• Characterization of Pro-Inflammatory Flagellin Proteins Produced by Lactobacillus ruminis and Related Motile Lactobacilli

      Neville, B. Anne; Forde, Brian M; Claesson, Marcus J.; Darby, Trevor; Coghlan, Avril; Nally, Kenneth; Ross, R Paul; O'Toole, Paul W.; Science Foundation Ireland; Irish Research Council for Science, Engineering and Technology; et al. (PLOS, 10/07/2012)
      Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.
    • Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus

      Romano, Stefano; Fernandez-Guerra, Antonio; Reen, F. Jerry; Glockner, Frank O.; Crowley, Susan P.; O'Sullivan, Orla; Cotter, Paul D.; Adams, Claire; Dobson, Alan D. W.; O'Gara, Fergal; et al. (Frontiers Media S. A., 30/03/2016)
      Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its microbiota. Our data suggest the presence of a sponge-specific lineage of Pseudovibrio. The reduction in genome size and the loss of some systems potentially used to successfully enter the host, leads to the hypothesis that P. axinellae strain AD2 may be a lineage that presents an ancient association with the host and that may be vertically transmitted to the progeny.
    • Complexes between linoleate and native or aggregated β-lactoglobulin: Interaction parameters and in vitro cytotoxic effect.

      Le Maux, Solene; Bouhallab, Said; Giblin, Linda; Brodkorb, Andre; Croguennec, Thomas; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; Irish Research Council for Science, Engineering and Technology; 08/RD/TMFRC/650 (Elsevier, 2013-11)
      Iron is essential for human health, but it sometimes causes an unpleasant taste, rusty colour and a decrease in the stability of food products. Previously, we found that ethanol-treated yeast (ETY) cells could remove iron from wine and juice, and reduce the fishy aftertaste induced by iron in wine–seafood pairings. However, the mechanism of iron sorption by ETY cells is undefined; thus, there is no indicator that can be used to estimate the iron sorption capacity of these cells. In this study, we showed that cell wall components are not mainly associated with iron sorption by investigating ETY cells with the cell wall removed. Moreover, plasma membrane permeability was correlated with the iron sorbing capacity of the cells. Microscopic analysis showed that iron accumulated within ETY cells. Proteinase-treated ETY cells had no iron sorbing capacity. On the basis of these results, we conclude that intracellular proteins are involved in iron sorption by ETY cells.
    • Composition of the early intestinal microbiota: Knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps

      Fouhy, Fiona; Ross, R Paul; Fitzgerald, Gerald F; STANTON, CATHERINE; Cotter, Paul D.; Irish Research Council for Science, Engineering and Technology; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; 11/PI/1137 (Landes Bioscience, 01/05/2012)
      The colonization, development and maturation of the newborn gastrointestinal tract that begins immediately at birth and continues for two years, is modulated by numerous factors including mode of delivery, feeding regime, maternal diet/weight, probiotic and prebiotic use and antibiotic exposure pre-, peri- and post-natally. While in the past, culture-based approaches were used to assess the impact of these factors on the gut microbiota, these have now largely been replaced by culture-independent DNA-based approaches and most recently, high-throughput sequencing-based forms thereof. The aim of this review is to summarize recent research into the modulatory factors that impact on the acquisition and development of the infant gut microbiota, to outline the knowledge recently gained through the use of culture-independent techniques and, in particular, highlight advances in high-throughput sequencing and how these technologies have, and will continue to, fill gaps in our knowledge with respect to the human intestinal microbiota.
    • Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

      Riboulet-Bisson, Eliette; Sturme, Mark H. J.; Jeffery, Ian B; O'Donnell, Michelle M.; Neville, B. Anne; Forde, Brian M; Claesson, Marcus J.; Harris, Hugh; Gardiner, Gillian E.; Casey, Patrick G.; et al. (PLOS, 17/02/2012)
      Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent
    • High-Throughput Sequencing Reveals the Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

      Fouhy, Fiona; Guinane, Caitriona M.; Hussey, Seamus; Wall, Rebecca; Ryan, C. Anthony; Dempsey, Eugene; Murphy, Brendan; Ross, R Paul; Fitzgerald, Gerald F; STANTON, CATHERINE; et al. (American Society for Microbiology, 04/09/2012)
      The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria (P = 0.0049) and significantly lower proportions of Actinobacteria (P = 0.00001) (and the associated genus Bifidobacterium [P = 0.0132]) as well as the genus Lactobacillus (P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants (P = 0.0049), but the Actinobacteria, Bifidobacterium, and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB-targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.
    • In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

      Fouhy, Fiona; O'Connell-Motherway, Mary; Fitzgerald, Gerald F; Ross, R Paul; STANTON, CATHERINE; van Sinderen, Douwe; Cotter, Paul D.; Irish Research Council for Science, Engineering and Technology; Science Foundation Ireland; Health Research Board; et al. (PLoS, 06/12/2013)
      Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.
    • In vivo activity of Nisin A and Nisin V against Listeria monocytogenes in mice

      Campion, Alicia; Casey, Patrick G.; Field, Des; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Programme for Research in Third-Level Institutions; Irish Research Council for Science, Engineering and Technology; Enterprise Ireland; Science Foundation Ireland (Biomed Central, 01/02/2013)
      Background: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. Results: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model. More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 105 cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. Conclusion: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.
    • Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

      Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B; Claesson, Marcus J.; Ross, R Paul; Scott, Karen P.; et al. (PLoS, 23/07/2013)
      Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.
    • Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

      Molloy, Evelyn M.; Field, Des; O'Connor, Paula M.; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Irish Research Council for Science, Engineering and Technology; Enterprise Ireland; Science Foundation Ireland (PLOS, 11/03/2013)
      It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.
    • Symposium review: Lactococcus lactis from nondairy sources: Their genetic and metabolic diversity and potential applications in cheese

      McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; Dairy Research Ireland; EU Marie Curie Actions Clarin Co-Fund; Irish Research Council for Science, Engineering and Technology; European Union (Elsevier, 2018-02-13)
      The widespread dissemination of species of the lactic acid bacteria (LAB) group in different environments testifies to their extraordinary niche adaptability. Members of the LAB are present on grass and other plant material, in dairy products, on human skin, and in the gastrointestinal and reproductive tracts. The selective pressure imparted by these specific environments is a key driver in the genomic diversity observed between strains of the same species deriving from distinct habitats. Strains that are exploited in the dairy industry for the production of fermented dairy products are often referred to as “domesticated” strains. These strains, which initially may have occupied a nondairy niche, have become specialized for growth in the milk environment. In fact, comparative genome analysis of multiple LAB species and strains has revealed a central trend in LAB evolution: the loss of ancestral genes and metabolic simplification toward adaptation to nutritionally rich environments. In contrast, “environmental” strains, or those from raw milk, plants, and animals, exhibit diverse metabolic capabilities and lifestyle characteristics compared with their domesticated counterparts. Because of the limited number of established dairy strains used in fermented food production today, demand is increasing for novel strains, with concerted efforts to mine the microbiota of natural environments for strains of technological interest. Many studies have concentrated on uncovering the genomic and metabolic potential of these organisms, facilitating comparative genome analysis of strains from diverse environments and providing insight into the natural diversity of the LAB, a group of organisms that is at the core of the dairy industry. The natural biodiversity that exists in these environments may be exploited in dairy fermentations to expand flavor profiles, to produce natural “clean label” ingredients, or to develop safer products.
    • β-lactoglobulin as a molecular carrier of linoleate: characterisation and effects on intestinal epithelial cells in vitro

      Le Maux, Solene; Giblin, Linda; Croguennec, Thomas; Bouhallab, Said; Brodkorb, Andre; Department of Agriculture, Food and the Marine; Irish Research Council for Science, Engineering and Technology; Teagasc Walsh Fellowship Programme; 08/RD/TMFRC/650 (American Chemical Society, 27/08/2012)
      The dairy protein β-lactoglobulin (βlg) is known to bind hydrophobic ligands such as fatty acids. In the present work, we investigated the biological activity in vitro of linoleate once complexed to bovine βlg. Binding of linoleate (C18:2) to bovine βlg was achieved by heating at 60 °C for 30 min at pH 7.4, resulting in a linoleate/βlg molar binding stoichiometry of 1.1, 2.1, and 3.4. Two types of binding sites were determined by ITC titrations. Binding of linoleate induced the formation of covalent dimers and trimers of βlg. The LD50 on Caco-2 cells after 24 h was 58 μM linoleate. However, cell viability was unaffected when 200 μM linoleate was presented to the Caco-2 cells as part of the βlg complex. The Caco-2 cells did not increase mRNA transcript levels of long chain fatty acid transport genes, FATP4 and FABPpm, or increase levels of the cAMP signal, in response to the presence of 50 μM linoleate alone or as part of the βlg complex. Therefore, it is proposed that βlg can act as a molecular carrier and alter the bioaccessibility of linoleate/linoleic acid.
    • β-Lactoglobulin-linoleate complexes: In vitro digestion and the role of protein in fatty acids uptake

      Le Maux, Solene; Brodkorb, Andre; Croguennec, Thomas; Hennessy, Alan A.; Bouhallab, Said; Giblin, Linda; Department of Agriculture, Food and the Marine; Irish Research Council for Science, Engineering and Technology; Teagasc Walsh Fellowship Programme; 08/RD/TMFRC/650 (Elsevier Inc and American Dairy Science Association, 2013-07)
      The dairy protein β-lactoglobulin (BLG) is known to bind fatty acids such as the salt of the essential longchain fatty acid linoleic acid (cis,cis-9,12-octadecadienoic acid, n-6, 18:2). The aim of the current study was to investigate how bovine BLG-linoleate complexes, of various stoichiometry, affect the enzymatic digestion of BLG and the intracellular transport of linoleate into enterocyte-like monolayers. Duodenal and gastric digestions of the complexes indicated that BLG was hydrolyzed more rapidly when complexed with linoleate. Digested as well as undigested BLG-linoleate complexes reduced intracellular linoleate transport as compared with free linoleate. To investigate whether enteroendocrine cells perceive linoleate differently when part of a complex, the ability of linoleate to increase production or secretion of the enteroendocrine satiety hormone, cholecystokinin, was measured. Cholecystokinin mRNA levels were different when linoleate was presented to the cells alone or as part of a protein complex. In conclusion, understanding interactions between linoleate and BLG could help to formulate foods with targeted fatty acid bioaccessibility and, therefore, aid in the development of food matrices with optimal bioactive efficacy