The flagellin of candidate live biotherapeutic Enterococcus...

来自 : 发布时间：2024-12-22

The flaGEllin of candidate live biotherapeutic Enterococcus gallinarum MRx0518 is a potent immunostimulant AbstractMany links between gut microbiota and disease development have been established in recent years, with particular bacterial strains emerging as potential therapeutics rather than causative agents. In this study we describe the immunostimulatory properties of Enterococcus gallinarum MRx0518, a candidate live biotherapeutic with proven anti-tumorigenic efficacy. Here we demonstrate that strain MRx0518 elicits a strong pro-inflammatory response in key components of the innate immune system but also in intestinal epithelial cells. Using a flagellin knock-out derivative and purified recombinant protein, MRx0518 flagellin was shown to be a TLR5 and NF-魏B activator in reporter cells and an inducer of IL-8 production by HT29-MTX cells. E. gallinarum flagellin proteins display a high level of sequence diversity and the flagellin produced by MRx0518 was shown to be more potent than flagellin from E. gallinarum DSM100110. Collectively, these data infer that flagellin may play a role in the therapeutic properties of E. gallinarum MRx0518. IntroductionEnterococcus gallinarum is a commensal Gram-positive species that sits within the Enterococcus casseliflavus clade of the Enterococcus 16鈥塖 rRNA phylogenic tree1,2. E. gallinarum and E. casseliflavus species are closely related, sharing over 99.8% nucleotide identity between their 16S rRNA genes3. E. gallinarum and E. casseliflavus are the only enterococcal species that are described as motile4,5 and unlike other members of the Enterococcus genus, they are infrequently linked with nosocomial infections6,7.In recent years, the role of the intestinal microbiota in cancer has received increasing attention because of its importance for immunotherapy efficacy聽(see review by Kroemer et al.8). Species in the Enterococcus genus have been identified as having potential uses in the growing field of oncobiotics9,10. Specifically, Enterococcus hirae has been shown to enhance cyclophosphamide efficacy by stimulating an anti-tumorigenic adaptive immune response following translocation to secondary lymphoid organs9. Routy et al. have also shown that E. gallinarum and several other enterococcal species were relatively overly abundant in patients who responded to immune checkpoint inhibitors (ICI)11. We have recently demonstrated that E. gallinarum MRx0518, a commensal strain that was isolated from a healthy human gut produces robust anti-tumorigenic effects after prophylactic oral dosing in murine models of breast, lung and renal carcinomas12.Flagellin from certain bacterial species are considered to be immunostimulatory and have also been exploited for their anti-tumorigenic and radioprotective potential (recently reviewed by Hajam et al.13). A Vibrio vulnificus flagellin expressed in an attenuated strain of Salmonella Typhimurium demonstrated tumour suppressive effects and decreased metastasis in murine models of orthotropic human colon cancer, when delivered intravenously14. Additionally, a Salmonella enterica flagellin derivative (CBLB502), is under investigation for the treatment of patients with advanced solid tumours15. Subcutaneous injection of this flagellin protein reduced tumour growth in a murine model of T-cell lymphoma through induction of pro-inflammatory cytokines and activation of cytotoxic lymphocytes16.Flagellin is a well-studied microbe-associated molecular pattern that is recognized by the transmembrane Toll-like receptor 5 (TLR5), which regulates the induction of downstream adaptive immune responses. TLR5 is expressed on the surface of a range of host cells including epithelial cells, endothelial cells, macrophages, dendritic cells (DCs) and T cells17,18,19. As a member of the TLR family, TLR5 forms an important link between the innate and adaptive immune systems and plays a role in the maintenance of gut homeostasis. TLR5 interacts with the extracellular monomeric form of bacterial flagellin of both Gram-negative and Gram-positive bacteria, leading to activation of the NF-魏B signalling pathway via the adaptor protein MyD88 and the serine kinase IRAK20,21,22. This can lead to systemic immune responses, stimulating the production of pro-inflammatory mediators including TNF-伪, IL-1尾, IL-6, IL-8, IL-12 and IL-23. A study by Cai et al. has shown that expression and activation of TLR5-associated pathways were elevated in breast carcinomas23. Furthermore, they demonstrated that flagellin activation of TLR5 in that context resulted in the local release of pro-inflammatory cytokines and anti-tumorigenic effects23.Historically, flagellin has been studied as a virulence-associated trait but is also recognized as a host colonisation factor. Bacterial flagellin is characterised by highly conserved N- and C-terminal domains (D0 and D1 domains) which have been shown to interact directly with TLR521,24. The hypervariable central region of flagellin (D2 and D3 domains) varies in size and structural organisation between species, and constitutes the main antigenic region of the protein24,25. Antigenic variation is thought to be one mechanism by which strains evolve to evade the host immune system26. Serologically distinct flagellins have been identified within bacterial species and have been used to track and type isolates27,28.Herein, we characterised the immunostimulatory potential of E. gallinarum MRx0518, a human commensal bacterium with demonstrated anti-tumorigenic properties12. This is the first study to examine the role of E. gallinarum flagellin as potential immunogens in the human gut. This work provides insights into the molecular effectors through which strain MRx0518 elicits an immunostimulatory response in human intestinal epithelial cells (IECs), macrophages and DCs and potentially exerts its anti-tumorigenic activity in vivo.Results Enterococcus gallinarum MRx0518 induces a strong immunostimulatory response in vitro To perform an initial assessment of the immunostimulatory potential of E. gallinarum MRx0518, we measured the cytokine responses of two key innate immune cell types, THP-1-derived macrophages and monocyte-derived DCs, after stimulation with live MRx0518 cells (Fig.聽1). We assessed a panel of pro- and anti-inflammatory cytokines involved in innate immunity and recruitment and activation of adaptive immune cells (IL-8,聽TNF伪, IL-6, IL-10, IL-12p70, IL-23 and聽IL-1尾). Both macrophages (Fig.聽1A) and DCs (Fig.聽1B) when unstimulated showed little to no production of the cytokines tested. As expected, a broadly consistent inflammatory profile was observed in both cell types in response to lipopolysaccharide (LPS), which was used as a pro-inflammatory response control (Fig.聽1A,B). However, while LPS induced production of pro-inflammatory cytokines IL-6, IL-8 and TNF伪 in both macrophages and DCs, LPS-mediated expression of IL-12p70 and IL-1尾 was lower in DCs in comparison to macrophages (Fig.聽1A,B). Compared to LPS, the cytokine production profiles were more consistent across the two cell types following E. gallinarum MRx0518 treatment (Fig.