The flagellin of candidate live biotherapeutic Enterococcus gallinarum MRx0518 is a potent immunostimulant.

Many 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-κB 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.

Bifidobacterium breve MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration.

Asthma is a phenotypically heterogeneous disease. In severe asthma, airway inflammation can be predominantly eosinophilic, neutrophilic, or mixed. Only a limited number of drug candidates are in development to address this unmet clinical need. Live biotherapeutics derived from the gut microbiota are a promising new therapeutic area. MRx0004 is a commensal Bifidobacterium breve strain isolated from the microbiota of a healthy human. The strain was tested prophylactically and therapeutically by oral gavage in a house dust mite mouse model of severe asthma. A strong reduction of neutrophil and eosinophil infiltration was observed in lung bronchoalveolar lavage fluid following MRx0004 treatment. Peribronchiolar and perivascular immunopathology was also reduced. MRx0004 increased lung CD4+CD44+ cells and CD4+FoxP3+ cells and decreased activated CD11b+ dendritic cells. Cytokine analysis of lung tissue revealed reductions of pro-inflammatory cytokines and chemokines involved in neutrophil migration. In comparison, anti-IL-17 antibody treatment effectively reduced neutrophilic infiltration and increased CD4+FoxP3+ cells, but it induced lung eosinophilia and did not decrease histopathology scores. We have demonstrated that MRx0004, a microbiota-derived bacterial strain, can reduce both neutrophilic and eosinophilic infiltration in a mouse model of severe asthma. This novel therapeutic is a promising next-generation drug for management of severe asthma.

Unusual genome complexity in Lactobacillus salivarius JCM1046.

BACKGROUND: Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046. RESULTS: JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM. CONCLUSION: Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

Subspecies diversity in bacteriocin production by intestinal Lactobacillus salivarius strains.

A recent comparative genomic hybridization study in our laboratory revealed considerable plasticity within the bacteriocin locus of gastrointestinal strains of Lactobacillus salivarius. Most notably, these analyses led to the identification of two novel unmodified bacteriocins, salivaricin L and salivaricin T, produced by the neonatal isolate L. salivarius DPC6488 with immunity, regulatory and export systems analogous to those of abp118, a two-component bacteriocin produced by the well characterized reference strain L. salivarius UCC118. In this addendum we discuss the intraspecific diversity of our seven bacteriocin-producing L. salivarius isolates on a genome-wide level, and more specifically, with respect to their salivaricin loci.

Production of multiple bacteriocins from a single locus by gastrointestinal strains of Lactobacillus salivarius.

Bacteriocins produced by Lactobacillus salivarius isolates derived from a gastrointestinal origin have previously demonstrated efficacy for in vivo protection against Listeria monocytogenes infection. In this study, comparative genomic analysis was employed to investigate the intraspecies diversity of seven L. salivarius isolates of human and porcine intestinal origin, based on the genome of the well-characterized bacteriocin-producing strain L. salivarius UCC118. This revealed a highly conserved megaplasmid-borne gene cluster in these strains involved in the regulation and secretion of two-component class IIb bacteriocins. However, considerable intraspecific variation was observed in the structural genes encoding the bacteriocin peptides. They ranged from close relatives of abp118, such as salivaricin P, which differs by 2 amino acids, to completely novel bacteriocins, such as salivaricin T, which is characterized in this study. Salivaricin T inhibits closely related lactobacilli and bears little homology to previously characterized salivaricins. Interestingly, the two peptides responsible for salivaricin T activity, SalTα and SalTß, share considerable identity with the component peptides of thermophilin 13, a bacteriocin produced by Streptococcus thermophilus. Furthermore, the salivaricin locus of strain DPC6488 also encodes an additional novel one-component class IId anti-listerial bacteriocin, salivaricin L. These findings suggest a high level of redundancy in the bacteriocins that can be produced by intestinal L. salivarius isolates using the same enzymatic production and export machinery. Such diversity may contribute to their ability to dominate and compete within the complex microbiota of the mammalian gut.

Genomic diversity of Lactobacillus salivarius.

Strains of Lactobacillus salivarius are increasingly employed as probiotic agents for humans or animals. Despite the diversity of environmental sources from which they have been isolated, the genomic diversity of L. salivarius has been poorly characterized, and the implications of this diversity for strain selection have not been examined. To tackle this, we applied comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) to 33 strains derived from humans, animals, or food. The CGH, based on total genome content, including small plasmids, identified 18 major regions of genomic variation, or hot spots for variation. Three major divisions were thus identified, with only a subset of the human isolates constituting an ecologically discernible group. Omission of the small plasmids from the CGH or analysis by MLST provided broadly concordant fine divisions and separated human-derived and animal-derived strains more clearly. The two gene clusters for exopolysaccharide (EPS) biosynthesis corresponded to regions of significant genomic diversity. The CGH-based groupings of these regions did not correlate with levels of production of bound or released EPS. Furthermore, EPS production was significantly modulated by available carbohydrate. In addition to proving difficult to predict from the gene content, EPS production levels correlated inversely with production of biofilms, a trait considered desirable in probiotic commensals. L. salivarius displays a high level of genomic diversity, and while selection of L. salivarius strains for probiotic use can be informed by CGH or MLST, it also requires pragmatic experimental validation of desired phenotypic traits.

Allelic variation of bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels.

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.