Living on the edge: A major role for cell surface molecules in probiotic-host interactions
Ingmar Claes, Sarah Lebeer, Jos Vanderleyden, Sigrid De Keersmaecker
Address: Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
Email:
Introduction:Recent studies suggest that microbe-associated molecular patterns (MAMPs) derived from intestinal bacteria may modulate specific diseases, like inflammatory bowel diseases (IBD) via stimulation of their respective pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). The final outcomeof a host cell response following an ancounter with microorganisms (beingpathogenic, probiotic or commensal) depends on thecombination of the distinct MAMPs that can interactwith the various PRRs and associated co-receptors thatfine-tune signalling. MAMPs are often components of the cell surface of bacteria and presumed key players in bacteria-host interactions.In our research group, various surface molecules of the probiotic Lactobacillus rhamnosus GG (LGG) are being studied as potential MAMPs. One example is lipoteichoic acid (LTA), an abundant molecule anchored to the cell wall of Gram-positive bacteria,which has been found to mediate inflammatory responses through TLR2.It consists of a glycerol-phosphate or ribitol-phosphate chain decorated with D-alanine ester or glycosyl substitutions, and extending into the cell wall.D-alanylation of LTA is directly related to the charge properties of this polymer, contributing to its function. To investigate the role of D-alanylation of LTA in probiotic interactions of LGG with the host, an LGG dltD mutant was constructed, which is completely devoid of D-alanine ester substitutions on LTA. ThisdltD mutant was subsequently tested in experimental mouse models for inflammatory bowel diseases (IBD).
Methods: Cell surface molecules ofLGG are mainly being studied by using in-house constructed site-specific knock-out mutants lacking key molecules.After construction of these mutants, they are first phenotypically characterized related to their changed cell surface properties and subsequently tested in in vitro assays for adhesion, biofilm formation and immunomodulation. In a next step, we use dextran sulfate sodium (DSS) to induce severe or mild chronic experimental colitis in mice. Mice receive phosphate buffered saline (PBS), LGG wild-type or a mutant via the drinking water.Macroscopic parameters, histological abnormalities and cytokine differences are scored to assess disease activity.
Results:Both in amild and a moresevere model, the dltD mutant was demonstrated to alleviate colitic parameters (e.g. body weight and macroscopic scoring) compared to PBS and wild-type treated mice. Furthermore, qRT-PCR analysis revealed that administration of dltD mutant bacteria to the colitic mice resulted in a downregulation of proinflammatory cytokines such as IL-12 and IFN-γ and toll-like receptor2 expression (TLR2).
Discussion:These results exemplify the importance of a singlesurface moleculefor the pro- or anti-inflammatory effects of LGG in experimental colitis. Further research involves revealing the molecular mechanisms underlying the positive effects of this altered cell surface and identifying the signalling pathways that are targeted.Ultimately, 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 on well defined molecular criteria.