| Research objectives:Vibrio alginolyticus is a halophilic Gram-negative Vibrio sp.,and it is widely distributed in the water environment.As one of the most pathogenic Vibrio spp.,V.alginolyticus has caused constant negative consequence to the sustainable development of marine fishing industry.On the other hand,as a food-borne opportunistic pathogen of human being,V.alginolyticus can cause acute gastrointestinal infection and septicemia,other than that,it could also lead to multiple organizational dysfunction after infecting immunocompromised patients,which is often life-threatening from neglecting the best time for treatment.However,suffering from being time-consuming and the tedious operation,so far it is still short of a specific real-time method detecting V.alginolyticus infection in animal or human being.In recent years,the related researches about Quorum sensing?QS?regulating bacterial infection have been increasing,which provided a novel research angle to the mechanism of V.alginolyticus infection.However,as a group of important QS signaling molecules,the details of N-acyl homoserine lactones?AHLs?produced by V.alginolyticus haven't been systematically profiled yet,which leads to a lack of researches about their effects to V.alginolyticus pathogenic progress.The above-mentioned aspects become the hinder of study on the infecting mechanism and the harm of V.alginolyticus.Therefore,the present research starts with the rapid detection of V.alginolyticus,followed by profiling and analyzing the detailed AHLs production of it,and finally focusing on the regulation of AHLs to V.alginolyticus functional regulation.The aim of this research is to lay a theoretical foundation for the prevention and treatment of V.alginolyticus infection.Research methods:1.By designing 3 pairs of specific primers targeting 8 base sequences in different regions of tox R gene in V.alginolyticus,followed by using Bst DNA polymerase,SYTO-9 fluorochrome and other commercial reagents as materials,an innovative Loop-mediated Isothermal Amplification?LAMP?method was prepared for the specific detection of varies concentration of V.alginolyticus in pure spent culture,mouse infection model and scallop leaching solution models.In the meantime,the LAMP results including its sensitivity and specifity were verified by 16s rDNA identification for the sensitivity evaluation.2.The detailed AHLs producing information were systematically detected and analyzed with biological method and chemical method.Using microbial sensor cross-feeding against V.alginolyticus assay,including Chromobacterium violaceum CV026 detecting short side-chain AHLs and Agrobacterium tumefaciens KYC55 detecting long side-chain AHLs,the producing range of AHLs generated by 47 V.alginolyticus strains were biologically detected.Using the High Performance Liquid Chromatography tandem Mass Spectrum?HPLC-MS/MS?assay,the profiling of AHLs,including 7 types of short side-chain AHLs?C4-HSL,3-OH-C4-HSL,C6-HSL,3-oxo-C6-HSL,C8-HSL,3-OH-C8-HSL,3-oxo-C8-HSL?and 7 types of long side-chain AHLs(C10-HSL,3-oxo-C10-HSL,C12-HSL,3-OH-C12-HSL,3-oxo-C12-HSL,3-OH-C14-HSL,3-oxo-C14-HSL),were detected on producing types and concentrations in the aim of expliciting the detailed information of AHLs.Based on their concentrations,AHLs were classified to verify the characteristics of them produced by V.alginolyticus and the dominant AHLs.3.Using the low-cost and easily prepared materials,3 important AHLs produced by V.alginolyticus,in the name of 3-oxo-C6-HSL,3-oxo-C10-HSL and C12-HSL,were artificially synthesized.To synthesize AHLs with carbonyl group on the 3rd carbon atom of its acyl side chain like 3-oxo-C6-HSL and 3-oxo-C10-HSL,3-oxo caproic acid and 3-oxo capric acid were separately synthesized with the following steps:n-butyric acid and n-caprylic acid were separately produced condensation reactions with Meldrum's acid,and then the products were de-condensed by methanol reflux,3-oxo caproic acid and 3-oxo capric acid were prepared by ester hydrolysis under the alkaline condition.Together under the limited temperature,3-oxo caproic acid,3-oxo capric acid and lauric acid were separately produced condensation reaction with L-homoserine lactone salt under the catalysis of EDC and triethylamine,and 3-oxo-C6-HSL,3-oxo-C10-HSL and C12-HSL were prepared.The chemical construction of the above-mentioned AHLs were predicted with 1H-NMR;and the molecular weight of them were calculated with MS;and lastly the purity of them were confirmed with HPLC.Based on the AHLs that were successfully synthesized,the biological activity of them were detected