Production of bioactive substances by intestinal bacteria as a basis for explaining probiotic mechanisms: Bacteriocins and conjugated linoleic acid

Abstract

The mechanisms by which intestinal bacteria achieve their associated health benefits can be complex and multifaceted. In this respect, the diverse microbial composition of the human gastrointestinal tract (GIT) provides an almost unlimited potential source of bioactive substances (pharmabiotics) which can directly or indirectly affect human health. Bacteriocins and fatty acids are just two examples of pharmabiotic substances which may contribute to probiotic functionality within the mammalian GIT. Bacteriocin production is believed to confer producing strains with a competitive advantage within complex microbial environments as a consequence of their associated antimicrobial activity. This has the potential to enable the establishment and prevalence of producing strains as well as directly inhibiting pathogens within the GIT. Consequently, these antimicrobial peptides and the associated intestinal producing strains may be exploited to beneficially influence microbial populations. Intestinal bacteria are also known to produce a diverse array of health-promoting fatty acids. Indeed, certain strains of intestinal bifidobacteria have been shown to produce conjugated linoleic acid (CLA), a fatty acid which has been associated with a variety of systemic health-promoting effects. Recently, the ability to modulate the fatty acid composition of the liver and adipose tissue of the host upon oral administration of CLA-producing bifidobacteria and lactobacilli was demonstrated in a murine model. Importantly, this implies a potential therapeutic role for probiotics in the treatment of certain metabolic and immunoinflammatory disorders. Such examples serve to highlight the potential contribution of pharmabiotic production to probiotic functionality in relation to human health maintenance.

Introduction

It is estimated that the healthy human adult gastrointestinal tract (GIT) harbors approximately 10¹³ microorganisms (Ventura et al., 2009). Initially developed at birth, the intestinal microbiotia is primarily comprised of high proportions of such microorganisms as bifidobacteria which are thought to be selected by breast or formula milk in the first weeks of life (Fanaro et al., 2003, Martin et al., 2009). Development of the adult microbiota is consistent with dominating populations of Firmicutes (including genera of Clostridia and lactic acid bacteria) and Bacteroidetes, while Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobia are present to a lesser extent (Eckburg et al., 2005). The complex intestinal biomass is predicted to consist of greater than 1000 different phylotypes comprised of both the permanent or autochthonous inhabitants which form the stable populations of the adult gut ecosystem, and the acquired allochthonous or transient flora, which are obtained from diet (such as probiotics) or from the environment. Probiotics are live microorganisms, most frequently species of lactobacilli and bifidobacteria, which when administered in adequate amounts, confer a health benefit on the host (FAO/WHO, 2001). Although such health benefits are strain-dependent (Helwig et al., 2006), a single daily oral administration of 10⁹ bacterial colony forming units (CFU) has generally been accepted as the optimal probiotic dose based on detection of the organism in human feces (Tannock, 2003). However, in some instances higher quantities may be required (Gionchetti et al., 2007, Larsen et al., 2006, Lee and Lee, 2009, Mimura et al., 2004), and thus the efficacious dosage may also be regarded as strain-dependent.

The intestinal microbiota is thought to have an integral role in human nutrition, metabolism, epithelial development, immune modulation, regulation of fat storage and protection against pathogens (Backhed et al., 2004, Claus et al., 2008, Corr et al., 2007, Lee et al., 2003, Martin et al., 2010, Martin et al., 2008b, Wells et al., 2010). Indeed, fluctuations in the composition of the intestinal ecosystem have been associated with various disease states including inflammatory immune disorders, obesity and cancer (Backhed, 2010, Davis and Milner, 2009, Scanlan et al., 2006). In this respect, probiotic interventions may beneficially influence the intestinal microbial ecology, intestinal homeostasis and host metabolism and, ultimately, the health of the host. Strain-specific microbial derived bioactive molecules, collectively termed “pharmabiotics” which include live or dead microorganisms as well as bacterial constituents and metabolites, may mediate many such interactions within the GIT (Shanahan et al., 2009). For example, production of antimicrobial peptides such as bacteriocins may beneficially modulate the intestinal ecology by the specific inhibition of pathogens (Corr et al., 2007).

