| Quorum sensing and quorum quenching are important discoveries in the field of microbial ecology in recent years.Microorganism establishes cell-cell communication connection through quorum sensing and quenching,which plays an important role in ecological competition and dynamic balance between microbes.Currently quorum quenching has become a hot area of research against bacterial infections.However,the organism is a holistic system.While quenching mechanism exerts an antibacterial effect,the role that affects intestinal microbiota is not yet known.Scientific research and evaluation are urgently needed to provide theoretical support for its application.Therefore,this study analyzed the changes of the diversity of intestinal flora and related functional genes under the condition of exogenous addition of quenching enzyme YtnP by high-throughput sequencing technology,and explored the body's immune response under its action,in order to reveal the relationship between the quorum quenching effect and the host intestinal flora,it provides a theoretical basis for the application of quorum induction qu enching strain or its quorum induction quenching enzyme in aquaculture.1.The effect of YtnP on the intestinal flora of grass carpIn the experiment,30 grass carp weighing 25±1g were randomly divided into two groups.The control group was injected with molecular sieve buffer,and the treatment group was injected with recombinant purified YtnP(25?g/m L,100?L)dissolved in molecularsieve.At 24h and 48h of the experiment,grass carp intestinal tissues and contents were mixed and homogenized,and 16S rRNA sequ encing was performed.The results showed that the OTU of control group and treatment group samples were 142±102 and 143±67,respectively;the difference o f Alpha diversity index between the groups was not significant(P>0.05).Beta diversity analysis showed that the distinction between groups was good.In addition,dominant bacteria in two groups were Actinomycetes,Fusobacterium,Proteobacteria and the abundance of Planctomycetes in the treat group increased significantly at the level of phylum.At the level of genus,dominant species in both groups were Cetobacterium and Nocardia,the abundance of Microbacterium in treatment gro up increased significantly(P<0.05).2.Effects of YtnP on intestinal microbiota of grass carp infected with A.hydrophila with spatio-temporal variation rules300 grass carp weighing 28±2g were randomly divided into blank control group(Contrgroup),negative control group:A.hydrophila group(AH group),positive control group,quorum quenching group(YtnP group)and co-infection group(AY group).Three parallels test were performed in each group,fish were injected with a volume of 100?L buffer(50%PBS,50%molecular sieve),a concentration of 3×10~6CFU/m L Aeromonas.Hydrophila ATCC7966,a concentration of 25?g/mL YtnP,a nd a mixture of AH and YtnP,respectively.Samples were taken for 16S r RNA sequencing on day1,day3,day5 and day7 after injection,and the samples on day 3were subjected to metagenomic sequencing.The difference of Alpha diversity index between the groups was not significant(P>0.05),Beta diversity(Pco A)showed that there were significant differences between groups(P<0.05).At the level of phylum,the abundance between groups showed spatio-temporal changes,mainly manifest as:(1)In the YtnP group,the abundance of Proteobacteria decreased first and then increased,while Actinomycetes and Fusobacterium increased first and then decreased;Acidobacteria and Cyanobacteria significantly increased;the abundance of Proteobacteria and Firmicutes of midgut increased in the YtnP group(P<0.05).(2)In the AY group,Proteobacteria increased first and then decreased,Actinomycetes,Fusobacterium,and Planctomycetes decreased and then increased,and all reached the highest/lowest level on the third day.At the level of genus:(1)In the YtnP group,Aeromonas.sp significantly decreased on the fifth day,Vibrio.sp significantly decreased;Rhodococcus overall increased significantly(P<0.05);(2)In the AY group,Aeromonas significantly decreased on fifth day,lactobacillus increased significantly on the 7th day,and Rhodococcus overall increased significantly(P<0.05).LEfse analysis showed that the biomarkers with significant differences at all levels between the groups(P<0.05),and AY group has the most dominant species.The analysis of PICRUSt 2 showed the variation trend of the functiona l/gene family of bacteria(confidence interval 95%,P<0.05),and the results showed that(1)lysozyme and antitoxin(Hig A,mazE)were significantly up-regulated in the YtnP group.