with cross-feeding method,which included the activity of 3-oxo-C6-HSL detection with microbial sensor C.violaceum CV026 and the acivity of 3-oxo-C10-HSL and C12-HSL detection with microbial sensor Escherichia coli MG4?pKDT17?.4.V.alginolyticus strains were routinely cultured for 0 h-120 h,and the proliferation curve were monitored and drawn with gradient dilution-plate count assay in order to screen the best culturing time.The biofilm forming condition were verified and classified within the optimized culturing time with semi-quantitative adhesive test.Considering both the 3-oxo-C10-HSL producing level and biofilm forming capacity,2 V.alginolyticus strains named N°24 and N°40 were screened.After routine culturing for 0 h-120 h,the biofilm formation period of these 2strains were detected with Fluorescence Labeling Microscopy?FLM?to obtain the general biofilm formation information of them.V.alginolyticus N°24 and N°40 were cultured under different environmental conditions to observed the effect of environmental factors to the biofilm formation,and the details were as follows:they were separately cultured for 36 h at 4°C,16°C,28°C and 40°C,and the intensity of their biofilm forming capacity were verified in order to discuss the effect of different temperatures,including cold,cool,warm or hot,to V.alginolyticus biofilm formation;then they were separately cultured for 36 h under 0.5%,1%,3.5%and 6.5%salinity environment at screened temperature?16°C or28°C?respectively,and the intensity of their biofilm forming capacity were verified in order to discuss the effect of different salinity,including oligohaline,reduced salinity,high salinity or rich salinity,and the relationship of salinity and temperature during V.alginolyticus biofilm forming progress;after the above mentioned procedures,they were treated with various concentration of 3-oxo-C10-HSL,including 1?M,2?M,5?M,10?M,40?M and 100?M,and cultured for 36 h at screened temperature?16°C or 28°C?respectively.The intensity of their biofilm forming capacity were verified and discussed with semi-quantitative adhesive test.Lastly,the 3D biofilm images of these 2 strains were reconstituted with Cofocal Laser Scaning Microscopy?CLSM?,which were further discussed about their differences in biofilm form and scale,based on which,5 specific parameters of biofilm matrix were analyzed with COMSTAT software,and the detailed information of 3-oxo-C10-HSL effect to V.alginolyticus biofim matrix were evaluated,including biomass,biofilm thickness,roughness and microclone colony number.Research results:1.The specific detection results for V.alginolyticus spent culture,mouse and scallop infection models were as follows:?1?The limit of detection of V.alginolyticus spent culture with concentration from 1×100 CFU/mL to 1×109 CFU/m L was1×102 CFU/m L under ESE-Quant-LAMP and qPCR-LAMP mood,respectively,which proved the high sensitivity of this LAMP assay in detecting V.alginolyticus pure culture.?2?V.alginolyticus was detected as the only positive result within 12 strains of Vibrio and non-Vibrio spent culture under ESE-Quant-LAMP mood;92 V.alginolyticus spent culture were all detected as the positive results under qPCR-LAMP mood.The results above mentioned proved this innovative LAMP method possessed excellent specificity in detecting V.alginolyticus spent culture,which was also confirmed by 16s rDNA identification.?3?Out of 6 mouse infection model samples which were detected for the remaining Vibrio in their blood,V.alginolyticus infection group was detected as the only positive result under qPCR-LAMP mood,accompanying with“S”type curve with different radian,which proved this LAMP method could be applied to detection of V.alginolyticus on mammal blood.?4?Out of 6 scallop infection samples which were detected for the remaining V.alginolyticus in their leaching solution,the infection groups were all detected as the positive results under ESE-Quant-LAMP mood,accompanying with the straight line in the control groups,which proved this LAMP method could be applied to real-time detection of V.alginolyticus on marine products.2.Firstly,several conditions of HPLC-MS/MS were optimized,which include:a.compounds for buffer solution A and B,flow rate,analyzing time and gradient elution conditions;b.curtain voltage and cone voltage,etc.;c.specific cleaving fragments of AHLs and optimized m/z.Based on these conditions,The linear relationship of each AHL based on 14 types of standard AHL compounds under HPLC-MS/MS optimized conditions was prepared with the linearity above 0.99.Combined the results of biological and chemical