Research On The Adaptation Mechanism In Bifidobacterium Longum NCC2705 Grown In Git

Bifidobacteria are Gram-positive, obligate anaerobic bacteria in the Actinomycetales branch of the high-G+C microorganisms. The gastrointestinal tracts (GIT) of healthy humans are colonized by a large, active and complex community of microbes, collectively termed intestinal microbiota. Bifidobacterium is considered to play an important role in the balance of normal intestinal flora. In addition, the contribution of Bifidobacterium to the host keeping the banlance of the GIT, inhibiting pathogenic bacterium, anti-aging and anti-cancer have been reported. By liberating lactic and acetic acids, Bifidobacteria prevent the colonization of potential bacterial pathogens in the colon, thereby maintaining a balance of normal intestinal flora. Many studies have provided evidence that certain strains of Bifidobacterium longum may be beneficial to the prevention and/or treatment of gastroenteritis, colonitis necrotizing enterocolitis, and chronic intestinal inflammation. However, there is few report on the interactions between bifidobacteria an the cells of GIT, especially the adaptation mechanism of them.In our research, the bacteria we utilizing is Bifidobacterium longum NCC2705 which was isolated from feces of infant. It became the important probiotics of Nestle Corporation. Genome sequencing projects of the bacteria had been completed by the researchers of Nestle Research Center in 2002. Thus B. longum NCC2705 had been the first bifidobacterium with clear genetic background.To investigate the molecular mechanisms underlying the adaptation of Bifidobacterium longum to the intestinal tract, we utilized a new model for rabbit intestinal culture of B. longum and reported the changes in proteomic profiles after incubation in the in vivo environment. By 2D-PAGE coupled with the software of ImageMaster 2D Elite Platnum, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses, proteomic profiles of B. longum NCC2705 grown in the in vivo and in vitro environments were compared. To confirm the results from the comparative proteomics experiments, total RNA was isolated from B. longum cultures and probed for expression of genes which encode the proteins that displayed at least a 3-fold change in volume by RT-PCR. Assessing the quantitative differences in relative volume by image analysis revealed 38 spots that exhibited a change of 3-fold or greater. These 38 spots represent 33 proteins. There are 19 up-regulated proteins, 14 down-regulated proteins, and 4 proteins with mobility changes were identified during intestinal growth. These identified proteins include key stress proteins, metabolism-related proteins, and proteins related to translation and so on.Then, some useful proteins which reflected the adaptation of B. longum NCC2705 to the intestine are analyzed. The expression of bile salt hydrolase (BSH) is increased 5.2 folds during intestinal growth. BSH catalyzes the hydrolysis of glycine- and/or taurineconjugated bile salts into amino acid residues and deconjugated bile salts. BSH might play a role in the molecular mechanisms for the initial interaction of probiotic with the intestinal environment. And it might play an important role in utilizing the liberated amino acids or increasing resistance to the toxic levels of bile salts in the gastrointestinal tract. The expression of elongation factor Tu (EF-Tu) is increased more than 5 folds during intestinal growth. EF-Tu is a new surface protein possessing the characteristics of an adhesion factor and that it is able to induce a proinflammatory response. The up-regulated of EF-Tu suggests that the protein contributes to the retention and attachment as a Bifidobacterium adhesinlike factor. The stress proteins are able to defend B. longum against the harmful ingredients of the gastrointestinal tract (GIT). These proteins involved in protein folding, assembly, and degradation (GroEL), trigger factor chaperone (Tig), and ATP-dependent Clp protease proteolytic subunit 2 (ClpP2). They showed increased synthesis during growth in vivo. Bifidobacteria colonize the lower GIT. They gain the energy mainly from the substrate phosphorylation of glycolysis. Bifidobacteria possess only one route for the metabolism of glucose, the fructose-6-phosphate phosphoketolase pathway. Three of nine enzymes of the bifid shunt were expressed at higher levels during intestinal growth in this study, including transaldolase, transketolase, and a probable ribose 5-phosphate isomerase. The proteins involved in the bifid shunt pathway are up-regulated significantly in the intestinal environment. It is suggested that B. longum NCC2705 can take full advantage of these enzymes to minimize crossfeeding of competitors.We also find that four proteins GlnA1, PurC, LuxS, and Pgk exhibit clear post-translational modification. Western blot analysis and Pro-Q Diamond staining revealed that Pgk and LuxS may become phosphorylated at Ser/Thr residues when growing in vivo. Pgk in B. longum NCC2705 may become phosphorylated to form the phosphoenzyme intermediate. And the phosphorylated autoinducer-2 production protein LuxS of B. longum NCC2705 (LuxS-P) is the active form of LuxS. It is probably that LuxS-P may play a key role in the regulation of quorum sensing.In the study above, we found several proteins which are important to the adaptation of B. longum in GIT. The phosphorylation of the LuxS suggested that the protein play an important role when growing in vivo. To confirm the existence of AI-2/luxS QS system in B. longum NCC2705, we clone and express the LuxS proteins of B. longum in E. coli and detect the fluorescence intensity of the cell-free condition media of the B. longum NCC2705 and the recombinant bacteria utilizing a bioluminescent bacterial reporter strain, Vibrio harveyi BB170, which produces light in response to AI-2. We found that the LuxS protein can be expressed in E. coli. The fluorescence of the V. harveyi increases after adding the cell-free culture fluids of B. logum NCC2705 and recombinant bacteria. There is significant positive correlation between the intensity of B. longum NCC2705 and the amount of AI-2 within certain limits. Consequently, the LuxS of B. longum NCC2705 is active, and the LuxS/AI-2-dependent QS system which can produces the quorum-sensing signal autoinducer-2.Our research has much significance for Bifidobacteria.spp living in the gastrointestinal tracts (GIT). It is the first time to utilize the rabbit model in the research on the physicological functions of B. longum. The technology Comparative Proteomics and Phosphorelated stain are used to find the changing of the protein expression. Additionally, we confirm the existence of the LuxS and AI-2 in B. longum NCC2705. Knowledge from the research can push the genetic vaccine development and create new research field of B.longum NCC2705.