Cormac G. M. Gahan

Bile Salt Hydrolase Activity in Probiotics

Probiotics are defined as “living microorganisms, which upon ingestion in certain numbers exert health benefits on the host beyond inherent basic nutrition” ([43][1]). Various studies have indicated that probiotics may alleviate lactose intolerance; have a positive influence on the intestinal

Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118.

The mechanisms by which probiotic strains enhance the health of the host remain largely uncharacterized. Here we demonstrate that Lactobacillus salivarius UCC118, a recently sequenced and genetically tractable probiotic strain of human origin, produces a bacteriocin in vivo that can significantly protect mice against infection with the invasive foodborne pathogen Listeria monocytogenes. A stable mutant of Lb. salivarius UCC118 that is unable to produce the Abp118 bacteriocin also failed to protect mice against infection with two strains of L. monocytogenes, EGDe and LO28, confirming that bacteriocin production is the primary mediator of protection against this organism. Furthermore, Lb. salivarius UCC118 did not offer any protection when mice were infected with a strain of L. monocytogenes expressing the cognate Abp118 immunity protein AbpIM, confirming that the antimicrobial effect is a result of direct antagonism between Lb. salivarius and the pathogen, mediated by the bacteriocin Abp118.

Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome

Bile salt hydrolases (BSHs) catalyze the “gateway” reaction in a wider pathway of bile acid modification by the gut microbiota. Because bile acids function as signaling molecules regulating their own biosynthesis, lipid absorption, cholesterol homeostasis, and local mucosal defenses in the intestine, microbial BSH activity has the potential to greatly influence host physiology. However, the function, distribution, and abundance of BSH enzymes in the gut community are unknown. Here, we show that BSH activity is a conserved microbial adaptation to the human gut environment with a high level of redundancy in this ecosystem. Through metagenomic analyses we identified functional BSH in all major bacterial divisions and archaeal species in the gut and demonstrate that BSH is enriched in the human gut microbiome. Phylogenetic analysis illustrates that selective pressure in the form of conjugated bile acid has driven the evolution of members of the Ntn_CGH-like family of proteins toward BSH activity in gut-associated species. Furthermore, we demonstrate that BSH mediates bile tolerance in vitro and enhances survival in the murine gut in vivo. Overall, we demonstrate the use of function-driven metagenomics to identify functional anchors in complex microbial communities, and dissect the gut microbiome according to activities relevant to survival in the mammalian gastrointestinal tract.

A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid

We observed that glutamate greatly enhances the survival of Listeria monocytogenes in gastric fluid, a phenomenon that is directly linked to glutamate decarboxylase activity (GAD). Glutamate‐mediated acid tolerance has been associated in other intestinal genera with the GAD system, in which glutamate is internalized and converted to γ‐aminobutyrate (consuming an intracellular proton) that is subsequently exchanged for another extracellular glutamate via a membrane‐located antiporter. Molecular analysis of L. monocytogenes LO28 revealed the presence of two glutamate decarboxylase homologues, designated gadA and gadB, that are differentially expressed. The gadB gene is co‐transcribed in tandem with an upstream gene, gadC, which encodes a potential glutamate/γ‐aminobutyrate antiporter. Expression of this transcript is upregulated in response to mild acid stress (pH 5.5). In contrast, expression of the monocistronic gadA message was weaker and was not induced by mild acid treatment. Non‐polar deletion mutations resulted in a dramatic decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order LO28 > ΔgadA > ΔgadB = ΔgadC > ΔgadAB for both stationary and logarithmic phase cultures. The exquisite sensitivity of the ΔgadAB mutant to ex vivo porcine and synthetic human gastric fluid demonstrates a clear role for the GAD system in facilitating survival of the organism in the stomach after ingestion and in other low‐pH environments. Furthermore, variations in levels of GAD activity between different strains of L. monocytogenes correlate significantly with levels of tolerance to gastric fluid. Sensitive strains, which include the sequenced L. monocytogenes EGD, exhibit reduced levels of GAD activity. It is clear from this study that expression of GAD by L. monocytogenes strains is an absolute requirement for survival in the stomach environment.

Contribution of Three Bile-Associated Loci, bsh, pva, and btlB, to Gastrointestinal Persistence and Bile Tolerance of Listeria monocytogenes

ABSTRACT Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes—bsh, pva, and btlB—previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and σB, the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes.

Bacterial stress response in Listeria monocytogenes: jumping the hurdles imposed by minimal processing

Minimal processing relies on the use of multiple sub-lethal stresses (or processes) to achieve a similar level of microbial control as that traditionally achieved using a single lethal stress. The benefit to the consumer is products which are less obviously processed than a frozen or canned, acidified or heavily salted food item. However, our increasing understanding of how bacteria can adapt to sub-lethal stresses in a manner which can render them less susceptible to additional insults, should be borne in mind when designing safety or extended shelf-life into a minimally processed product. Listeria monocytogenes is a target organism for many minimally processed food manufacturers because of its ability to tolerate adverse conditions such as low Aw and low temperature. In this communication we use L. monocytogenes as a model system to describe some of the consequences of stress adaptation in terms of improved survival in minimally processed foods and, importantly, the consequences in terms of the virulence of the target organism.

Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut.