聽1A,B). Both LPS and MRx0518 significantly induced IL-8 production in macrophages and DCs (p鈥?lt;鈥?.0001). MRx0518 also induced production of all other pro-inflammatory cytokines tested in both cell types. Significantly higher levels of TNF伪 (p鈥?lt;鈥?.001 and p鈥?lt;鈥?.01), IL-6 (p鈥?lt;鈥?.001 and p鈥?lt;鈥?.001) IL-12p70 (p鈥?lt;鈥?.05 and p鈥?lt;鈥?.001) and IL-23 (p鈥?lt;鈥?.001 and p鈥?lt;鈥?.05) in comparison to untreated cells were observed in macrophages and DCs. IL-1尾 production was also induced by MRx0518 stimulation in both cells types but only reached statistical significance in macrophages (p鈥?lt;鈥?.05). In addition to up-regulating the production of pro-inflammatory cytokines, MRx0518 treatment also induced a significant increase in IL-10 levels in comparison to LPS-treated and untreated cells. Of note, the variation observed in cytokine production by DCs (Fig.聽1B) is most likely attributable to inherent donor heterogeneity. Overall, the data indicate that strain MRx0518 has a clear and potent immunostimulatory effect on host immune cells by inducing the production of a range of pro- and anti-inflammatory cytokines associated with both innate and adaptive immunity.Figure 1Cytokine production by THP-1-derived macrophages and monocyte-derived dendritic cells in response to E. gallinarum MRx0518. IL-8, TNF伪, IL-6, IL-10, IL-12p70, IL-23, IL-1尾 concentrations (pg/ml) in (A) THP-1-derived macrophages and (B) monocyte-derived DCs cell-free supernatants after 1鈥塰 contact with E. gallinarum MRx0518 (MRx0518LV). A multiplicity of infection (MOI) of 10:1 was employed for both stimulation assays. A MOI of 1:1 was employed for IL-8 detection in macrophages, due to saturation of the assays at the MOI of 10:1. The effect of LPS on cytokines production is also shown as a positive control. The graphs represent an average of at least three biological replicates. Statistical comparisons were performed with GraphPad Prism (La Jolla, CA, USA) using ordinary one-way ANOVA analysis followed by Tukey鈥檚 multiple comparison tests. Statistically significant differences with the untreated control are shown on the graphs as *(p鈥?lt;鈥?.05), **(p鈥?lt;鈥?.01), ***(p鈥?lt;鈥?.001) and ****(p鈥?lt;鈥?.0001).Full size image E. gallinarum MRx0518 treatment affects gene expression in human intestinal epithelial cellsIECs represent one of the primary points of contact between commensal bacteria and the host in the gut. A Transwell庐 co-culture system was employed to assess the transcriptional response of the mucin-secreting cell line HT29-MTX to treatment with MRx0518, using a Human Transcriptome Microarray (Fig.聽2). Treatments with metabolically active cells (MRx0518LV), inactivated cells (MRx0518HK) or culture supernatants (MRx0518SN) were found to induce distinct host responses. Treatment with MRx0518SN elicited the largest number of differentially expressed genes, inducing upregulation of 275 genes, 228 of which were not upregulated by other MRx0518 treatments (Supplementary Table聽S1). MRx0518LV and MRx0518HK induced the upregulation of 106 and 63 genes respectively that were not upregulated in MRx0518SN-treated cells. Similarly, the MRx0518SN also induced the downregulation of the largest number of genes in IECs (Fig.聽2). Only 14 upregulated genes and one downregulated gene were common to all treatment groups (Supplementary Table聽S1). MRx0518HK cells had the least impact on IEC transcription levels (Fig.聽2). Despite MRx0518SN treatment inducing the largest number of differentially expressed genes in IECs, pathway enrichment analysis of the transcriptomic data indicated that MRx0518LV had the largest impact on physiological pathways, with over-representation of pathways involved in innate inflammatory responses, interferon signalling and apoptosis (Supplementary Fig.聽S1). Of particular interest was the upregulation of CCL20 (~20-fold) and CXCL8 (~5-fold) in MRx0518LV- and MRx0518SN-treated cells, both of which play a role in immune cell recruitment (Table聽1). NFKBIA and TNFAIP3, genes involved in regulating NF-魏B signalling, were also significantly upregulated in MRx0518LV-treated cells. ICAM1 was significantly upregulated in MRx0518LV-treated cells, but not in MRx0518SN-treated cells (Table聽1). HT29-MTX cells demonstrated modest upregulation of CXCL1 expression (2.41-fold) in response to MRx0518LV, which was not observed with other treatments.Figure 2Transcriptomic analysis of the response of HT29-MTX cells to E. gallinarum MRx0518 treatments. Venn diagrams showing (A) up- and (B) down-regulated genes in HT29-MTX cells after 3鈥塰 contact with MRx0518 live (MRx0518LV), heat-killed (MRx0518HK) and culture supernatant (MRx0518SN) (MOI 100:1 or equivalent). Each treatment was compared to its respective control (cell culture media or YCFA). Diagrams were generated with InteractiVenn62.Full size imageTable 1 Immunomodulatory genes selected for further analysis. Genes were filtered based on a fold change鈥夆墺鈥?.5, p鈥?lt;鈥?.05, coding transcripts only and the presence of a gene symbol. n.s.: not significant.聽Full size tableA protein in MRx0518 culture supernatant activates NF-魏B and TLR5 reporter cellsThe immunostimulatory response observed in macrophages, DCs and IECs following stimulation with MRx0518 appears to be exerted, in part, through NF-魏B signalling. In order to confirm activation of this signalling pathway, we examined the effect of MRx0518 treatments (MRx0518LV, MRx0518HK and MRx0518SN) on NF-魏B and TLR5 reporter cell lines (Fig.聽3). NF-魏B activation was assessed by measuring the expression of the secreted embryonic alkaline phosphatase (SEAP) reporter gene. MRx0518LV did not activate NF-魏B reporter cells but activated TLR5 reporter cells (p鈥?lt;鈥?.0001) (Fig.聽3A,B). The lack of SEAP detection in NF-魏B reporter cells is likely due to the growth of MRx0518 in the cell culture media during the 22鈥塰 incubation which possibly impacted the viability of the reporter cells rather than genuine absence of signalling activation. MRx0518HK induced a strong response in the TLR5 reporter cells, which was slightly higher than that observed in the NF-魏B reporter cells (p鈥?lt;鈥?.0001 for both cell lines in comparison to untreated cells) (Fig.聽3A,B). Both reporter cell lines were activated by MRx0518SN, to the same extent as their respective positive controls (Fig.聽3A,B). Overall, MRx0518SN was the most potent stimulant of both NF-魏B and TLR5. These results, combined with the transcriptional response of HT29-MTX cells to MRx0518SN, prompted us to investigate the active component in this fraction. In an effort to identify the nature of molecules responsible for the observed host response, we treated MRx0518SN with a range of enzymes (i.e. DNase, proteases and apyrase). Trypsin treatment had the greatest effect on activation of NF-魏B and TLR5 reporter cells, while other enzymatic treatments had smaller effects (data not shown). TLR5 activation was completely abolished by trypsin treatment, whereas low but detectable activation remained in the NF-魏B reporter cells (p鈥?lt;鈥?.0001 when compared to MRx0518SN) (Fig.聽3C,D). These assays established that a molecule of proteinaceous nature was present in MRx0518SN which was most likely responsible for TLR5-mediated NF-魏B activation. However, residual NF-魏B activation (Fig.