Effect Of Extrinsic N-Acyl-Homoserine Lactone Signal Molecules And Intestine Microflora On Quorum Sensing In Bacteroides Fragilis

BackgroundAutoinducer conducts the communication among bacteria. Bacteria can survey the amount changes in other bacteria according to the concentration of this signal molecules; it is a course of Quorum-sensing (QS). The signaling pathway is involved in various of physicial activities, such as bioluminescence, virulence gene expression, biofilms formation, exocellular enzyme and polysaccharides synthesis, antibiotics production, etc. Different bacteria apply unique QS mechanism in regulating biological activities. There are three classes of QS mechanism, modified polypeptides acting as signal regulatory molecular in gram-positive bacteria, AHL produced by LuxI/LuxR system doing the same activity in gram-negative bacteria, and the third one in both gram-positive and gram-negative bacteria is normally supposed to the secondary signal molecule. There is a great interest in research of QS in recent years but details about the QS in anaerobic bacteria have not been mentioned yet.Bacteroides fragilis accounts for1%-2%of human intestine micro flora and also is the main anaerobic bacteria causing gastrointestinal infection. Their large amount presentation and formation of biofilm are induced by a multiple factors, among which is their information communication mechanism so as to keep a coincidence of a whole biological activity in human.There are few researches about Bacteroides fragilis in the following aspects, the relations between QS and biofilm formation, whether it produces the QS signal molecules AHL and the contribution of AHL and intestine microflora to its biological activity. This research focused on the deep study of AHL in the direction of their influence factors and their presentation in bacterial QS based on UPLC-MS. Another part of the research approached whether Bacteroides fragilis have QS circulation and the impaction of QS related gene expression in Bacteroides fragilis by AHL originated from both extrinsic and other closely related enterobacteria in the way of molecular biology. This research, as the base for further study in QS mechanism, established the technique and methods for systemic research of intestinal microecological signal transduction and a clue for intestinal microecology pharmaceutics according to the Bacteroides fragilis QS signal molecule.Part I Establishment of detection for QS signal molecule AHLMethods:A total of6AHL standard substance were detected and analyzed by UPLC-MS(Quadrupole Time-of-Flight, Q-TOF) metabonomics in the parameters of solvent selection, moblie phase optimization, filter membrane aperture, electrospray ionization mode in mass spectrometry, chromatographic column selection, conditional optimization in mass spectrometry, preparation for AHL extraction and separation, and the ionogenic feature of AHL in mass spectrometry.Results:Technological platform for bacterial QS signaling molecule AHL detection was established in the following parameters:positive ion electrospray mode, chromatographic column of Aquity C181.7μm2.1mm×100mm, Methyl Cyanides as solvent. The lactonic ring of AHL was kept unspoiled featuring as apiece m/s of102.05or both102.05and74.05during the mass spectrometry assay (N-Tetradecanoyl-DL-homoserine lactone'm/s is102.05). The extraction solvent composed of trichloromethane, methanol and water showed the sensitivity at5ng/ml for bacterial culture extraction.Part II Detection of QS signal molecule AHL in Bacteroides fragilis based on UPLC-MSMethods:All of the experimental bacteria including Bacteroides fragilis, bacillus bifidus, Escherichia coli, Enterococcus faecium, Vibrio parahaemolyticus, Enterobacteria cloacae, Acinetobacter baumannii, Salmonella typhosa, were ensured by sequencing their16SrRNA. Extracted bacterial culture from Bacteroides fragilis and other experimental bacteria were analyzed by UPLC-MS (Q-TOF-MS) to determine the presentation of AHL following the established parameters in this study, AHL standard substance as a control.Results:16SrRNA in all of the experimental bacteria shared the more than98%sequence homology with GenBank database. The AHL standard substance solving in different bacterial culture showed a featured piece of m/s of102.05or