Characterization of Lactobacillus pili and the niche-adaptation factors of intestinal Lactobacillus ruminis

The mammalian gut microbiota is composed of autochthonous species that permanently colonize the host intestine, and of allochthonous species that are only transiently able to occupy the intestinal environment. In this thesis research, Lactobacillus ruminis and Lactobacillus rhamnosus GG were investigated as paradigms for each type of microbe–host interaction, with special emphasis on the in vitro characterization of their adaptation factors in the host gastrointestinal tract (GIT). As one of the best-characterized probiotics, L. rhamnosus GG is of great interest to researchers and the probiotic industry. According to previous genome analyses, L. rhamnosus GG has two pilus operons: spaCBA encoding the well-studied SpaCBA pili and spaFED putatively encoding SpaFED pili. However, the expression of SpaFED pili in L. rhamnosus GG under laboratory conditions has not thus far been reported. Nevertheless, in vivo expression of spaFED cannot be ruled out, either. Therefore, in this study, aimed at exploring the role of SpaFED pili in host adhesion and immunomodulatory effects, a nisin-induced expression system was used for the generation of SpaFED or SpaF-deleted pili in Lactococcus lactis NZ9000. The results revealed that SpaFED pili were essential in mediating lactococcal adhesion to intestinal cell lines (Caco-2 and HT-29), to certain extracellular matrix (ECM) proteins, and to porcine mucins, the tip pilin SpaF playing a central role as a focal adhesion factor. With regard to immunomodulation, SpaFED pili appeared to dampen the immune responses, which was largely attributed to the SpaF pilin, in human embryonic kidney (HEK) 293blue cells expressing toll-like receptor (TLR) 2, and in Caco-2 cells. However, while encountering human peripheral blood monocyte-derived dendritic cells (moDCs), neither immune response enhancement nor immune dampening by SpaFED pili was observed. For the autochthonous L. ruminis species, the mammalian GIT is so far the only identified niche. Consistent with genomic analyses, transmission electron microscopy (TEM) demonstrated that pili in L. ruminis ATCC 25644 of human origin consisted of the tip (LrpC), basal (LrpB), and backbone (LrpA) pilins, assembled in a pattern similar to that in SpaCBA and SpaFED pili of L. rhamnosus GG. Analogously to dissecting the characteristics of SpaFED pili in L. rhamnosus GG, recombinant L. lactis strains were constructed, producing either LrpCBA or LrpC-lacking pili of L. ruminis. Recombinant LrpCBA pili mediated lactococcal adherence to ECM proteins and intestinal epithelial cells, and also dampened TLR2-dependent NF-κB signaling and IL8 production in HEK cells, whereas L. ruminis itself induced evidently elevated immune responses. When incubated with Caco-2 cells, L. ruminis and recombinant lactococcal constructs expressing pili with and without LrpC pilin lowered IL-8 production. Biofilm formation in L. ruminis was not attributed to LrpCBA pili. Subsequently, a novel L. ruminis strain (GRL1172) was isolated from porcine feces and demonstrated to be flagellated and piliated. To expand our knowledge of the autochthony-promoting properties of this gutdwelling species, we analyzed the abilities of this new isolate and three other L. ruminis strains (a human isolate, ATCC 25644, and two bovine isolates, ATCC 27780 and ATCC27781) to adhere to host cells and extracellular matrix proteins, to inhibit pathogen binding and growth, to maintain epithelial barrier functions, and to modulate immune responses in vitro. The results indicated that the strains shared several characteristics, such as binding to ECM proteins and HT-29 cells, inhibition of pathogen adhesion and growth, maintenance of epithelial barrier functions in epithelial cells, and activation of innate immune responses to various extents. In conclusion, this thesis study demonstrated the adhesiveness of SpaFED and LrpCBA pili, which may respectively promote the gut retention of L. rhamnosus GG (in addition to the SpaCBA pili) and of L. ruminis. Moreover, pilus-mediated dampening of innate immune responses might be a strategy for these two gut bacterial species to induce immune tolerance in the host. Additionally, L. ruminis inhibited enteropathogen adhesion and growth, as well as maintained intestinal barrier function, which could be regarded as beneficial to the host and which may in turn favor the persistent residence of L. ruminis.