聽3C) also suggested that other molecules in MRx0518SN that are not affected by trypsin digestion may contribute to NF-魏B signalling. Flagellin is the only known TLR5 ligand, and both expression profiling and phenotypic observations indicated that MRx0518 expresses flagellin in its late log growth phase and is motile under in vitro conditions (Supplementary Fig.聽S2). NanoLC-MS/MS analysis confirmed the presence of flagellin in high abundance in MRx0518SN (Supplementary Table聽S2), strongly suggesting that flagellin was the molecule responsible for the observed NF-魏B activation in the reporter assays.Figure 3Activation of NF-魏B and TLR5 pathway by E. gallinarum MRx0518 treatments. NF-魏B (A) and TLR5 (B) activation after 22鈥塰 incubation with E. gallinarum MRx0518 (MRx0518LV), heat-killed MRx0518 (MRx0518HK) and culture supernatant (MRx0518SN) in HEK-Blue鈩?hTLR5 and THP1-Blue鈩?NF-kB reporter cell lines. A MOI of 10:1 was used with MRx0518LV and a 100:1 MOI equivalent was used with MRx0518HK and MRx0518SN. Heat-killed Listeria monocytogenes (HKLM) and Salmonella Typhimurium flagellin (FLA-ST) were used as positive controls for each cell line and YCFA was included as a negative control for MRx0518SN. NF-魏B (C) and TLR5 (D) activation after 22鈥塰 incubation with E. gallinarum MRx0518 culture supernatant (MRx0518SN) and trypsin-treated supernatant (MRx0518Trypsin) (MOI 100:1 equivalent). Each graph represents an average of at least three biological replicates. Statistical comparisons were performed with GraphPad Prism using ordinary one-way ANOVA analysis followed by Tukey鈥檚 (A,B), Dunnett鈥檚 (C) or Sidak鈥檚 (D) multiple comparison tests. Statistically significant differences with the relevant control are shown on the graphs as ****(p鈥?lt;鈥?.0001).Full size imageFlagellin is responsible for the activation of NF-魏B and TLR5 reporter cellsAn insertion mutation in the flagellin gene (fliC) in E. gallinarum MRx0518 (strain MRx0518 fliC::pORI19) was generated. Genetic manipulations in E. gallinarum had not been described in the literature prior to this study, it was therefore necessary to develop a transformation protocol for strain MRx0518. Electrocompetent cells were successfully generated by growing bacterial cultures in sub-inhibitory concentrations of glycine29 followed by mutanolysin and lysozyme treatments to further weaken the cell wall peptidoglycan layer (see Material and Methods for details). The fliC gene was disrupted by homology-driven insertion of the suicide plasmid pORI1930. Insertion of pORI19 within the fliC gene was confirmed by DNA sequencing and the non-motile phenotype of the resulting mutant strain was confirmed in vitro (Supplementary Fig.聽S2). Both MRx0518SN and MRx0518 fliC::pORI19 culture supernatant (fliCSN) were tested in the NF-魏B and TLR5 reporter assays along with culture supernatant from an additional E. gallinarum strain, DSM100110 (DSM100110SN) (Fig.聽4A,B). Strain DSM100110 is a murine isolate which was chosen for its highly-motile phenotype in vitro (Supplementary Fig.聽S2). A significant reduction of NF-魏B activation (approximately 75% compared to MRx0518SN) was observed for fliCSN-treated NF-魏B reporter cells (p鈥?lt;鈥?.0001) (Fig.聽4A). The presence of additional stimulatory molecules in fliCSN may have contributed to the observed residual activation of NF-魏B signalling, as previously noted for trypsinized-MRx0518SN (Fig.聽3C). Inactivation of the flagellin gene completely abolished TLR5 activation (no observable difference with the YCFA culture medium control) and was significantly reduced in comparison to MRx0518SN (p鈥?lt;鈥?.0001) (Fig.聽4B). Interestingly DSM100110SN induced very little activation of the TLR5 reporter cells (not statistically significant when compared to YCFA). Analysis by nanoLC-MS/MS of the protein content of DSM100110SN revealed the absence of flagellin (data not shown), which explains the observed lack of TLR5 activation elicited by this strain (Fig.聽4B).Figure 4Flagellin plays a role in E. gallinarum MRx0518 immunostimulatory effect. NF-魏B (A) and TLR5 (B) reporter assays with MRx0518 (MRx0518SN), MRx0518 fliC::pORI19 (fliCSN) and DSM100110 (DSM100110SN) culture supernatants (MOI 100:1 equivalent). NF-魏B (C) and TLR5 (D) reporter assays with a range of concentrations of E. gallinarum MRx0518 and DSM100110 purified recombinant flagellins (FliCMRx0518 and FliCDSM100110). The 鈥楥ontrol鈥?bar corresponds to the empty vector control. Each graph represents an average of at least three biological replicates. Reporter cells were incubated with treatments for 22鈥塰. Statistical comparisons were performed with GraphPad Prism using ordinary one-way ANOVA analysis followed by Tukey鈥檚 (A,B) or Sidak鈥檚 (C,D) multiple comparison tests. Statistically significant differences with the relevant control are shown on the graphs as *(p鈥?lt;鈥?.05), ***(p鈥?lt;鈥?.001) and ****(p鈥?lt;鈥?.0001).Full size imageThe lack of flagellin in DSM100110SN limited our ability to determine the immunogenic potential of its flagellin in supernatant-stimulation assays. Flagellins from strains MRx0518 and DSM100110 were therefore overexpressed and purified as N-terminally his-tagged recombinant proteins (Supplementary Fig.聽S3) and used to perform a dose-response assay. Both recombinant flagellins were capable of activating NF-魏B and TLR5 reporter cells (Fig.聽4C,D). Lower protein concentrations stimulated a strong response in TLR5 reporter cells in comparison to the NF-魏B reporter cells. This was perhaps unsurprising as flagellin is known to interact directly with TLR520 and this TLR5 reporter cell line is expected to be 20鈥?00 fold more responsive than cell lines that express basal levels of TLR5, as indicated by the manufacturer. Purified FliCMRx0518 showed comparable levels of activation to MRx0518SN in both reporter cell lines, with saturation levels dropping at concentrations less than 1鈥塶g/ml. FliCDSM100110 was weakly active in the NF-魏B reporter assay at the concentrations tested (Fig.聽4C). At concentrations greater than 5鈥塶g/ml, both recombinant proteins induced comparable responses in TLR5 reporter cells, which were not significantly different (Fig.聽4D). However, at lower concentrations聽FliCMRx0518 was significantly more stimulatory than FliCDSM100110 at equivalent concentrations (p鈥?lt;鈥?.0001 for tests at 0.2 and 1鈥塶g/ml and p鈥?lt;鈥?.05 for 0.04鈥塶g/ml). The same trend was observed in the NF-魏B reporter cells (p鈥?lt;鈥?.0001 for 0.8, 4 and 20鈥塶g/ml).FliCMRx0518 and FliCDSM100110 display sequence divergence and reside in distinct clusters of a FliC phylogenetic treeFliCMRx0518 displayed a higher capacity to stimulate both TLR5 and NF-魏B than FliCDSM100110 at low concentrations. This prompted the further examination of the flagellar loci and particularly the FliC protein sequences of both strains. Organisationally-conserved 40-kb motility loci were identified in the genome sequences of strains MRx0518 and DSM100110, both of which encode 47 contiguous genes (Fig.