Intestinal organoids to model Salmonella infection and its impact on progenitors.

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.

The Salmonella virulence protein PagN contributes to the advent of a hyper-replicating cytosolic bacterial population.

Salmonella 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.

Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis.

Salmonella 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.

IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica.

Salmonella is a genus of Gram-negative bacteria in the Enterobacteriaceae family causing various illnesses. The ability of the different serovars of Salmonella enterica subsp. enterica to infect a host and to induce pathology relies in part on their cellular and molecular interactions with the intestinal epithelium. In the current study, an in vitro approach using non-polarized or polarized IPEC-1 porcine intestinal epithelial cells were used in order to assess the relation between adhesion, invasion, and induction of the immune response as a function of the serotype of Salmonella. Five serovars, Choleraesuis (host-adapted), Typhimurium (ubiquitous), Typhisuis (host-restricted), which are relevant for pig infection, and Dublin and Gallinarum, which are host-restricted or host-adapted, were studied. A strong variation was observed in the percentages of adhesion and invasion amongst the S. enterica serovars used to interact with the non-polarized and polarized cells. Subsequently, differences were identified between serovars in terms of immune response induced. Serovars Typhimurium and Typhisuis induced a strong innate immune response four and half hours after the beginning of cell stimulation while Choleraesuis, Gallinarum, and Dublin did not. A strong inflammatory response could limit the spread of the porcine serovars to the gut while, with a weak response, bacteria may not be constrained by the immune response enabling severe systemic diseases. Different repertoires of adhesion factors and of secreted protein effectors between Salmonella serovars interacting with IPEC-1 cells probably explains the differences in their early pathogenic behaviours.

Gene expression in the chicken caecum is dependent on microbiota composition.

Gut microbiota is of considerable importance for each host. Despite this, germ-free animals can be obtained and raised to sexual maturity and consequences of the presence or absence of gut microbiota on gene expression of the host remain uncharacterised. In this study, we performed an unbiased study of protein expression in the caecum of germ-free and colonised chickens. The major difference between these two groups was in the expression of immunoglobulins which were essentially absent in the germ-free chickens. Microbiota also caused a minor decrease in the expression of focal adhesion and extracellular matrix proteins and an increase in the expression of argininosuccinate synthase ASS1, redox potential sensing, fermentative metabolic processes and detoxification systems represented by sulfotransferases SULT1C3 or SULT1E1. Since we also analysed expression in the caecum of E. coli Nissle and E. faecium DSM7134 mono-associated chickens, we concluded that at least immunoglobulin expression and expression of cystathionine synthase (CBS) was dependent on microbiota composition with E. coli Nissle stimulating more immunoglobulin and PIGR expression and E. faecium DSM7134 stimulating more CBS expression. Gut microbiota and its composition therefore affected protein expression in the chicken caecum though except for immunoglobulin production, the remaining differences were unexpectedly low.

An Updated View on the Rck Invasin of Salmonella: Still Much to Discover.

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.

Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis.

Salmonella Gallinarum and Salmonella Enteritidis are genetically closely related however associated with different pathologies. Several studies have suggested that S. Gallinarum is less invasive in vitro than S. Enteritidis. In this study we confirm that the S. Gallinarum strains tested were much less invasive than the S. Enteritidis strains tested in cells of avian or human origin. In addition, the S. Gallinarum T3SS-1-dependent ability to invade host cells was delayed by two to three hours compared to S. Enteritidis, indicating that T3SS-1-dependent entry is less efficient in S. Gallinarum than S. Enteritidis. This was neither due to a decreased transcription of T3SS-1 related genes when bacteria come into contact with cells, as transcription of hilA, invF and sipA was similar to that observed for S. Enteritidis, nor to a lack of functionality of the S. Gallinarum T3SS-1 apparatus as this apparatus was able to secrete and translocate effector proteins into host cells. In contrast, genome comparison of four S. Gallinarum and two S. Enteritidis strains revealed that all S. Gallinarum genomes displayed the same point mutations in each of the main T3SS-1 effector genes sipA, sopE, sopE2, sopD and sopA.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates.

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.

Salmonella enteritidis Rck-mediated invasion requires activation of Rac1, which is dependent on the class I PI 3-kinases-Akt signaling pathway.

The Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed.

Expression of Toll-like receptor 4 and downstream effectors in selected cecal cell subpopulations of chicks resistant or susceptible to Salmonella carrier state.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1ß (IL-1ß), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1ß, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

Ability of Bacillus cereus group strains to cause food poisoning varies according to phylogenetic affiliation (groups I to VII) rather than species affiliation.

Cytotoxic activity levels of culture filtrates and toxin distributions varied according to the phylogenetic group (I to VII) within the Bacillus cereus group, suggesting that these groups are of different clinical significance and are more suitable than species affiliations for determining food poisoning risk. A first-line, simple online tool (https://www.tools.symprevius.org/Bcereus/english.php) to assign strains to the different phylogenetic groups is presented.

