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Abstract The gut microbiota exerts profound influence on poultry immunity and metabolism through mechanisms that yet need to be elucidated. Here we used conventional and germ-free chickens to explore the influence of the gut microbiota on transcriptomic and metabolic signatures along the gut-lung axis in poultry. Our results demonstrated a differential regulation of certain metabolites and genes associated with innate immunity and metabolism in peripheral tissues of germ-free birds. Furthermore, we evidenced the gut microbiota’s capacity to regulate mucosal immunity in the chicken lung during avian influenza virus infection. Finally, by fine-analysing the antiviral pathways triggered by the short-chain fatty acid (SCFA) butyrate in chicken respiratory epithelial cells, we found that it regulates interferon-stimulated genes (ISGs), notably OASL, via the transcription factor Sp1. These findings emphasize the pivotal role of the gut microbiota and its metabolites in shaping homeostasis and immunity in poultry, offering crucial insights into the mechanisms governing the communication between the gut and lungs in birds.

Abstract In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.

Abstract Selection for increased resistance to Salmonella colonisation and excretion could reduce the risk of foodborne Salmonella infection. In order to identify potential loci affecting resistance, differences in resistance were identified between the N and 61 inbred lines and two QTL research performed. In an F2 cross, the animals were inoculated at one week of age with Salmonella enteritidis and cloacal swabs were carried out 4 and 5 wk post inoculation (thereafter called CSW4F2 and CSW4F2) and caecal contamination (CAECF2) was assessed 1 week later. The animals from the (N × 61) × N backcross were inoculated at six weeks of age with Salmonella typhimurium and cloacal swabs were studied from wk 1 to 4 (thereafter called CSW1BC to CSW4BC). A total of 33 F2 and 46 backcross progeny were selectively genotyped for 103 and 135 microsatellite markers respectively. The analysis used least-squares-based and non-parametric interval mapping. Two genome-wise significant QTL were observed on Chromosome 1 for CSW2BC and on Chromosome 2 for CSW4F2, and four suggestive QTL for CSW5F2 on Chromosome 2, for CSW5F2 and CSW2BC on chromosome 5 and for CAECF2 on chromosome 16. These results suggest new regions of interest and the putative role of SAL1.

ABSTRACTSalmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella-containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor β1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.

Abstract Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt‐derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.

ABSTRACTSalmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S. Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S. Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S. Typhimurium.

ABSTRACT: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the main bacterial disease in poultry leading to significant economic losses worldwide. Antibiotic treatments favor the emergence of multidrug-resistant bacteria, and preventive measures are insufficient to control the disease. There is increasing interest in using the potential of bacteriophages, not only for phage therapy but also for prevention and biocontrol. This study aimed to evaluate the efficacy of a phage cocktail administered in ovo to prevent avian colibacillosis in chicks. When 4 different phages (REC, ESCO3, ESCO47, and ESCO58), stable under avian physiological conditions, were combined and inoculated at 17 embryogenic days (ED), they were transmitted to the newly hatched chicks. In a second trial, the 4-phage cocktail was inoculated into the allantoic fluid at ED16 and after hatch 1-day-old chicks were challenged with the O2 APEC strain BEN4358 inoculated subcutaneously. Two phages (REC and ESCO3) were still detected in the ceca of surviving chicks at the end of the experiment (7-days postinfection). Chicks that received the phages in ovo did not develop colibacillosis lesions and showed a significant decrease in intestinal BEN4358 load (8.00 × 107 CFU/g) compared to the challenged chicks (4.52 × 108 CFU/g). The majority of the reisolated bacteria from the ceca of surviving chicks had developed full resistance to ESCO3 phage, and only 3 were resistant to REC phage. The partially or complete resistance of REC phage induced a considerable cost to bacterial virulence. Here, we showed that phages inoculated in ovo can partially prevent colibacillosis in 1-wk-old chicks. The reduction in the APEC load in the gut and the decreased virulence of some resistant isolates could also contribute to control the disease.

Abstract Background Currently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains. Results These methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence. Conclusions Loss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

ABSTRACT The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus (Nouzillyvirus). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro, this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E. coli. Used in ovo, this phage combination allowed embryos to survive APEC infection. Thus, this phage combination represents a promising treatment for avian colibacillosis.

ABSTRACT Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. IMPORTANCE Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by S. Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

Abstract Background Salmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication resulting from consumption of contaminated poultry products. Genetic selection of animals that are more resistant to Salmonella carriage and modulation of the gut microbiota are two promising ways to decrease individual Salmonella carriage. The aims of this study were to identify the main genetic and microbial factors that control the level of Salmonella carriage in chickens (Gallus gallus) under controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines N and 61 were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition, using a 16S rRNA gene high-throughput sequencing approach. Results Our results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Among the 617 operational taxonomic units (OTU) observed, more than 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between the high and low Salmonella carriers, with 39 differentially abundant OTU. Using metagenome functional prediction based on 16S data, several metabolic pathways that are potentially associated to microbiota taxonomic differences (e.g. short chain fatty acids pathways) were identified between high and low carriers. Conclusions Overall, our findings demonstrate that the caecal microbiota composition differs between genetic lines of chickens. This could be one of the reasons why the investigated lines differed in Salmonella carriage levels under experimental infection conditions.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRβ repertoire and the influences of microbial colonisation on tissue-resident TCRβ+ cells. The results reveal that the microbiome is an important driver of TCRβ diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRβ sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRβ clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRβ repertoire.