聽5). Gene organisation was similar to that of other motile enterococci4 and both strains share 69.3% nucleotide (nt) identity (ID) over the length of the operon. Indels are present between the two loci, which result in changes to the start and stop sites of a number of homologous genes but are not predicted to result in the formation of pseudogenes. Each strain encodes a single FliC protein which are 360 amino acid (aa) and 361 aa long in MRx0518 and DSM100110 respectively and share 77.2% aa identity (Fig.聽S4 and Supplementary Dataset聽S1). Modelling of the structure of FliCMRx0518 revealed the presence of three domains (Fig. S4), as predicted using the Phyre2 server31 (data not shown).Figure 5Sequence alignment of the flagellar loci of E. gallinarum MRx0518 and E. gallinarum DSM100110. A linear comparison of the BLASTN matches between the flagellar loci of of E. gallinarum strains MRx0518 and DSM100110. Vertical grey-coloured blocks between sequences indicate regions of shared nucleotide ID. The gradient of the grey colour corresponds to the percentage of shared nt ID. The genes in each element are coloured according to their function, as follows: blue (biosynthesis), green (chemotaxis), grey (other function) and yellow (hypothetical proteins).Full size imagePhylogenetic analyses indicated that the FliC proteins of E. gallinarum and E. casseliflavus branch closely to the FliC proteins of motile lactobacilli (Supplementary Fig.聽S5, Supplementary Dataset聽S1)32,33. In order to assess the level of diversity within the flagellin of these closely related species, the FliC sequences of 15 E. gallinarum and 3 E. casseliflavus strains derived from the 4D Pharma plc and DSMZ culture collections (Supplementary Table聽S3), together with those available in public databases (11 E. gallinarum and 27 E. casseliflavus), were assessed by comparative analyses. The FliC proteins of E. gallinarum varied in length from 352 aa to 361 aa, while the E. casseliflavus FliC proteins varied between 357 aa and 361 aa. Between 75.82% and 100% aa ID was observed among the examined proteins, with several E. gallinarum FliC proteins displaying higher levels of sequence homology to E. casseliflavus FliC, than to each other (Supplementary Fig.聽S4, Supplementary Dataset聽S1). The highest level of sequence divergence both within and between the E. gallinarum and E. casseliflavus FliC proteins was observed in the D2 region whereas the D0 and D1 regions were more highly conserved (Fig.聽S4). The regions known to be critical for TLR5 interaction in other bacterial species21,34 were found to be conserved (residues 87鈥?6) in all strains examined. Three distinct clusters were present within the FliC-based Maximum Likelihood phylogenetic tree shown in Fig.聽6, with two well-supported E. gallinarum clusters evident (E. gallinarum_1 and E. gallinarum_2) and the majority of E. casseliflavus strains grouping together. Interestingly, strains MRx0518 and DSM100110 were resident in distinct clusters, each of which broadly represent their sources of isolation (Fig.聽6).Figure 6Phylogenetic analysis of the FliC protein of E. gallinarum and E. casseliflavus. The FliC sequences of selected strains (Supplementary Table聽S3) were aligned with MUSCLE59. The evolutionary history was inferred by using the maximum likelihood method based on the Le_Gascuel_2008 model61, using MEGA7 software60. Statistical support (above 60%) was estimated with bootstraps and is indicated at branch nodes. Well-defined clades are indicated. The type strain Lactobacillus mali DSM20444 was used as an outgroup for analyses. The origins of strains are indicated where the information was available on the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov).Full size imageInactivation of the flagellin gene abolishes MRx0518 immunogenic effects in IECsIn order to confirm the involvement of flagellin in the observed immunostimulatory effects of strain MRx0518, the impact of MRx0518SN and fliCSN together with FliCMRx0518 recombinant flagellin on gene expression and cytokine production levels in IECs were tested. The changes in IEC gene expression following stimulation with MRx0518SN, fliCSN and FliCMRx0518 were investigated using a targeted panel of qPCR primers, designed based on the transcriptional profiles of IECs in response to the聽MRx0518 treatments described above (Table聽1, Fig.聽7A). The expression of NFKBIA was unchanged, despite a slight upregulation being observed in the microarray-derived data (1.61-fold). MRx0518SN significantly induced the expression of the CCL20 and CXCL8 genes (p鈥?lt;鈥?.0001 and p鈥?lt;鈥?.05 respectively compared to the YCFA treatment)聽(Fig. 7A), which was consistent with the upregulation previously observed. fliCSN had no effect on expression levels of the five genes tested. Co-culture of HT29-MTX cells with fliCSN did not induce the stimulatory response observed with MRx0518SN which strongly supports the role of flagellin as a major effector of MRx0518 immunogenicity. This was further confirmed by co-culturing HT29-MTX cells with recombinant MRx0518 flagellin. The addition of FliCMRx0518 led to a significant (p鈥?lt;鈥?.05) upregulation in the expression of all five genes in the panel, with fold changes higher than those observed with MRx0518SN (Fig.聽7A). The levels of IL-8 secreted by HT29-MTX cells stimulated with MRx0518SN, fliCSN, DSM100110SN and FliCMRx0518 was measured in cell-free supernatants following 24鈥塰 co-culture (Fig.聽7B). MRx0518SN induced a significant release of IL-8 in IECs in comparison to cells treated with YCFA (p鈥?lt;鈥?.0001). The inactivation of the flagellin gene in the MRx0518 strain reduced IL-8 secretion to levels comparable to those observed with YCFA. Treatment of cells with recombinant flagellin strongly stimulated IL-8 secretion in comparison to the untreated and YCFA groups (p鈥?lt;鈥?.0001) but also in comparison to cells treated with MRx0518SN (p鈥?lt;鈥?.001). In contrast, DSM100110SN had no observable impact on IL-8 stimulation (Fig.聽7B).Figure 7Inactivation of the flagellin gene in E. gallinarum MRx0518 abolishes its immunostimulatory profile. (A) Changes in gene expression in HT29-MTX after 24鈥塰 co-culture with MRx0518 and MRx0518 fliC::pORI19 culture supernatants (MRx0518SN and fliCSN) (MOI 100:1 equivalent) and 1鈥壜礸/ml purified MRx0518 recombinant flagellin (FliCMRx0518), measured by qPCR (fold change compared to the YCFA or the empty vector control, as appropriate). Statistical comparisons were performed with GraphPad Prism on 螖CT values using two-way ANOVA followed by Tukey鈥檚 multiple comparison tests. Statistically significant differences in comparison to the YCFA or empty vector control group as appropriate are shown on the graph as *(p鈥?lt;鈥?.05), **(p鈥?lt;鈥?.01) and ****(p鈥?lt;鈥?.0001). (B) IL-8 concentrations (pg/ml) detected by ELISA assay in HT29-MTX cell-free supernatant after 24鈥塰 co-culture with MRx0518 (MRx0518SN), MRx0518 fliC::pORI19 (fliCSN) and DSM100110 (DSM100110SN) culture supernatants (MOI 100:1 equivalent), and 1鈥壜礸/ml purified MRx0518 recombinant flagellin (FliCMRx0518). YCFA was included as a negative control. Statistical comparisons were performed with GraphPad Prism using an ordinary one-way ANOVA followed by Tukey鈥檚 multiple comparison test. Statistically significant differences with the