both102.05and74.05in electrospray ionization quadrupole time-of-flight (Q-TOF) mass spectrometry (N-Tetradecanoyl-DL-homoserine lactone'm/s is102.05), while AHL was detected in none of the Bacteroides fragilis and other bacteria.Part III Study on Bacteroides fragilis culture featureMethods:The survival of Bacteroides fragilis was recorded in the condition of different period, temperature and after10times freezing and thawing, when preserved in both anaerobic glycerol broth and common glycerol broth. Its viability and survival rate were compared when exposed in the air for5min,10min,30min,60min,90min,120min,150min,180min,24h.Results:Bacteroides fragilis was cultured successfully when persevered at either-20℃or-80℃for as long as3months in both anaerobic broth and common broth, and survived even after freezing and thawing for10times. It was still live when exposed in the air for180min but dead for24h. Its survival rate after exposed in air for5min, lOmin,30min,60min was84.1%,68.1%,46.4%and34.8%respectively.Part Ⅳ The impact of extrinsic N-Acyl-Homoserine Lactone signal molecules and intestine microflora on Bacteroides fragilis biological activityMethods:Bacteroides fragilis was monitored by OD value during a rang of culture period with the culture supernatant from5common intestinal bacteria and2AHL standard substances in the anaerobic blood broth.Results:Standard Bacteroides fragilis reached the log phase after culturing for8h,6h,6h,6h,10h, and10h, and stable phase after10h,8h,8h,8h,24h,and24h, in standard anaerobic hemoculture, anaerobic hemoculture added with N-Dodecanoyl-DL-homoserine lactone, culture supernatant from Enterococcus faecium, Staphylococcus aureus, Enterococcus faecium, N-(β-Ketocaproyl)-DL-homoserine lactone respectively. It took standard Bacteroides fragilis the same time in reaching the log phase and stable phase in anaerobic hemoculture added with other culture supernatant as in standard one.Part Ⅴ Detection of Bacteroides fragilis QS related gene expression in different culture condition by fluorescent quantified PCRMethods:Bacteroides fragilis QS related gene luxI, luxR and subtypes were detected by PCR. The mRNA of luxR subtypes in Bacteroides fragilis was quantitatived by real-time fluorescent quantitative PCR and further compared in the impact of intestine microflora and2AHLs.Results:A total of8luxR-homologous genes, except luxR8, were detected in Bacteroides fragilis, while none of luxI and other lux genes were detected. This result indicated the8luxR-homologous genes were involved in class Ⅰ QS circulation. LuxR subtypes expression is different in period of culture, reaching the highest level after6h or4h culture while the lowest after1h or8h., with the except of luxR1that was expressed in low quality all the period and luxR8of non-presentation. LuxR expression was up-regulated by N-Dodecanoyl-DL-homoserine lactone, Enterococcus faecium and Staphylococcus aureus, on the contrary, down-regulated by N-(β-Ketocaproyl)-DL-homoserine lactone and Enterococcus faecium.ConclusionsIn this study, the bacterial QS Class Ⅰ signal molecule AHL detection platform based on UPLC-MS technique was established, class Ⅰ signal molecule AHL and QS related genes in Bacteroides fragilis was determined, and the impact of different extrinsic AHL and enterobacteria on the biological activity and luxR gene expression was further explored, so it arrived at conclusions as follows.1. Bacteroides fragilis have class Ⅰ QS. LuxR regulatory factors and their subtypes were involved in this system and may play roles in QS when conjuncted with extrinsic signal molecule.2. Neither LuxⅠ gene presented nor N-Acyl-Homoserine Lactone (AHL) was produced in Bacteroides fragilis.3. UPLC-MS (Q-TOF-MS) technique is useful in detection of QS Class Ⅰ signal molecule AHL with the advantage of high accuracy, high sensitivity, good repeatability, easy operate, high efficiency. The lactonic ring of AHL was kept unspoiled featuring as apiece m/s of102.05or both102.05and74.05in Q-TOF-MS.4. Different extrinsic AHLs and common enterobacteria secretions produced different effect on QS in Bacteroides fragilis.5. Bacteroides fragilis was tolerant to oxygen to some extent.6. The extraction solvent composed of trichloromethane, methanol and water was optimized in extraction QS Class I signal molecule AHL,...