methods,the details of AHLs produced by V.alginolyticus were as follows:?1?The detection results of the scale of short and long side-chain AHLs with microbial sensing strains revealed that there were a very low concentration of short side-chain AHLs or even no such AHLs could be produced by V.alginolyticus while the producing scale of long side-chain AHLs was wide,and there was a significant concentration difference between them.?2?There were at least 6 types and at most 11 types of AHLs that 47 V.alginolyticus strains could produce,and the scale was from nM level to?M level,which confirmed a rich diversity both on the producing types and concentration.?3?Within the above-mentioned AHLs,shor side-chain AHLs were generally at low concentration except 3-OH-C4-HSL while there was a huge difference on the concentration of long side-chain AHLs produced by different V.alginolyticus strains.?4?C8-HSL,3-oxo-C8-HSL and 3-OH-C14-HSL were the 3 types of AHLs that were not detected in 47 strains,which revealed the V.alginolyticus could possibly not produce these3 types of AHLs.?5?3-oxo-C10-HSL among 14 types of detecting AHLs was the dominant AHL that V.alginolyticus produced,which the most concentration was over 5?M and had a difference between strains.3.Three important types of AHLs that V.alginolyticus could produce,namely 3-oxo-C6-HSL,3-oxo-C10-HSL and C12-HSL,were successfully artificially synthesized under general laboratory environment using low-cost materials,and the AHLs could induce the micro community color of microbial sensor to change,which was positively related to the cross-fed culturing time and AHL concentration,and the above-mentioned phenomenon confirmed the good biological activity of these 3 AHLs.4.The biofilm forming pattern of V.alginolyticus and the regulation of different factors were as follows:?1?Most of the detecting V.alginolyticus strains could form weak biofilms,and the synthesis of biofilm matrix turned into a dynamic and rapid speed after culturing for 12h,and then reached to a peak after 60 h,followed by going into the collapsing period after84 h.?2?Biofilm formation of V.alginolyticus could be increased at lower temperature such as 16°C,and on the contrary,it could be inhibited by culturing at high temperature such as 40°C.?3?The effect of salinity to V.alginolyticus biofilm formation could be influenced by temperature,which brought to a result that there are a clear increase of V.alginolyticus biofilm formation under high salinity environment at lower temperature.?4?3-oxo-C10-HSL could regulate V.alginolyticus biofilm formation,the regulating pattern of which was bidirectional,and that means when the concentration of 3-oxo-C10-HSL in the environment is in a medium level,V.alginolyticus biofilm formation could be increased,other than that,much lower or high concentration of 3-oxo-C10-HSL could not influence V.alginolyticus biofilm formation,or could even inhibite this progress,which might be related to the feed-back mechanism of QS system.Research conclusion:1.An innovative LAMP method with high sensitivity and excellent specificity was established,which could be applied to both field diagnose and laboratory research for the rapid detection of V.alginolyticus.2.Profiling of diverse AHLs produced by V.alginolyticus were systematically detected,and the most dominant AHL among all the producing AHLs were confirmed.3.A low-cost and easy preparing chemical method was established for the artificial synthesis of AHL compounds,the products of 3-oxo-C6-HSL,3-oxo-C10-HSL and C12-HSL have excellent biological activity.4.3-oxo-C10-HSL could regulate the biofilm formation progress of V.alginolyticus,which could also be influenced by environment factors,such as temperature and salinity.Significance of research:This research has filled up the blank of rapid detection technology for the identification of V.alginolyticus,and it is not only the very first systematial study on the profiling of AHLs production of V.alginolyticus,but it is also the first to confirm and artificially synthesize the dominant AHL produced by V.alginolyticus,which has broken through the hinder of purifying and separating difficulties of naturally produced AHLs.Based on the above-mentioned base,this research illuminated the key effect of 3-oxo-C10-HSL in the progress of V.alginolyticus biofilm formation,and revealed the close relationship between QS system and V.alginolyticus biological functions,which provided an important perspective for studies on the mechanism of V.alginolyticus infection in the future. |
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