Analysis of the Peptidoglycan Hydrolase Complement of Lactobacillus casei and Characterization of the Major γ-D-Glutamyl-L-Lysyl-Endopeptidase

Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties

Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

Peer reviewe

The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG

Peer reviewe

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

BACKGROUND: A distinctive feature of type 2 diabetes is inability of insulin-secreting beta-cells to properly respond to elevated glucose eventually leading to beta-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of beta-cell dysfunction. PRINCIPAL FINDINGS: We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon. CONCLUSION: The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms

Cued to act on impulse: more impulsive choice and risky decision making by women susceptible to overeating after exposure to food stimuli

There is increasing evidence that individual differences in tendency to overeat relate to impulsivity, possibly by increasing reactivity to food-related cues in the environment. This study tested whether acute exposure to food cues enhanced impulsive and risky responses in women classified on tendency to overeat, indexed by scores on the three factor eating questionnaire disinhibition (TFEQ-D), restraint (TFEQ-R) and hunger scales. Ninety six healthy women completed two measures of impulsive responding (delayed discounting, DDT and a Go No-Go, GNG, task) and a measure of risky decision making (the balloon analogue risk task, BART) as well as questionnairemeasures of impulsive behaviour either after looking at a series of pictures of food or visually matched controls. Impulsivity (DDT) and risk-taking (BART) were both positively associated with TFEQ-D scores, but in both cases this effect was exacerbated by prior exposure to food cues. No effects of restraint were found. TFEQ-D scores were also related tomore commission errors on the GNG, while restrained women were slower on the GNG, but neither effect was modified by cue exposure. Overall these data suggest that exposure to food cues act to enhance general impulsive responding in women at risk of overeating and tentatively suggest an important interaction between tendency for impulsive decision making and food cues thatmay help explain a key underlying risk factor for overeating

Factors determining the probiotic effect of Lactobacillus rhamnosus GG in experimental colitis

Probiotics are defined as live micro-organisms that, when administered in adequate amounts, can confer a health benefit on the host . During the last decades, several clinical and experimental studies have been published on the health-promoting capacities of probiotics, which are often applied as functional food products. An important challenge in probiotic research is to develop and validate assays to determine the health-promoting capacities of probiotics as the effects are generally more subtle compared to pharmaceutical drugs or only observed in preventive set-ups. It is thought that probiotics can exert beneficial effects by (i) restoring microbial imbalances, (ii) enhancing the epithelial barrier function, and/or (iii) modulating immune responses. A crucial factor for successful application is the choice of the probiotic strain for each specific purpose. Lactobacillus rhamnosus GG (LGG) is one of the best documented probiotic strains with proven health benefits that include the prevention and relieve of acute diarrhea in children, the prevention of antibiotic-associated diarrhea and the prevention of atopic disease. However, the effects of LGG against f inflammatory bowel diseases (IBD) are less clear. IBD diseases such as Crohn s disease (CD) and ulcerative colitis (UC), are chronic idiopathic diseases that involve inflammation of the intestinal tract. IBD patients are very interested in the application of probiotics.The aim of this PhD research was to get a better insight in the molecular mechanisms behind the probiotic mode of action of LGG in relation to gut inflammation by using both in vitro and in vivo experiments. At the Centre of Microbial and Plant Genetics (CMPG), we have developed various genetic and molecular tools specifically for LGG, including the construction of knock-out mutants to study the effector molecules of this probiotic strain. By comparison of the immunomodulatory capacity of mutants that lack a certain surface molecule with the capacity of LGG wild-type, the role of these surface molecules is studied in situ with live bacteria.Lipoteichoic acid (LTA) is generally regarded as the Gram-positive pro-inflammatory counterpart of Gram-negative lipopolysaccharides (LPS). In the present work, we investigated the impact of a dedicated dltD gene knock-out mutation, resulting in removal of the D-alanine residues of LTA, on the anti-inflammatory efficacy of LGG in a murine experimental colitis model. In the mouse model, the probiotic efficacy of LGG was significantly enhanced by the dltD mutation. These results highlight the impact of a single gene on the overall pro- or anti-inflammatory effects of LGG in experimental colitis. Although the absence of a pro-inflammatory LTA on the dltD mutant could explain why the dltD mutant is less pro-inflammatory, it does not fully explain why this mutant is actually able to alleviate colitis symptoms. This observation indicated, therefore, the existence of LGG molecules that can confer anti-colitis effects. As the dltD mutant was found to have different amounts of the major secreted proteins (Msp s) in its cell culture supernatant compared to LGG wild-type, Msp1 and Msp2 were further investigated for their potential anti-colitis role.Both Msp1 (p75) and Msp2 (p40) were previously shown to play a key role in interaction with intestinal epithelial cells by promoting homeostasis. As they showed homology to peptidoglycan (PG) hydrolases, their role in the physiology of LGG was first investigated. We found that these proteins have crucial physiological roles by acting as a PG hydrolase involved in bacterial cell separation (Msp1) or as an indispensible enzyme which possibly initiates cell septum formation (Msp2). Subsequently, we studied the importance of these two proteins and PG for eliciting anti-inflammatory effects in intestinal epithelial cells. Surprisingly, we found that Msp2 interacts with Toll like receptor (TLR) 2. How these interactions relate to the anti-inflammatory effect of Msp2 in a disease situation remains elusive.Ultimately, we hope that disclosing the genetic factors supporting probiotic action of this prototypical strain will contribute to delineating the optimal conditions driving the best performance of LGG and to the screening and selection of novel probiotic strains based on well-defined molecular criteria.status: publishe

Anti-inflammatory potential of probiotics: lipoteichoic acid makes a difference

Lipoteichoic acid (LTA) mutants of lactobacilli suppress inflammation in animal models of experimental colitis. The fact that a single mutation of an administered Lactobacillus strain can result in enhanced probiotic efficacy is surprising given the genetic diversity and complexity of the intestinal niche, but at the same time exciting from a microbiological, immunological and gastroenterological point of view. In this Opinion article, we discuss the possible impacts of LTA modification in probiotic bacteria in the context of the current knowledge regarding the proinflammatory capacity of LTA, structure-activity relationships of LTA, intestinal LTA recognition in healthy and colitis conditions and anti-inflammatory molecules of lactobacilli.status: publishe

Functional mechanisms of probiotics

Probiotics are the live microorganisms which when ingested in adequate amounts confer health benefits. The strains most frequently used as probiotics include Lactic acid bacteria, bifidobacteria and yeast Saccharomyces boulardii. However, several other bacterial strains are being investigated for potential probiotic value viz. Enterococcus, Streptococcus, Bacillus, among others. Significant therapeutic potential of probiotics has been demonstrated in several in vitro studies and that involving animal models and humans. Despite intense focus on probiotics research the mechanisms responsible for health benefits are not yet completely understood. Several important mechanisms have been proposed such as improvement of gut epithelial barrier function, Immunomodulatory effects, degradation of toxin receptors, competition for nutrients, production of inhibitory substances, antiproliferative effects, blocking of adhesion sites and modulation of gut microbiota. Bacterial cell components such as DNA or peptidoglycan may also be involved in functional mechanism of probiotics. Effectiveness of a probiotic for potential application as prophylactic or treatment agent for certain ailment is determined by its ability to possess all or most of these characteristic features. The current article describes the general functional mechanisms of probiotics