relevant control are shown on the graphs as ***(p鈥?lt;鈥?.001) and ****(p鈥?lt;鈥?.0001).Full size imageDiscussionEnterococcus gallinarum MRx0518 is a candidate live biotherapeutic, isolated from a healthy human faecal sample. Oral delivery of this strain has demonstrated anti-tumour efficacy in murine models of breast, lung and renal carcinomas12. Only a limited number of studies have characterised strains of this species in any detail, and fewer still that have examined the interactions of E. gallinarum strains with the immune system35,36. To begin to understand how strain MRx0518 interacts with the host, we examined its effect upon IECs, macrophages and DCs, host cells which have distinct roles in the innate immune response. DCs are capable of priming T cells at distal sites and stimulating a homing response to drive T cell accumulation to sites of inflammation. Macrophages tend to act locally to maintain homeostasis and can induce secondary activation of T cells. Both cell types come into direct contact with luminal bacteria in the gut but can also play a role in anti-tumour immunity at distal tumour sites. MRx0518LV elicited a strong and consistent pro-inflammatory signature in both macrophages and DCs, at levels similar to or higher than those elicited by the control inflammatory stimulant LPS. MRx0518LV significantly elevated levels of TNF伪, a known regulator of IL-6 and IL-8 production, in both DCs and macrophages. Similarly, a study by van den Bogert and colleagues found that E. gallinarum HSIEG1 is also capable of inducing cytokine secretion in vitro35. IL-10, a cytokine well-described for its anti-inflammatory and tolerogenic effects, was also induced by MRx0518LV treatment and can actively suppress the expression of IL-6, IL-12, IL-1尾 and TNF伪. Given the elevation of both pro- and anti-inflammatory cytokines in response to MRx0518 treatment, it is noteworthy that IL-10 and IL-6 are reciprocal cytokines, both utilising the activity of transcription factor聽STAT3 to alter cellular responses with broadly opposing effects37. IL-10 is not a pan-inhibitory cytokine of inflammatory responses; it is known to activate and increase CD8伪 cytotoxic capacity which may be significant for anti-tumour responses38,39.High levels of IL-8 production were observed in macrophages and DCs in response to MRx0518LV exposure. Additionally, MRx0518LV, MRx0518SN and purified flagellin all induced expression of CCL20 and CXCL8 in HT29-MTX cells; the products of which are implicated in the recruitment of immune cells and the subsequent activation of the adaptive branch of the immune system. Similarly, Salmonella-derived flagellin has been shown to stimulate expression of the CCL20 gene in Caco-2 cells40 and E. coli flagellin has been shown to induce secretion of IL-8 and CCL20 in HT29鈥?9A and Caco-2 cells41.Flagellin is a potent immunostimulant which acts through TLR5 and has been exploited in recent years for its capacity as a vaccine adjuvant and its anti-tumorigenic efficacy13. Purified MRx0518 flagellin showed significant activation of TLR5 in reporter cells and proved more potent than DSM100110 flagellin at equivalent nanomolar concentrations. Given the body of work that is emerging regarding the role of TLRs and their associated ligands in anti-cancer therapies, the potential contribution of flagellin to MRx0518 anti-tumour activity warrants further investigation. The administration of S. Typhimurium flagellin has been shown to reduce tumour growth and cell proliferation in colon and breast cancer cells23,42. An elegant study by聽Cai et al. demonstrated that 80% of breast carcinoma聽tissues tested were found to be positive for TLR5 expression and that聽TLR5-signalling was also聽upregulated in breast carcinomas23. They concluded that flagellin-mediated TLR5 activation is involved in modulation of the tumour microenvironment and mediates its anti-tumorigenic effect through pro-inflammatory cytokine induction. A flagellin from V. vulnificus expressed in an attenuated strain of S. Typhimurium was shown to be effective in tumour growth reduction in several murine cancer models when delivered intravenously14. Interestingly, Zheng et al. showed that Salmonella and flagellin demonstrate complementarity to recruit and activate immune cells, through colonization of the tumour site and interaction with TLR5 respectively14.Activation of TLRs on the surface of tumours may require transport or delivery of flagellin to distal聽tumour sites which could be achieved through translocation of the bacteria or their components from the gut. Manfredo-Viera et al. recently demonstrated that E. gallinarum was able to translocate from the murine gut to induce an autoimmune response in immunocompromised mice36. Translocation of another enterococcal species E. hirae to secondary lymphoid organs has been shown to enhance efficacy of a chemotherapeutic agent9. Additionally, E. gallinarum was found to be overly abundant in patients who responded to treatment with anti-PD111, suggesting a potential role for this species in patient responsiveness to ICI treatments. Studies are currently ongoing to investigate the ability of MRx0518 to translocate from the gut to extra-intestinal sites. Testing聽the translocation聽potential of MRx0518-derivatives, including flagellin, is also underway using FliCMRx0518-directed antibodies.Inter- and intra-species comparative analysis of the FliC proteins of E. gallinarum and E. casseliflavus indicate that the D0 and D1 domains are highly conserved while the majority of variability lies within the D2 domains, as observed for flagellin of other species25. The sequence divergence displayed in the FliC sequence in E. gallinarum is comparable with that of C. difficile43 and E. coli44 and is less than that reported for P. aeruginosa45 and B. thuringiensis46. Antigenic variation in the FliC sequence may contribute to differences in immunogenic potential of E. gallinarum strains. However, it is yet to be determined to what extent the variance observed in the D2 domains of FliCMRx0518 and FliCDSM100110 contributes to the immunogenic profiles of these strains.Inactivation of flagellin in MRx0518 resulted in complete abrogation of TLR5-mediated activation of NF-魏B. However, some residual activity remained in the NF-魏B reporter cells when treated with fliCSN. This suggests the involvement of additional or complementary bacterial effectors present in culture supernatants. Of particular interest was the identification of enolase as the most abundant protein in the MRx0518SN (Supplementary Table聽S2) as enolase has been shown to play a role in host-interactions in lactic acid bacteria, through plasminogen binding47. Small molecules such as ATP and CpG DNA, can act synergistically with flagellin to trigger host immune responses48,49,50. The potential contribution of these molecules to the observed immunogenic effects of MRx0518 warrants further investigation.Taken together, these data demonstrate that E. gallinarum MRx0518, and more specifically its flagellin, is a strong immunostimulant of both immune and intestinal epithelial cells. Importantly FliCMRx0518 displays higher potency than FliCDSM100110. The extent