Abstract Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.

Abstract Background Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear. Results Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of β1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in β1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-β1 integrin antibody, supporting the hypothesis that β1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization. Conclusions Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.

Summary Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super‐shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super‐shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal‐to‐animal cross‐contaminations and reinfections are reduced. We hypothesized that unlike super‐shedders, low‐shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic‐treated chicks are more prone to become super‐shedders; (ii) super or low‐shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low‐ and super‐shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host–cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

Super shedding occurs when a small number of individuals from a given host population shed high levels of a pathogen. Beyond this general definition, various interpretations of the shedding patterns have been proposed to identify super shedders, leading to the description of the super shedding phenomenon in a wide range of pathogens, in particular enteric pathogens, which are of considerable interest. Several underlying mechanisms may explain this observation, including factors related to the environment, the gut microbiota, the pathogen itself (i.e., genetic polymorphism), and the host (including immune factors). Moreover, data suggest that the interplay of these parameters, in particular at the host–pathogen–gut microbiota interface, is of crucial importance for the determination of the super shedding phenotype in enteric pathogens. As a phenomenon playing an important role in the epidemics of enteric diseases, the evidence of super shedding has highlighted the need to develop various control strategies.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

The immune system plays a crucial role in sustaining animal health. Its development is markedly influenced by early microbial colonization of the gastrointestinal tract.

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens.

Salmonella Typhimurium expresses on its outer membrane the protein Rck which interacts with the epidermal growth factor receptor (EGFR) of the plasma membrane of the targeted host cells. This interaction activates signaling pathways, leading to the internalization of Salmonella. Since EGFR plays a key role in cell proliferation, we sought to determine the influence of Rck mediated infection on the host cell cycle. By analyzing the DNA content of uninfected and infected cells using flow cytometry, we showed that the Rck-mediated infection induced a delay in the S-phase (DNA replication phase) of the host cell cycle, independently of bacterial internalization. We also established that this Rck-dependent delay in cell cycle progression was accompanied by an increased level of host DNA double strand breaks and activation of the DNA damage response. Finally, we demonstrated that the S-phase environment facilitated Rck-mediated bacterial internalization. Consequently, our results suggest that Rck can be considered as a cyclomodulin with a genotoxic activity.

Background Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus—T-VEC—has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent.Methods/Results We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model.Conclusion These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Dendritic cells (DCs) play a key role in immunity and are highly potent at presenting antigens and orienting the immune response. Depending on the environmental signals, DCs could turn the immune response toward immunity or immune tolerance. Several subsets of DCs have been described, with each expressing various surface receptors and all participating in DC-associated immune functions according to their specific skills. DC subsets could also contribute to the vicious circle of inflammation in immune diseases and establishment of immune tolerance in cancer. They appear to be appropriate targets in the control of inflammatory diseases or regulation of autoimmune responses. For all these reasons, in situ DC targeting with therapeutic antibodies seems to be a suitable way of modulating the entire immune system. At present, the field of antibody-based therapies has mainly been developed in oncology, but it is undergoing remarkable expansion thanks to a wide variety of antibody formats and their related functions. Moreover, current knowledge of DC biology may open new avenues for targeting and modulating the different DC subsets. Based on an update of pathogen recognition receptor expression profiles in human DC subsets, this review evaluates the possibility of inducing tolerant DCs using antibody-based therapeutic agents.

To establish an infection, Salmonella has to interact with eukaryotic cells. Invasion of non-phagocytic cells (i.e., epithelial, fibroblast and endothelial cells) involves either a trigger or a zipper mechanism mediated by the T3SS-1 or the invasin Rck, respectively. Another outer membrane protein, PagN, was also implicated in the invasion. However, other unknown invasion factors have been previously suggested. Our goal was to evaluate the invasion capability of a Salmonella Typhimurium strain invalidated for the three known invasion factors. Non-phagocytic cell lines of several animal origins were tested in a gentamicin protection assay. In most cells, we observed a drastic decrease in the invasion rate between the wild-type and the triple mutant. However, in five cell lines, the triple mutant invaded cells at a similarly high level to the wild-type, suggesting the existence of unidentified invasion factors. For the wild-type and the triple mutant, scanning-electron microscopy, confocal imaging and use of biochemical inhibitors confirmed their cellular uptake and showed a zipper-like mechanism of internalization involving both clathrin- and non-clathrin-dependent pathways. Despite a functional T3SS-1, the wild-type bacteria seemed to use the same entry route as the mutant in our cell model. All together, these results demonstrate the existence of unknown Salmonella invasion factors, which require further characterization.