Probiotics interact with the intestinal epithelial cells directly via cell components such as DNA, lipoteichoic acids and cell-surface polysaccharides (Ghadimi et al., 2010, Jijon et al., 2004, Lebeer et al., 2010, Pedersen et al., 2005) but also indirectly, through the production of bioactive metabolites (Heuvelin et al., 2009, Menard et al., 2004, Tao et al., 2006, Yan et al., 2007). Microbial-derived peptides and polysaccharides can activate signaling pathways and influence factors such as cytokine secretion and gut permeability, thereby enhancing epithelial barrier function. For example, a bioactive peptide secreted by Bifidobacterium infantis increased transepithelial resistance of human T84 cells by altering the expression of tight junction proteins (claudins and occludin) and cytokines (IFN-γ) via a mitogen-activated protein (MAP)-kinase-dependent mechanism (Ewaschuk et al., 2008). B. infantis conditioned medium also reduced colonic permeability and improved colitis in IL-10 deficient mice (Ewaschuk et al., 2008). Furthermore, the anti-inflammatory activity mediated by a bioactive polysaccharide PSA produced by the gut commensal Bacteriodes fragilis protected mice against Helicobacter hepaticus induced colitis (Mazmanian et al., 2008). Therefore, certain members of the intestinal microbiota can enhance gut barrier function and maintain intestinal homeostasis indicating a potential therapeutic role for probiotics in the prevention and treatment of gastrointestinal diseases.

In addition to having local effects in the gut, the intestinal microbiota is thought to possess the capacity to influence the overall metabolic homeostasis of the host. Indeed, germ-free mice exhibited altered metabolic profiles of the colon, liver and kidney compared with conventional animals (Claus et al., 2008). The colon of germ-free animals was characterized by an increase in raffinose and reduced levels of amino acids, ammonia, 5-aminovalerate and short chain fatty acids (SCFA) as well as other metabolites involved in energy pathways (Claus et al., 2008). Moreover, elevated levels of taurine, trimethylamine-N-oxide (TMAO) and bile acids were evident in the liver of germ-free animals. In addition, higher levels of choline, betaine, myo-inositol and scyllo-inositol in the kidneys of germ-free mice were linked with a hypertonic environment which was associated with renal dysfunction in previous studies (Claus et al., 2008). Indeed the extensive metabolic capability of intestinal microorganisms, such as bifidobacteria, is thought to provide them with a competitive advantage within the GIT. For example, it was recently suggested that the predominance of Bifidobacterium breve populations characteristic of the infant microbiota may be attributed to an operon dedicated to the degradation of starch, amylopectin and pullulan exclusively present in this species (Kleerebezem and Vaughan, 2009). This undoubtedly contributes to the ability of bifidobacteria to colonize early in life given that human milk and colostrum contain a wide variety of complex sugars. It has also been established that interventions with such probiotic strains can modulate the metabolic phenotype of the host in a very specific manner. For instance, the administration of Lactobacillus paracasei or Lactobacillus rhamnosus (in the presence or absence of galactosyl oligosaccharide prebiotics) increased bifidobacteria and reduced Clostridium perfringens and Staphylococcus aureus populations of germ-free mice inoculated with a model of human baby microbiota compared with control animals (Martin et al., 2008a, Martin et al., 2008b). The resulting alterations in lipid metabolism involved a decrease in hepatic triglycerides and an increase in long chain poly unsaturated fatty acids (PUFA) and correlated with a reduction in plasma lipoprotein levels. The modulation of amino acid, methylamine and SCFA metabolism as well as glycogenesis and gluconeogenesis was also reported upon exposure to the probiotics (Martin et al., 2008a, Martin et al., 2008b). As discussed in greater detail below, the gut microbiota may also modulate the fatty acid composition of the liver, brain and adipose tissue of the host via synthesis of bioactive fatty acids such as conjugated linoleic acid (CLA) (Wall et al., 2010, Wall et al., 2009). Anti-inflammatory and anti-cancer properties are among the wide array of health-promoting effects associated with isomers of CLA (Cook et al., 1993, Ha et al., 1987). Therefore, production of such bioactive metabolites may also be considered an important probiotic trait.

It is thus evident that the commensal microbiota has an almost infinite potential for metabolite production, many of these are pharmabiotic substances which can positively influence human health. In this review, we specifically examine the ability of the gut flora to produce antimicrobial peptides, namely bacteriocins, and bioactive fatty acids such as CLA and discuss their contribution to probiotic functionality.