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

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Julkaisun nimi: Characterization of Lactobacillus pili and the niche-adaptation factors of intestinal Lactobacillus ruminis
Tekijä: Yu, Xia
Muu tekijä: Helsingin yliopisto, eläinlääketieteellinen tiedekunta
Opinnäytteen taso: Väitöskirja (artikkeli)
Tiivistelmä: 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 GIT. L. rhamnosus GG has two pilus operons: spaCBA encoding the well-studied SpaCBA pili and spaFED putatively encoding SpaFED pili. The expression of SpaFED pili in L. rhamnosus GG under laboratory conditions has not thus far been reported. In this study, 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, to certain extracellular matrix 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 HEK 293-blue cells expressing TLR2, and in Caco-2 cells. While encountering human peripheral blood monocyte-derived dendritic cells, neither immune response enhancement nor immune dampening by SpaFED pili was observed. Consistent with genomic analyses, transmission electron microscopy demonstrated that pili in gut autochthonous L. ruminis ATCC 25644 of human origin consisted of the tip (LrpC), basal (LrpB), and backbone (LrpA) pilins. 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 IL-8 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. Subsequently, a novel L. ruminis strain was isolated from porcine feces and demonstrated to be flagellated and piliated. We analyzed the abilities of this new isolate and three other L. ruminis strains 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 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.not available
URI: URN:ISBN:978-951-51-3972-6
Päiväys: 2018-01-19
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