of the activity of MRx0518 flagellin in vivo remains to be determined. In this context, MRx0518 derivatives are currently being investigated in murine cancer models in order to shed light on their influence聽on聽the previously established therapeutic effect of E. gallinarum MRx0518.Material and MethodsBacterial strains, plasmids and culture conditionsE. gallinarum strains were routinely cultured in Yeast extract, Casitone, Fatty Acid media (YCFA, E O Laboratories, Bonnybridge, Scotland, UK) at 37鈥壜癈 in an anaerobic cabinet (Don Whitley Scientific, Shipley, England, UK). Late log phase cultures were grown for approximately 3鈥塰 (10% inoculum). E. coli strains were grown in Luria-Bertani broth at 20鈥壜癈 or 37鈥壜癈 in aerobic conditions with shaking (180鈥?00鈥塺pm). Growth media were supplemented with erythromycin (20鈥壩糶/ml for E. gallinarum and 100鈥壩糶/ml for E. coli), ampicillin (100鈥壩糶/ml) and kanamycin (25鈥?0鈥壩糶/ml) (Sigma-Aldrich, Gillingham, England, UK), where appropriate. Bacterial strains and plasmids used in this study are listed in Supplementary Table聽S3.Preparation of bacterial fractions for co-culture assaysLate log phase bacterial cultures were centrifuged at 5000 x g for 5鈥塵in at room temperature to generate bacterial fractions. Pelleted cells were washed once in phosphate-buffered saline (PBS) (Sigma-Aldrich) and resuspended in antibiotic-free cell culture media to the appropriate dilution (live fraction, MRx0518LV). Culture supernatants were harvested and filtered through a 0.22 渭m pore size filter and diluted in water to provide equivalents for the live fraction described above (supernatant fraction, MRx0518SN). Bacterial cultures were heat-inactivated for 40鈥塵in at 80鈥壜癈 and prepared as described above for the live fraction (heat-killed fraction, MRx0518HK). Viable cell counts were determined by spread plating. When required, culture supernatants were digested with 500鈥壩糶/ml trypsin or an equivalent volume of Hank鈥檚 balanced salt solution (HBSS) (Thermo Fisher Scientific, Waltham, MA, USA) as a mock digestion control for 1鈥塰 at 37鈥壜癈, followed by inactivation with 10% (v/v) foetal bovine serum (FBS) (Sigma-Aldrich).Immortalised cell lines and growth conditionsTHP-1 cells (Public Health England, Salisbury, England, UK) were routinely grown in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100鈥壩糶/ml streptomycin (cRPMI). HT29-MTX-E12 cells (Public Health England) were routinely cultured in Dulbecco鈥檚 Minimal Eagle鈥檚 Medium (DMEM) supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 4.5鈥塵g/ml glucose, 8.9鈥壩糶/ml L-alanine, 15鈥壩糶/ml L-asparagine, 13.3鈥壩糶/ml L-aspartic acid, 14.7鈥壩糶/ml L-glutamic acid, 7.5鈥壩糶/ml glycine, 11.5鈥壩糶/ml L-proline, 10.5鈥壩糶/ml L-serine, 100 U/ml penicillin, 100鈥壩糶/ml streptomycin and 0.25鈥壩糶/ml amphotericin B (cDMEM). Cells were seeded into assay vessels and cultured for nine days, following which they were washed twice with HBBS and placed into co-culture medium (cDMEM without antibiotic and supplemented with 5鈥壩糶/ml apo-transferrin and 0.2鈥壩糶/ml sodium selenite). HEK-Blue鈩?hTLR5 cells (InvivoGen, San Diego, CA, USA) were grown in DMEM supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 4.5鈥塵g/ml glucose, 100 U/ml penicillin, 100鈥壩糶/ml streptomycin, 100鈥壩糶/ml Normocin鈩?(InvivoGen), 30鈥壩糶/ml blastocydin and 100鈥壩糶/ml xeocin to 90% density. THP1-Blue鈩?NF-kB cells (InvivoGen) were grown in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100鈥壩糶/ml streptomycin, 25鈥塵M HEPES, 100鈥壩糶/ml Normocin鈩? 10鈥壩糶/ml blastocydin. All reagents were supplied by Sigma-Aldrich unless otherwise specified. Immortalised cell lines were routinely grown at 37鈥壜癈 in 5% CO2 atmosphere.Immortalised and primary cells stimulationTHP-1 cells were differentiated into macrophages by the addition of 5鈥塶g/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) to the culture media for 48鈥塰. Cells were plated in 96-well plates (200,000 cells/well) in cRPMI without PMA, antibiotics and FBS and incubated for 3鈥塰. Treatments (live bacteria at a MOI of 10:1 or a MOI of 1:1 for IL-8 detection as saturation was obtained with the 10:1 MOI) and controls (50鈥塶g/ml LPS or PBS) were then added and incubated for 1鈥塰 at 37鈥壜癈 under anaerobic conditions. Culture medium was then replaced with cRPMI and incubated for 24鈥塰 under standard growth conditions. Cell-free supernatants were then harvested, centrifuged for 3鈥塵in at 10,000 x g at 4鈥壜癈 and stored at 鈭?0鈥壜癈 for cytokine detection. Human PBMCs, obtained from STEMCELL Technologies (Vancouver, Canada) from healthy donors, were used to isolate primary monocyte populations by negative selection using a Human Monocyte Isolation kit. Monocytes were then differentiated into immature dendritic cells by incubation with 20鈥塶g/ml recombinant human IL-4 and 50鈥塶g/ml recombinant human GM-CSF for 8 days at 37鈥壜癈 in a 5% CO2 atmosphere in cRPMI supplemented with 55鈥壩糓 2-mercaptoethanol. Immature dendritic cells were recovered, washed, resuspended in cRPMI medium without antibiotics and plated in 96-well plates (200,000 cells/well). Treatments (live bacteria at a MOI of 10:1) and controls (100鈥塶g/ml LPS or cRPMI) were added to the cells and incubated for 1鈥塰 at 37鈥壜癈 under anaerobic conditions. Culture medium was then replaced with cRPMI and incubated for 17鈥塰 under standard culture conditions. Cell-free supernatants were then harvested, centrifuged for 3鈥塵in at 10,000 x g at 4鈥壜癈 and stored at 鈭?0鈥壜癈 prior to cytokine detection.Cytokine quantificationCytokine quantification was conducted using a ProcartaPlex multiplex immunoassay following the manufacturers recommendations (Thermo Fischer Scientific, Waltham, MA, USA). Briefly, 50鈥壜祃 of cell-free co-culture supernatants (CFS) were used for cytokine quantification using a MAGPIX庐 MILLIPLEX庐 system (Merck, Darmstadt, Germany) with the xPONENT software (Luminex, Austin, TX, USA). Data was analysed using the MILLIPLEX庐 analyst software (Merck) using a 5-parameter logistic curve and background subtraction to convert mean fluorescence intensity to pg/ml values.Transcriptional analysis using microarraysHT29-MTX cells were cultured in 24-well Transwell庐 (Corning, Corning, NY, USA), and incubated with treatments of MRx0518LV, MRx0518HK and MRx0518SN at a MOI of 100:1 (or equivalent) for 3鈥塰 at 37鈥壜癈 under anaerobic conditions. Cells were washed and lysed, and RNA was isolated from lysate using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was converted to cDNA using a GeneChip鈩?High Throughput WT PLUS Kit, which was then hybridized to a GeneChip鈩?Human Transcriptome Array 2.0. Microarray chips were washed and stained using a GeneChip鈩?Fluidics Station 450 instrument and the GeneChip鈩?Expression Wash, Stain and Scan kit, and then scanned using a GeneChip鈩?Scanner 3000 instrument (Thermo Fischer Scientific). Data analysis was carried out using Transcriptome Analysis Console 4.0 software (Thermo Fischer Scientific). Data were normalized using the Robust Multiarray Average algorithm, and fold changes were calculated using the normalized log2-transformed values of treated cells relative to respective controls. Data were filtered using cut-offs of p鈥?lt;鈥?.05, fold change of 鈭?.5 and 鈮?.5, and the presence of a gene symbol and coding variants. Pathway analysis was carried out using MetaCore鈩?