Moleculaire analyse van de interactie tussen het probioticum Lactobacillus rhamnosus GG en intestinale epitheelcellen

Probiotics are defined as “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host”. Growing numbers of intervention trials show that certain bacterial strains or mixtures of strains, generally lactobacilli and bifidobacteria, can confer specific health benefits. Many of the interactions between probiotic bacteria and the host are thought to be mediated by molecular structures, known as microbe-associated molecular patterns (MAMPs), which can be recognized by the host through specific pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Unfortunately, in human intervention trials, there is often a lack of attention to mechanisms of actions. Nevertheless, we believe that for an optimized application of probiotics, it is imperative to understand the mechanisms of interaction with the host in detail. Lactobacillus rhamnosus GG is one of the best described probiotic strains with proven health benefits including promotion of gastro-intestinal health, prevention of respiratory tract infections and modulation of the immune system. In this PhD research, we aimed to develop a platform for the elucidation of PRR-mediated probiotic mechanisms, in specific with intestinal epithelial cells (IECs). We chose to focus on previously described cell wall components of L. rhamnosus GG: lipoteichoic acid (LTA), the spaCBA pili and the major secreted proteins Msp1 and Msp2. Earlier research indicated that the promiscuous TLR-2 might be an important PRR for L. rhamnosus GG. First, we developed TLR reporter cell lines in a simple HEK293T model cell to analyze whether the bacterial cell wall compounds could activate TLR-mediated NF-κB signaling. An important innovative step in this PhD thesis was the development of a stable TLR-2/TLR-6 expressing cell line. Hereby, we aimed to investigate the roll of bacterial cell wall compounds as MAMPs by their co-incubation with these reporter cell lines under different forms: purified fractions (with or without chemical modification), recombinant proteins, and mutant strains that allow to investigate the presentation of MAMPs in their cellular context. We also aimed to elucidate structure-activity relationships (SAR) of MAMP-PRR interactions in a second step. In a third step, we aimed to confirm the initial findings in a more complex setting in IEC lines.LTA is generally regarded as the Gram-positive counterpart of Gram-negative lipopolysaccharides. In this work, we used highly purified LTA and a mutant with a modified LTA structure to show that LTA is an important MAMP of L. rhamnosus GG for the interaction with TLR-2, only when present in a heterodimer with TLR-6, resulting in NF-κB activation. The SAR analysis confirmed that the acyl chains of the glycolipid anchor are critical for binding to the receptor. These findings could be confirmed in the Caco-2 IEC line, as LTA induced the pro-inflammatory chemokine IL-8. These data support a key role for LTA in immune stimulatory actions of L. rhamnosus GG.SpaCBA pili are proteinaceous protrusions of the cell surface that play a crucial role in the excellent adherence capacity of L. rhamnosus GG. We analyzed isogenic single and double mutants of L. rhamnosus GG for their capacity to interact with TLR-2/TLR-6. The data suggest that the SpaCBA pili, when presented on live cells, negatively interfere with TLR-2/TLR-6 mediated NF-κB induction by other ligands, such as LTA. To gain better insights in the immunomodulatory role of L. rhamnosus GG, we optimized a purification protocol to obtain native SpaCBA pili in pure form. Surprisingly, the first data with these purified pili show that they can significantly activate NF-κB signaling in the TLR-2/TLR-6 cell line when presented under this form at a concentration of 5 µg/ml. The major secreted proteins Msp1 and Msp2 were previously shown to promote homeostasis in IECs, dependent on the Akt signaling pathway. Here, we show that 10-100 µg/ml of the recombinant proteins could induce TLR-2/TLR-6 mediated NF-κB signaling. Moreover, the purified native glycosylated Msp1 did also induce NF-κB activation in the TLR-2/TLR-6 cell line, indicating that the glycosylation does not play a major role in this interaction. Whether Msp1/2 mediated TLR-2/TLR-6 signaling is also involved in Akt activation, remains under investigation. In this PhD work, we contributed to better molecular insights on MAMP-TLR2/6 interactions of L. rhamnosus GG, although many details remain to be unraveled. Nevertheless, we are convinced that the unique set-up with the use of both proteins (recombinant and purified) and mutant strains can help elucidate MAMP-PRR interactions and SAR. This platform could further help to clarify probiotic modes of action and could help the design of novel applications, not only in L. rhamnosus GG, but also in other strains.nrpages: 165status: publishe