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Research on the Adaptation Mechanism in Bifidobacterium Longum NCC2705 Grown in Git

Author: JiangZuo
Tutor: HuangLiuYu；YuanJing
School: PLA Military Academy of Medical Sciences
Course: Preventive Medicine in Military
Keywords: Bifidobacterium longum NCC2705 Comparative Proteomics Phosphorylation Quorum sensing AI-2 signaling molecules Bioluminescence analysis
CLC: R371
Type: Master's thesis
Year: 2010
Downloads: 141
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Abstract

The actinomycetes bacteria Branch Bifidobacterium genus Bifidobacterium genus, gram-positive bacteria, GC% content is high, obligate anaerobic. In a variety of memory in the healthy human intestinal bacteria, bifidobacteria play physiological function is most important. Bacteria, inhibit the growth of pathogenic bacteria, anti-aging, anti-tumor, and provide a variety of nutrients for the human body to play a role in maintaining the balance of intestinal flora. Bifidobacterium regulation of intestinal pH, and rely on adhesion ability to inhibit foreign invasion of a variety of enteric pathogens and opportunistic pathogen invasion, prevention and treatment of gastroenteritis, colon through the synthesis of lactic acid and acetic acid and other substances , necrotizing enterocolitis, and chronic intestinal infection and other diseases of the gastrointestinal tract. However, reported very little research on the interaction of bifidobacteria and intestinal cells, especially bifidobacteria in the intestinal environment adaptability mechanism is no report. Bifidobacterium longum NCC2705 as used in the present study were isolated from the faeces of healthy infants, is a strain of Bifidobacterium associated with a human body is very important. Nestle researchers in Switzerland in 2002 and completed the sequencing of the genome of the bacterium, making it the first clear genetic background Bifidobacterium facilitate functional genomics and proteomics research. Research Bifidobacterium longum NCC2705 in intestinal adaptation mechanism, we use the rabbit as an animal model, the rabbit colon and rabbits in vitro culture of Bifidobacterium longum NCC2705 comparative proteomics research. Using two-dimensional gel electrophoretic separation of protein electrophoresis results were analyzed using Image Master 2D Elite Platnum software. Study found a total of 38 protein spots were a more than 3-fold change in expression abundance in after rabbits culture, Bifidobacterium longum the NCC2705 genome-wide protein, while there are four pairs of proteins in the glue chart position migration, may be a result of post-translational modification of the protein. Since then, we have these 38 protein spots differentially expressed protein spots and displacement, MALDI-TOF-MS and / or ESI-MS/MS identified by mass spectrometry, and by semi-quantitative RT-PCR validation. The results showed that the 38 protein spots represent 33 proteins, compared to culture in vitro, in vivo culture Bifidobacterium longum 19 upregulation and 14 downregulation four protein position migration. These differences in protein include stress protein and energy metabolism related proteins, as well as translation-related protein. Bifidobacterium longum NCC2705 intestinal adaptability key protein analysis: in vivo environment, culture, bile salt hydrolase increased 5.2 times than in vitro expression abundance. Bile salt hydrolase enzyme that catalyzes the hydrolysis of glycine and taurine conjugation of bile salts, and the release of amino acids to the formation of bile acids and bile salts inactivation probiotics to avoid the toxic effects of bile salts. Enzyme in the early probiotic and environmental interactions in vivo, as well as in the process of the body's intestinal utilization of free amino acids and intestinal anti-bile salt poisoning has played an important role. Elongation factor EF-Tu in vivo cultured expression abundance increased more than five-fold. It is a novel surface protein, the main role in the process of protein synthesis, having the characteristics of adhesion factor and induced proinflammatory. Upregulation in vivo culture process shows that the protein plays a role in the process of bacterial colonization and adhesion. In addition, stress proteins include: stress protein (GroEL) protein folding, aggregation, and degradation of the trigger factor chaperone (Tig) and ATP-dependent Clp proteolytic subunit 2 protein (ClpP2) in the rabbit vivo cultured expression abundance were significantly increased, the main role is to avoid intestinal Bifidobacterium longum by various harmful factors able to survive under the harsh conditions of the intestine. Bifidobacterium was a stranger in the gastrointestinal tract, through its unique Bifid Shunt pathway of glycolysis. In the present study, this pathway enzymes three enzymes in the Bifidobacteria after vivo cultivation elevated expression abundance, which are aldose COMT, ketose transferring enzyme and ribose 5 - phosphate isomerase , which showed that Bifidobacterium longum NCC2705 full use of the rapid growth of these enzymes to participate in the intestinal flora compete. 4 protein in the present study the position of migration are: GlnA1 PurC, by LuxS in Pgk, further use of the Pro-Q Diamond phosphorylated protein gel staining and Western Blot method to verify the migration phosphorylation of the protein spots and Analysis. The results show that the two the pgk and the LuxS protein phosphorylation. Bifidobacterium longum NCC2705 in the Pgk phosphorylation and play a role in formation of phosphorylated intermediates; LuxS is a key enzyme in the density sensor system signaling molecules AI-2 production during the in vivo environment, the protein phosphorylation enhanced. We speculate that phosphorylation may be in the form of the LuxS an active form that LuxS-P, the enzyme in this form in Bifidobacterium density induction system plays a key role. Through the front of proteomics research, we found a number of proteins that play a key role in bifidobacteria to adapt to the intestinal environment process, these proteins enhance Bifidobacterium surrounding adverse environmental adaptability. In addition, we also found that the four positions of the migration of the protein occurs AC signal molecules AI-2 production, wherein the bacterial species closely related LuxS protein phosphorylation This phenomenon shows that adapt to the intestinal environment in Bifidobacterium may play a role in the process of exchange of information with other flora. Thus, we LuxS protein in Bifidobacterium longum NCC2705 functional, bacteria can secrete the activity of signaling molecules AI-2 were studied. We use the expression of the AI-2-specific Vibrio harveyi BB170 report of system testing Bifidobacterium longum NCC2705 and LuxS bacteria in the AI-2 secretion and activity. Experiments found that Bifidobacterium longum NCC2705 luxS gene can be expressed in E. coli. Were detected by the recombinant bacteria in the expression of Bifidobacterium longum NCC2705, and LuxS AI-2 and found that Bifidobacterium longum NCC2705, and recombinant bacteria can secrete signaling molecules AI-2 Vibrio harveyi BB170 issued the fluorescence, and the AI- 2 secretion amount within a certain range and the Bifidobacterium flora density positive correlation. This result indicates that the Bifidobacterium longum NCC2705 LuxS protein having activity, and there is dependent on luxS/AI-2 QS system capable of secreting the quorum sensing signal molecules AI-2. This study, interpretation of Bifidobacterium longum NCC2705 the adaptive mechanisms in the gut, the first time the use of rabbit intestinal model of bifidobacteria in the intestinal physiological changes, combined proteomics and phosphorylation staining method The protein expression of bacteria in the body, outside the conditions under a valid comparison. In addition, this study for the first time for the quorum sensing signal molecules of the Bifidobacterium longum NCC2705 AI-2 exists and the generation process of the functional activity of the key enzyme LuxS research and verification. These important conclusions and results provide important theoretical basis for Bifidobacterium molecular biology research and Bifidobacterium carrier genetically engineered vaccine research.

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