(Clarivate Analytics, Philadelphia, PA, USA).NF-魏B and TLR5 reporter assaysTHP1-Blue鈩?NF-kB and HEK-Blue鈩?hTLR5 cells (InvivoGen, San Diego, CA, USA), grown to 90% density were washed once with phosphate-buffered saline (PBS) (Sigma-Aldrich, Gillingham, England, UK) and resuspended in growth media without antibiotic at a density of 160,000 and 500,000 cells/ml, respectively. MRx0518LV was added at a MOI of 10:1, MRx0518HK was used at a MOI of 100:1 and a 100:1 MOI equivalent volume was used for the supernatant fractions. Recombinant proteins were added at concentrations of 0.006鈥?00鈥塶g/ml. Positive controls for each reporter assay, Salmonella Typhimurium flagellin (FLA-ST) and heat-killed L. monocytogenes (HKLM) (InvivoGen), were used at 20鈥塶g/ml concentrations and a MOI of 200:1 respectively. Cells were then incubated at 37鈥壜癈 in a 5% CO2 atmosphere for 22鈥塰. QUANTI-Blue鈩?(InvivoGen) was added聽to cells, plates were incubated for a further 2鈥塰 and the optical density at 655鈥塶m was recorded. Graphs show results from averaged technical replicates and at least three independent experiments.Transcriptional analysis of the flagellar loci of E. gallinarum MRx0518Total RNA was extracted from late-log phase cultures of strain MRx0518, treated聽with RNAprotect (Qiagen), using the RNeasy Mini kit (Qiagen) according to the manufacturer鈥檚 protocol with minor modifications. Briefly, mechanical cell lysis was performed using Lysing Matrix B and a MP Fast-Prep-24 tissue and cell homogenizer (MP Biomedicals, Santa Ana, CA, USA) with oscillations set at 6鈥塵/s. Cells were disrupted for two 20鈥塻 cycles with a 1鈥塵in rest on ice between cycles. RNA quality was assessed using a Tapestation (Agilent Technologies, Santa Clara, CA, USA) with the Agilent RNA Screentape (Agilent Technologies). The absence of RNA degradation was confirmed, and all samples had a minimum RNA Integrity Numbers鈥夆墺鈥?. MICROBExpress kit (Thermo Fischer Scientific) was used to deplete rRNA species and the absence of 16鈥塖 and 23鈥塖 rRNA species was assessed using an Agilent Tapestation with the Agilent RNA Screentape (Agilent Technologies). RNA samples depleted in rRNA were sent to GATC Biotech for strand-specific library preparation and Illumina sequencing was performed to produce 50鈥塨p single-end reads. An average of 18,705,633 raw reads were generated per RNA-Seq library. Raw reads were trimmed using Trimmomatic51 and quality filtered (an average of 18,245,365.6 reads/library passed QC) reads were aligned (99.05% of total clean reads mapped) to the MRx0518 genome using Bowtie52. Data generated from three biological replicates were merged using BAMtools53 and subsequently used to calculate the expression levels of the motility loci of strain MRx0518 using Geneious R11 (Biomatters, Auckland, New Zealand). The read numbers associated with each gene were expressed in RPKM (reads per kilobases per million reads) scores54.Motility assaysMotility in vitro was assessed using BBL鈩?Motility Test Medium supplemented with 0.005% (w/v) 2,3,5-triphenyltetrazolium chloride (BD, Sparks, MD, USA). In brief, a fresh colony was stab-inoculated in 20鈥塵l equilibrated media and incubated for 48鈥塰 at 37鈥壜癈 in anaerobic conditions. All assays were performed in triplicate.Protein identification by nanoLC-MS/MSSample preparation and protein identification by LC-MS/MS were performed by Aberdeen Proteomics (University of Aberdeen, UK). In brief, 40鈥塵l culture supernatants were concentrated down to 0.5鈥塵l and washed with ultrapure water. Proteins were precipitated using a ReadyPrep 2-D Cleanup Kit (Bio-Rad) and resuspended in 100鈥壜祃 50鈥塵M ammonium bicarbonate. Proteins were incubated with porcine trypsin (Promega, Madison, WI, USA) for 16鈥塰 at 37鈥壜癈 and the resulting supernatants were dried by vacuum centrifugation and dissolved in 0.1% trifluoroacetic acid. Peptides were further desalted using 碌-C18 ZipTips (Merck). Peptides were then eluted into a 96-well microtiter plate, dried by vacuum centrifugation and dissolved in 10鈥壜祃 LC-MS loading solvent (2% acetonitrile, 0.1% formic acid). Peptides were separated and identified by nanoLC-MS/MS (Q Exactive hybrid quadrupole-Orbitrap MS system) (Thermo Fischer Scientific) using a 15-cm PepMap column, 60-minute LC-MS acquisition method and an injection volume of 5鈥壜祃. Data analysis was performed with Proteome Discoverer (Thermo Fischer Scientific) and the workflow included the Mascot Server as the search engine with the following parameters: enzyme鈥?鈥塼rypsin, maximum mixed cleavage sites鈥?鈥?, precursor mass tolerance鈥?鈥?0 ppm, dynamic modifications鈥?鈥塷xidation (M), static modifications鈥?鈥塩arbamidomethyl (C). Identified peptides were matched against a strain-specific protein sequence database, which was constructed based on the sequenced genome of E. gallinarum MRx0518 (3068 sequences).Recombinant flagellin expression and purificationE. gallinarum MRx0518 and DSM100110 full-length fliC genes were amplified by PCR using primer pairs DC022/DC023 and DC024/DC025, respectively (Supplementary Table聽S3). Gene products were then cloned into the pQE-30 vector (Supplementary Table聽S3) (Qiagen) using BamHI and SalI restriction sites. The resulting constructs, which add 12 amino acid residues (MRGSHHHHHHGS) to the N-terminal end of the proteins, were then transformed into E. coli M15 pREP4 (Supplementary Table聽S3) (Qiagen) for over-expression. Expression of recombinant proteins were induced according to the manufacturer鈥檚 instructions, by adding 0.1鈥塵M IPTG for 18鈥塰 at 20鈥壜癈 with shaking (200鈥塺pm). E. coli cells were lysed by sonication and the recombinant proteins were purified using Ni-NTA columns (Qiagen). An empty vector control was also expressed and purified in parallel, to provide a control for the potential effect of residual contaminants and endotoxins. Endotoxins were removed using Pierce鈩?High Capacity Endotoxin Removal Spin Column (Thermo Fischer Scientific) according to the manufacturer鈥檚 instructions. Residual endotoxin levels were quantified using Pierce鈩?LAL Chromogenic Endotoxin Quantification Kit (Thermo Fischer Scientific) and shown to be suitable for co-culture assays (Supplementary Fig.聽S3). Protein concentrations were measured using Pierce鈩?BCA Protein Assay Kit (Thermo Fischer Scientific) and the purity of each recombinant protein preparations was assessed by SDS-PAGE (Bio-Rad, Hercules, CA, USA) (Supplementary Fig.聽S3).Sequencing and annotation of the flagellar loci and fliC genes of E. gallinarum and E. casseliflavus strainsThe flagellar loci of E. gallinarum strains MRx0518 and DSM100110 and the fliC genes of E. gallinarum and E. casseliflavus were sequenced as part of ongoing bacterial genome sequencing projects carried out by Diversigen (Houston, TX, USA), GATC Biotech (Konstanz, Germany) and MicrobesNG (Birmigham, England, UK) on behalf of 4D pharma Research Ltd (for additional details see Supplementary Table聽S3). MicrobesNG (http://www.microbesng.uk) is supported by the BBSRC (grant number BB/L024209/1). The 鈥淩apid Annotation using Subsystem Technology鈥?