.g., Lactococcus, Enterococcus, Spiroplasma), but patterns were heterogeneous (Shannon diversity index: range 1.1-2.7) and diverse to each other (Bray-Curtis dissimilarity index ≥30%). However, when larvae were challenged with the MDR-Ec with or without administering bacteriophages the microbiota showed a non-significant reduction of the diversity during the 28-day experiments. In conclusion, the Z. morio larvae model promises to be a feasible and high-throughput approach to study novel gut decolonization strategies for MDR-Ec reducing the number of subsequent confirmatory mammalian experiments. [ABSTRACT FROM AUTHOR]

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection. IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) is the etiologic agent of a non-contagious arthropod-borne disease transmitted to wild and domestic ruminants. BTV induces a large panel of clinical manifestations ranging from asymptomatic infection to lethal hemorrhagic fever. Despite the fact that BTV has been studied extensively, we still have little understanding of the molecular determinants of BTV virulence. In our report, we have performed a comparative yeast two-hybrid (Y2H) screening approach to search direct cellular targets of the NS4 virulence factor encoded by two different serotypes of BTV: BTV8 and BTV27. This led to identifying Wilms' tumor 1-associated protein (WTAP) as a new interactor of the BTV-NS4. In contrast to BTV8, 1, 4 and 25, NS4 proteins from BTV27 and BTV30 are unable to interact with WTAP. This interaction with WTAP is carried by a peptide of 34 amino acids (NS422−55) within its putative coil-coiled structure. Most importantly, we showed that binding to WTAP is restored with a chimeric protein where BTV27-NS4 is substituted by BTV8-NS4 in the region encompassing residue 22 to 55. We also demonstrated that WTAP silencing reduces viral titers and the expression of viral proteins, suggesting that BTV-NS4 targets a cellular function of WTAP to increase its viral replication. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication. [ABSTRACT FROM AUTHOR]

Bluetongue (BT) is a non-contagious animal disease transmitted by midges of the Culicoides genus. The etiological agent is the BT virus (BTV) that induces a variety of clinical signs in wild or domestic ruminants. BT is included in the notifiable diseases list of the World Organization for Animal Health (OIE) due to its health impact on domestic ruminants. A total of 27 BTV serotypes have been described and additional serotypes have recently been identified. Since the 2000s, the distribution of BTV has changed in Europe and in the Mediterranean Basin, with continuous BTV incursions involving various BTV serotypes and strains. These BTV strains, depending on their origin, have emerged and spread through various routes in the Mediterranean Basin and/or in Europe. Consequently, control measures have been put in place in France to eradicate the virus or circumscribe its spread. These measures mainly consist of assessing virus movements and the vaccination of domestic ruminants. Many vaccination campaigns were first carried out in Europe using attenuated vaccines and, in a second period, using exclusively inactivated vaccines. This review focuses on the history of the various BTV strain incursions in France since the 2000s, describing strain characteristics, their origins, and the different routes of spread in Europe and/or in the Mediterranean Basin. The control measures implemented to address this disease are also discussed. Finally, we explain the circumstances leading to the change in the BTV status of France from BTV-free in 2000 to an enzootic status since 2018. [ABSTRACT FROM AUTHOR]

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. [ABSTRACT FROM AUTHOR]

We present the complete genome sequence of Pseudomonas canadensis . The strain (Pcan-CK-23) was isolated from Zophobas morio (superworm) larvae. The genome consisted of a 6,424,469 bp chromosome with a GC content of 60.3% and 5,973 genes. Pcan-CK-23 can be used as a reference genome for further studies with P. canadensis ., Competing Interests: The authors declare no conflict of interest.