Sigrid De Keersmaecker

Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens

How can probiotic bacteria transduce their health benefits to the host? Bacterial cell surface macromolecules are key factors in this beneficial microorganism–host crosstalk, as they can interact with host pattern recognition receptors (PRRs) of the gastrointestinal mucosa. In this Review, we highlight the documented signalling interactions of the surface molecules of probiotic bacteria (such as long surface appendages, polysaccharides and lipoteichoic acids) with PRRs. Research on host–probiotic interactions can benefit from well-documented host–microorganism studies that span the spectrum from pathogenicity to mutualism. Distinctions and parallels are therefore drawn with the interactions of similar molecules that are presented by gastrointestinal commensals and pathogens.

Genes and Molecules of Lactobacilli Supporting Probiotic Action

SUMMARY Lactobacilli have been crucial for the production of fermented products for centuries. They are also members of the mutualistic microbiota present in the human gastrointestinal and urogenital tract. Recently, increasing attention has been given to their probiotic, health-promoting capacities. Many human intervention studies demonstrating health effects have been published. However, as not all studies resulted in positive outcomes, scientific interest arose regarding the precise mechanisms of action of probiotics. Many reported mechanistic studies have addressed mainly the host responses, with less attention being focused on the specificities of the bacterial partners, notwithstanding the completion of Lactobacillus genome sequencing projects, and increasing possibilities of genomics-based and dedicated mutant analyses. In this emerging and highly interdisciplinary field, microbiologists are facing the challenge of molecular characterization of probiotic traits. This review addresses the advances in the understanding of the probiotic-host interaction with a focus on the molecular microbiology of lactobacilli. Insight into the molecules and genes involved should contribute to a more judicious application of probiotic lactobacilli and to improved screening of novel potential probiotics.

Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG

ABSTRACT Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

ABSTRACT Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.

Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

ABSTRACT Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.

Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria.

The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.

Brominated furanones inhibit biofilm formation by Salmonella enterica serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium is a main cause of bacterial food-borne diseases. As Salmonella can form biofilms in which it is better protected against antimicrobial agents on a wide diversity of surfaces, it is of interest to explore ways to inhibit biofilm formation. Brominated furanones, originally extracted from the marine alga Delisea pulchra, are known to interfere with biofilm formation in several pathogens. In this study, we have synthesized a small focused library of brominated furanones and tested their activity against S. enterica serovar Typhimurium biofilm formation. We show that several furanones inhibit Salmonella biofilm formation at non-growth-inhibiting concentrations. The most interesting compounds are (Z)-4-bromo-5-(bromomethylene)-3-alkyl-2(5H)-furanones with chain lengths of two to six carbon atoms. A microarray study was performed to analyze the gene expression profiles of Salmonella in the presence of (Z)-4-bromo-5-(bromomethylene)-3-ethyl-2(5H)-furanone. The induced genes include genes that are involved in metabolism, stress response, and drug sensitivity. Most of the repressed genes are involved in metabolism, the type III secretion system, and flagellar biosynthesis. Follow-up experiments confirmed that this furanone interferes with the synthesis of flagella by Salmonella. No evidence was found that furanones act on the currently known quorum-sensing systems in Salmonella. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. Conclusively, this work demonstrates that particular brominated furanones have potential in the prevention of biofilm formation by Salmonella serovar Typhimurium.

Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine

Probiotic bacteria are administered as live microorganisms to provide a health benefit to the host. Insight into the adaptation factors that promote the survival and persistence of probiotics in the gastrointestinal tract (GIT) is important to understand their performance. In this study, the role of the long galactose‐rich exopolysaccharides (EPS) of the prototypical probiotic strain Lactobacillus rhamnosus GG (LGG) was investigated. In a competition experiment with wild type, the isogenic EPS mutant CMPG5351 exhibited a reduced persistence in the murine GIT, especially in the lower parts of the intestine. This was surprising as our previous in vitro studies had shown an increased adhesion capacity for this EPS mutant. Follow‐up assays indicated that this mutant is more sensitive towards host innate defence molecules, such as the LL‐37 antimicrobial peptide and complement factors. This suggests that EPS forms a protective shield for LGG against these molecules in the GIT. Moreover, culturing LGG wild‐type in subinhibitory concentrations of host defence factors such as LL‐37 resulted in increased production of EPS, indicating that bacterial EPS production is modulated in the host to fine‐tune the balance between adhesion and immune evasion. These observations are of interest in understanding the dynamics of adaptation of probiotics to the host environments.

A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella Typhimurium LT2.

BackgroundMetabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. Salmonella enterica subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem.ResultsHere, we describe a community-driven effort, in which more than 20 experts in S. Typhimurium biology and systems biology collaborated to reconcile and expand the S. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for S. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches.ConclusionTaken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.