(RAST) database was used for automated annotation of open reading frames55,56,57 followed by manual curation of the gene annotations in Geneious R11. The flagellar locus and fliC of strain MRx0518 was used as a reference sequence for all comparative analyses and homologs (as determined by BLASTp similarity searches) within additional strains were identified and extracted from the draft genomes of available E. gallinarum or E. casseliflavus genomes downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/).Comparative analysis of the flagellar loci of E. gallinarum strains MRx0518 and DSM100110Nucleotide alignments were generated using a local BLAST v 2.7.1+鈥塱nstallation which were then visualised and analysed for gene conservation and sequence synteny using EasyFig. 2.2.258.Phylogenetic analysesFliC protein sequences were downloaded from the NCBI protein database or were derived from sequence data available for the strains outlined in Table聽S5, using BLASTP-based homology searches against the聽FliCMRx0518 sequence. Protein sequences were aligned using MUSCLE59 and evolutionary analyses were conducted in MEGA760. Phylogenies were inferred using the Maximum Likelihood method based on the Le_Gascuel_2008 model61. A discrete Gamma distribution was used for the multispecies FliC tree (Fig.聽S5), to model evolutionary rate differences among sites (5 categories (+G)). The rate-variation model allowed for some sites to be evolutionarily invariable ([+I]). The trees with the highest log likelihood are displayed and the reliability of the groups were evaluated by bootstrap testing with 1,000 re-samplings. The FliC of Lactobacillus mali DSM20444 (accession number KRN11091.1) was used as an outgroup during E. gallinarum and E. casseliflavus interspecies phylogenetic analyses.Generation of an E. gallinarum MRx0518 flagellin gene insertion mutantThe flagellin insertion mutant was created using the non-replicative plasmid pORI1930 (Supplementary Table聽S3). An internal fragment of E. gallinarum MRx0518 fliC gene was amplified using primers DC020 and DC021 (Supplementary Table聽S3) and cloned into pORI19. Restriction enzymes and Quick Ligase (New England Biolabs, Ipswich, MA, USA) were used according to the manufacturer鈥檚 instructions. This construct was propagated in E. coli EC101 by chemical transformation (Supplementary Table聽S3) and isolated using the Genopure Plasmid Maxi Kit (Roche Diagnostics, Basel, Switzerland) from a 500-ml culture. Isolated plasmid DNA was concentrated using 0.3鈥塎 sodium acetate pH 5.2 and ethanol down to 20鈥壩糽. A protocol was developed to prepare E. gallinarum MRx0518 electrocompetent cells, which was adapted from a previously published method29. In brief, E. gallinarum MRx0518 was grown for 18鈥塰 in GM17 broth, supplemented with 0.5鈥塎 sucrose and 3% (w/v) glycine (Sigma-Aldrich). Cells were then washed twice with 0.5鈥塎 sucrose and 10% (v/v) glycerol and treated with 10鈥壩糶/ml lysozyme and 10 U/ml mutanolysin (Sigma-Aldrich) for 30鈥塵in at 37鈥壜癈. E. gallinarum MRx0518 cells were then transformed by electroporation with 10鈥壩糶 of plasmid DNA and recovered in BHI broth before plating on selective BHI agar. Plasmid insertion was confirmed for successful transformants by PCR amplification and sequencing (GATC Biotech, Konstanz, Germany) using primers listed in Supplementary Table聽S3. In vitro motility of the flagellin insertion mutant was assessed as described for strain MRx0518.Gene expression profiling by qPCRHT29-MTX cells were cultured in Transwell庐 and incubated with bacterial culture supernatant (MOI 100:1 equivalent) or recombinant flagellin (1鈥壜礸/ml) for 24鈥塰. Mammalian RNA was isolated as described above. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific). qPCR analysis was carried out using the primers detailed in Supplementary Table聽S3 and Power SYBR鈩?Green PCR Master Mix (Thermo Fischer Scientific).IL-8 ELISAIL-8 secretion was quantified from HT29-MTX co-culture supernatants after 24鈥塰 of treatment (with bacterial culture supernatant at a MOI 100:1 equivalent or 1鈥壜礸/ml recombinant flagellin) using the Human IL-8 (CXCL8) standard ABTS ELISA development kit (Peprotech, Rocky Hill, NJ, USA) according to the manufacturer鈥檚 instructions. The motility loci of E. gallinarum MRx0518 and DSM100110 have been deposited under GenBank accession numbers MK210233 and MK176551, respectively. The fliC genes of the E. gallinarum and E. casseliflavus strains outlined in Supplementary Table聽S3 have been deposited under GenBank accession numbers MK142539-MK142553 and MK174384- MK174386 respectively. 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BMC Bioinformatics 16, 169, https://doi.org/10.1186/s12859-015-0611-3 (2015).Article聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsWe would like to thank Dr. Riboulet-Bisson and Professor Benachour (U2RM Universit茅 de Caen, France) for their technical advice on聽the genetic manipulation of enterococcal species. We are grateful to Professors Paul W. O鈥橳oole and Joseph Petrosino for their critical review of the manuscript.Author informationAuthor notesDelphine L. Laut茅-Caly and Emma J. Raftis contributed equally.Affiliations4D Pharma Research Ltd, Life Science Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS, United KingdomDelphine L. Laut茅-Caly,聽Emma J. Raftis,聽Philip Cowie,聽Emma Hennessy,聽Amy Holt,聽D. Alessio Panzica,聽Christina Sparre,聽Beverley Minter,聽Eline Stroobach聽 聽Imke E. MulderAuthorsDelphine L. Laut茅-CalyView author publicationsYou can also search for this author in PubMed聽Google ScholarEmma J. RaftisView author publicationsYou can also search for this author in PubMed聽Google ScholarPhilip CowieView author publicationsYou can also search for this author in PubMed聽Google ScholarEmma HennessyView author publicationsYou can also search for this author in PubMed聽Google ScholarAmy HoltView author publicationsYou can also search for this author in PubMed聽Google ScholarD. Alessio PanzicaView author publicationsYou can also search for this author in PubMed聽Google ScholarChristina SparreView author publicationsYou can also search for this author in PubMed聽Google ScholarBeverley MinterView author publicationsYou can also search for this author in PubMed聽Google ScholarEline StroobachView author publicationsYou can also search for this author in PubMed聽Google ScholarImke E. MulderView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsD.L.C. and E.J.R. contributed equally to this work. D.L.C., E.J.R. and I.E.M. conceived the study and D.L.C., E.J.R., P.C., E.H. designed the assays, D.L.C., E.J.R., P.C., E.H., C.S., A.H., D.A.P., B.M. and E.S. performed the experiments, D.L.C., E.J.R., P.C. and E.H. interpreted the data and D.L.C., E.J.R. and P.C. wrote the paper.Corresponding authorCorrespondence to Emma J. Raftis.Ethics declarations Competing Interests All authors were employees of 4D Pharma Research Ltd while engaged in the research project. This work was supported by funding provided by 4D Pharma PLC. 4D Pharma Research Ltd owns a family of patent applications which are pending internationally and derived from International Patent Publication No. WO2017/085520 and UK Patent Application No. 1804384.4. 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