Significance It is known that the gastrointestinal microbiota influences adiposity and weight gain in the host. However the mechanisms by which gut microorganisms coordinate host physiological processes are currently unclear. We demonstrate that a single, widely distributed function of the gut microbiota, bile salt hydrolase (BSH) activity, significantly influences lipid metabolism, weight gain, and cholesterol levels in the host. In our study microbial BSH activity was shown to direct expression of host signalling pathways with known roles in lipid metabolism, circadian rhythm, and epithelial cell function. The work defines the significant impact of in situ bile hydrolysis on host metabolism and indicates how this finding may be exploited as a potential intervention strategy for the control of obesity and metabolic syndrome. Alterations in the gastrointestinal microbiota have been implicated in obesity in mice and humans, but the key microbial functions influencing host energy metabolism and adiposity remain to be determined. Despite an increased understanding of the genetic content of the gastrointestinal microbiome, functional analyses of common microbial gene sets are required. We established a controlled expression system for the parallel functional analysis of microbial alleles in the murine gut. Using this approach we show that bacterial bile salt hydrolase (BSH) mediates a microbe–host dialogue that functionally regulates host lipid metabolism and plays a profound role in cholesterol metabolism and weight gain in the host. Expression of cloned BSH enzymes in the gastrointestinal tract of gnotobiotic or conventionally raised mice significantly altered plasma bile acid signatures and regulated transcription of key genes involved in lipid metabolism (Pparγ, Angptl4), cholesterol metabolism (Abcg5/8), gastrointestinal homeostasis (RegIIIγ), and circadian rhythm (Dbp, Per1/2) in the liver or small intestine. High-level expression of BSH in conventionally raised mice resulted in a significant reduction in host weight gain, plasma cholesterol, and liver triglycerides, demonstrating the overall impact of elevated BSH activity on host physiology. In addition, BSH activity in vivo varied according to BSH allele group, indicating that subtle differences in activity can have significant effects on the host. In summary, we demonstrate that bacterial BSH activity significantly impacts the systemic metabolic processes and adiposity in the host and represents a key mechanistic target for the control of obesity and hypercholesterolemia.

Bile Stress Response in Listeria monocytogenes LO28: Adaptation, Cross-Protection, and Identification of Genetic Loci Involved in Bile Resistance

ABSTRACT Bile is one of many barriers that Listeria monocytogenes must overcome in the human gastrointestinal tract in order to infect and cause disease. We demonstrated that stationary-phase cultures of L. monocytogenes LO28 were able to tolerate concentrations of bovine, porcine, and human bile and bile acids well in excess of those encountered in vivo. Strain LO28 was relatively bile resistant compared with other clinical isolates of L. monocytogenes, as well as with Listeria innocua, Salmonella enterica serovar Typhimurium LT2, and Lactobacillus sakei. While exponential-phase L. monocytogenes LO28 cells were exquisitely sensitive to unconjugated bile acids, prior adaptation to sublethal levels of bile acids or heterologous stresses, such as acid, heat, salt, or sodium dodecyl sulfate (SDS), significantly enhanced bile resistance. This adaptive response was independent of protein synthesis, and in the cases of bile and SDS adaptation, occurred in seconds. In order to identify genetic loci involved in the bile tolerance phenotype of L. monocytogenes LO28, transposon (Tn917) and plasmid (pORI19) integration banks were screened for bile-sensitive mutants. The disrupted genes included a homologue of the capA locus required for capsule formation in Bacillus anthracis; a gene encoding the transcriptional regulator ZurR; a homologue of an Escherichia coli gene, lytB, involved in isoprenoid biosynthesis; a gene encoding a homologue of the Bacillus subtilis membrane protein YxiO; and a gene encoding an amino acid transporter with a putative role in pH homeostasis, gadE. Interestingly, all of the identified loci play putative roles in maintenance of the cell envelope or in stress responses.

The relationship between acid stress responses and virulence in Salmonella typhimurium and Listeria monocytogenes.

All pathogenic bacteria possess the ability to evade or surmount body defenses (stresses, as experienced by the bacterium) long enough to cause a sufficient reaction, which is then manifested as a disease or illness. While opportunistic pathogens will only cause illness in the event of a predisposing weakness in these defenses, many pathogens must take on and overcome intact defenses. This is particularly true of gastrointestinal pathogens such as Listeria monocytogenes and Salmonella spp., which must circumvent many different stresses in order to arrive at the site of infection. These include the acid barrier of the stomach, the physical barrier of the epithelial cells lining the gastrointestinal tract, and various immune defenses including the initial onslaught of macrophages. Thus, these organisms have developed elaborate systems for sensing stress, and for responding to those stresses in a self-protective fashion. One well characterised adaptive response is to acid stress, the so-called acid tolerance response (ATR). The ATR is a complex phenomenon, involving a number of changes in the levels of different proteins and presumably, many allied events at the level of gene regulation. A number of molecular approaches have identified numerous interesting chromosomal loci involved both in sensing and responding to stress and in virulence. The identity of some of these genes, and their impact on stress responses and virulence will be discussed.

Analysis of the Role of OpuC, an Osmolyte Transport System, in Salt Tolerance and Virulence Potential of Listeria monocytogenes

ABSTRACT The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori+ RepA− vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake inListeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.