(2)the functions of enterobactin biosynthesis and catechol degradation were significantly up-regulated in the AY group.In addition,metagenomic sequencing analysis showed that AY significantly increased 37 Unigenes(P<0.05)co mpared with the AH group which was related to the steady state of intestinal-symbiotic bacteria.3.Temporal and Spatial Changes of YtnP's Immune Response to Grass CarpOn the 1st,3rd,5th and 7th day after injection,the intestine,head kidney,and spleen were taken to extract RNA and reverse transcribe to cDNA in control group,AH group,YtnP group,AY group,followed by detecting the changes of related immune factors.The results showed that:(1)intestinal tissue samples analysis results showed that AY group significantly increased IFN-?1 on the first day,AY and YtnP groups significantly increased the expression of the multifunctional factors IL-15 and IL-6 on the fifth day,while AH group mainly inhibited their expression(P<0.05).All three groups can significantly up-regulate the expression of pro-inflammatory factors IL-1?and TNF-?,while YtnP inhibits the expression of TNF-?at 7th day;in addition,AY group and YtnP group on day1,3 can significantly up-regulate the inhibitory factor IL-10(P<0.05);(2)Analysis results of head and kidney tissue samples showed that IFN-?1 and IL-15 were up-regulated and then down-regulated in the YtnP group,while their expression was suppress ed in the AY group;AH group could significantly up-regulate the expression of pro-inflammatory factors IL-1?and TNF-?,while AY inhibited their expression(P<0.05);YtnP could significantly up-regulate the inhibitory factor IL-10 on the third day(P<0.05);(3)The analysis results of spleen tissue samples showed that the IL-6 of three groups showed a significant upward trend,and the AY group could s ignificantly inhibit its expression on the fifth day;IL-15 of three groups were down-regulated and then up-regulated;all three groups could significantly up-regulate the expression of pro-inflammatory factors IL-1?,TNF-?,IL-8,and anti-inflam matory factors IL-10 and TGF-?1,the trends were all down-regulated and then up-regulated,and YtnP group had up-regulated IL-10 up to 28 times(P<0.01).The to tal level of gene changes indicated that the YtnP group and AY group had enha nced anti-inflammatory effects.4.Modulation of YtnP on grass carp immunity in vitroFour grass carps weighing 500±50g were selected and leukocytes of head kidney,spleen and gill were separated by Percoll,followed by treated with(2ng/m L,20ng/m L.200ng/m L)YtnP at the final concentration.Adherent cells were collected after 12h,and RNA reverse transcription into cDNA was extracted to study changes of related immune factors.The results showed that(1)In the head kidney leukocytes,YtnP had a significant regulatory effect,especially IFN-?1 was significantly up-regulated by up to 212 times(P<0.001),and the multifunctional factors IL-6 and IL-15 were significantly up-regulated at different concentrations.Proin flammatory factors IL-1?,IL-8 and TNF-?were significantly up-regulated(P<0.01)at 2ng/m L and 20ng/m L concentrations,and the proinflammatory effects at 200ng/mL concentrations were reduced.The expression of anti-inflammatory factors TG F-?1,IL-10,IL-21 increased with the increase of YtnP concentration,and IL-10 was up-regulated from no significant to extreme significantly(P<0.001).(2)In spleen leukocytes,YtnP inhibited the expression of pro-inflammatory and anti-inflammatory factors at the same time,and the effect of inhibiting IL-1?,IL-10,IL-15 was obvious,but 2ng/m L YtnP significantly increased IFN-?1 and IL-6(P<0.05);(3)Ing ill leukocytes,200ng/mL YtnP induced IL-10 and IL-6 to be significantly up-reg ulated(P<0.01).Comprehensive analysis shows that YtnP can improve the anti-infective ability of grass carp and the balance of intestinal microecology,revealing the relations hip between YtnP and the host intestinal flora and immunity,laying a theoretical foundation for its scientific application. |
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