TolC, but not AcrB, is involved in the invasiveness of multidrug-resistant Salmonella enterica serovar Typhimurium by increasing type III secretion system-1 expression.

The AcrAB-TolC efflux system is involved in multidrug and bile salt resistances. In addition, this pump has recently been suggested to increase the invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) into host cells in vitro and could therefore have an important clinical relevance for multidrug-resistant strains. The aim of this study was to investigate the role of the TolC outer membrane channel and the AcrB transporter in the interaction of multidrug-resistant S. Typhimurium strains with eukaryotic cells, especially in relation to the expression of the type III secretion system-1 (TTSS-1) required for Salmonella invasion. Deletion of tolC led to a reduced transcription of the Salmonella pathogenicity island-1 genes sipA, invF and hilA, demonstrating that all genes required for TTSS-1 biosynthesis are down-regulated in this mutant. Consequently, tolC mutants secreted smaller amounts of the TTSS-1 effector proteins SipA and SipC, and invasion tests performed with one mutant showed that it was significantly less able to invade HT-29 epithelial cells than its parental strain. This control seems specific to the TTSS-1 among the three TTSS of Salmonella as no down-regulation of expression of TTSS-2 or flagella was observed in this mutant. By contrast, acrB mutants behaved as their parents except that they secrete a slightly greater amount of SipA and SipC proteins. These data indicate that TolC but not AcrB mediates the uptake of multidrug-resistant S. Typhimurium into target host cells. Therefore, this role of TolC in the invasion of the intestine in addition to its role in bile salt resistance reinforces the interest of targeting TolC for fighting multidrug-resistant Salmonella.

Avirulence of viable but non-culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models.

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.

Virulence and cord blood mononuclear cells cytokine production induced by perinatal listeria monocytogenes strains from different phylogenetic lineages.

Some of the phylogenetic lineages of Listeria monocytogenes are more likely to cause invasive disease in humans than are strains from other phylogenetic lineages. This suggests that strains belonging to these lineages display different levels of pathogenicity. To investigate this, we carried out a plaque-forming assay with HT-29 cells to evaluate the virulence of six perinatal strains from the three lineages that compose the species. All of the strains were largely over the 3.34 cutoff (between 4.29 and 5.97 mean log) with the HT-29 model and can therefore be considered to be equally virulent. We also explored part of the immune response of cord blood mononuclear cells by measuring cytokine production. All strains induced the production of similar amounts of TNF-alpha and IL-1beta. High concentrations of IL-6 and IL-8 were produced (between 6,000 and 17,000 pg/ml), whereas little or no IFN-gamma or IL-12 was produced. Thus, there is no difference between the strains from the three genetic lineages in terms of virulence or cytokine response. Given the epidemiological distribution of the serotypes responsible for human listeriosis and the genetic structure of the L. monocytogenes species, our results suggest: (i) that all strains from lineage I (serotypes 1/2b and 4b), a genetically homogeneous subpopulation, have a similar level of pathogenicity, and (ii) that lineage II (serotypes 1/2a and 1/2c), which is genetically more heterogeneous, is composed of strains with different levels of pathogenicity. The ones responsible for invasive diseases, particularly perinatal infections, display a similar level of pathogenicity to lineage-I strains and are not virulence-attenuated strains that can only infect the most immunocompromised hosts, whereas the other lineage-II strains are probably less pathogenic for humans.

Establishment and characterization of partially differentiated chicken enterocyte cell clones.

Three enterocyte cell clones were established in vitro from the intestine of a PA12 hen embryo. These cells exhibited epithelioid morphology and grew as monolayers. The cells were continuously propagated in culture up to 250 passages. Gradual increase in growth rate with time and in anchorage-independent growth in both agar and agarose showed that the three cell clones spontaneously transformed in vitro. The clones were heteroploid with one marker chromosome. Interestingly, they had features of partly differentiated enterocytes, especially microvilli, junctions connecting adjacent cells (tight junctions, desmosomes, hemidesmosomes, gap junctions), villin and cytokeratins. In addition, cells expressed brush border enzyme activity and transepithelial resistance. The fact that the levels of dipeptidyl peptidase IV (DPP-IV) and alkaline phosphatase activities fluctuated according to culture time and that MHC class II was induced by activation of cells with interferon suggested that the state of differentiation of the 3 cell clones could be modified in vitro. These clones are the first established avian enterocyte cell clones to be described. Because each cell clone exhibited differences in the level of differentiation and sensitivity to Salmonella infection, their use will allow comparative investigations concerning markers of differentiation of avian enterocytes and infection by host-adapted bacteria and parasites.