Background: Recent studies have demonstrated an effect of the gut microbiota on brain development and behavior leading to the concept of the microbiota-gut-brain axis. However, its effect on behavior in birds is unknown. The aim of the present study was to determine the effect of the absence of gut microbiota on emotional reactivity in birds by comparing germ-free (GF) quails to those colonized (COL) with gut microbiota.Material and Methods: From hatching, the quails of both groups GF (n = 36) and COL (n = 36) were reared in sterile isolators. The COL quails were colonized at day 2 by introducing a pool of droppings from conventional adult females into the drinking water and feed. The quails were weighed individually on day 2, 6, and 14. From day 8, emotional reactivity was assessed in each group in the isolators through several behavioral tests.Results: GF quails showed a considerable decrease in emotional reactivity demonstrated by spending less time in tonic immobility during the tonic immobility test (242 s ± 31 vs. 331 s ± 32, p ≤ 0.05), traveling a shorter distance (3,897 cm ± 242 vs. 4,827 cm ± 278, p ≤ 0.05) at a lower velocity (6.55 cm/s ± 0.4 vs. 8.1 cm/s ± 0.5, p ≤ 0.05) during the social separation test and spending more time near an object at the beginning of the novel object test (33.7 s ± 6.4 vs. 18.5 s ± 4.1, p ≤ 0.05). No difference in growth was found between the 2 groups.Conclusion: For the first time, this study demonstrates that the absence of gut microbiota reduces emotional reactivity in Japanese quails in situations of fear and social perturbation without influence on growth.

Salmonella is a facultative intracellular Gram-negative bacterium, responsible for a wide range of food- and water-borne diseases ranging from gastroenteritis to typhoid fever depending on hosts and serotypes. Salmonella thus represents a major threat to public health. A key step in Salmonella pathogenesis is the invasion of phagocytic and non-phagocytic host cells. To trigger its own internalization into non-phagocytic cells, Salmonella has developed different mechanisms, involving several invasion factors. For decades, it was accepted that Salmonella could only enter cells through a type three secretion system, called T3SS-1. Recent research has shown that this bacterium expresses outer membrane proteins, such as the Rck protein, which is able to induce Salmonella entry mechanism. Rck mimics natural host cell ligands and triggers engulfment of the bacterium by interacting with the epidermal growth factor receptor. Salmonella is thus able to use multiple entry pathways during the Salmonella infection process. However, it is unclear how and when Salmonella exploits the T3SS-1 and Rck entry mechanisms. As a series of reviews have focused on the T3SS-1, this review aims to describe the current knowledge and the limitations of our understanding of the Rck outer membrane protein. The regulatory cascade which controls Rck expression and the molecular mechanisms underlying Rck-mediated invasion into cells are summarized. The potential role of Rck-mediated invasion in Salmonella pathogenesis and the intracellular behavior of the bacteria following a Salmonella Rck-dependent entry are discussed.

Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14+ monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients.

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

In order to understand the causes of the low virulence of Listeria monocytogenes field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and in vivo virulence. Molecular analyses revealed the same mutations in the picA, inlA and inlB genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain L. monocytogenes species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single trans complementation with the [inlA.sub.EGDe], [inlB.sub.EGDe] or [plcA.sub.EGDe] genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the [plcA.sub.EGDe] gene on the chromosome and trans complementation with either the [inlA.sub.EGDe] or the [inlB.sub.EGDe] gene restored the ability to form plaques, but only partly restored the in vivo virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed in vivo. These results indicate that the low virulence of L. monocytogenes strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

The folding and insertion of β-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) β-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella.

Salmonella enterica serotype Senftenberg (S. Senftenberg) has recently become more frequent in poultry flocks. Moreover some strains have been implicated in severe clinical cases. To explain the causes of this emergence in farm animals, 134 S. Senftenberg isolates from hatcheries, poultry farms and human clinical cases were analyzed. Persistent and non-persistent strains were identified in chicks. The non-persistent strains disappeared from ceca a few weeks post inoculation. This lack of persistence could be related to the disappearance of this serotype from poultry farms in the past. In contrast, persistent S. Senftenberg strains induced an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis, but a weaker systemic infection than S. Enteritidis in chicks and mice. An in vitro analysis showed that the low infectivity of S. Senftenberg is in part related to its low capacity to invade enterocytes and thus to translocate the intestinal barrier. The higher capacity of persistent than non-persistent strains to colonize and persist in the ceca of chickens could explain the increased persistence of S. Senftenberg in poultry flocks. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGD[DELTA]prfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix [alpha]H. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.