| Campylobacter jejuni(C.jejuni),a Gram-negative food-borne pathogen,is the leading cause of gastroenteritis worldwide.Human infection with C.jejuni can cause diarrhea,vomiting,abdominal pain,fever,and other symptoms.Some cases can lead to serious autoimmune diseases such as Guillain-Barre syndrome or Miller Fisher Syndrome,which impose a heavy burden on human health and the world economy.The type VI secretion system(T6SS)is a potential virulence factor.It is a bacteriophage-like structure mounted upside down on the bacterial cell membrane,through which effector proteins can be directly injected into host cells and exert a biological effect.The prevalence of T6SS in C.jejuni varies in different countries and different host sources.However,the prevalence characteristics of C.jejuni T6SS were only sporadically reported,and systematic studies were lacking in China.In this study,a multiplex PCR detection method and a Western Blot detection method were established.Prevalence and distribution characteristics of T6SS in C.jejuni isolated from different hosts in Eastern China were studied,and combined with whole-genome sequencing and bioinformatics analysis techniques,the distribution characteristics of the T6SS gene cluster were analyzed.The phenotype was analyzed to initially understand the potential role of T6SS in C.jejuni.This study aims to provide data support for the study of virulence factors of C.jejuni and to make new attempts to analyze the pathogenic mechanism of C.jejuni from different host sources.1.Establishment of a detection method for C.jejuni T6SSThe structure of 1038 strains of C.jejuni T6SS gene cluster obtained from the NCBI database was analyzed,and two detection target genes were determined.The software was used to design primers,and the multiplex PCR detection method of C.jejuni T6SS was established by optimizing the reaction conditions and sensitivity experiments.Using prokaryotic expression technology,inducing expression and purification of recombinant Hcp.Lymphocyte hybridoma technology was used to prepare Hcp monoclonal antibody,and a Western Blot detection method was established for C.jejuni T6SS.The results showed that the multiplex PCR method could specifically amplify the hcp(483 bp)and vgrG(758 bp)gene fragments of positive strains such as ATCC 43431 and ATCC 33560,while the negative strains such as 81-176 and NCTC 11168 had no bands.Compared with the sequence published in GeneBank,the homology of the product sequencing results was greater than 99%.The optimal annealing temperature of this method is 50℃,and the lowest detection limit is 10-3ng of DNA.Compared with the traditional method,the T6SS gene cluster of isolates screened is more complete by this method.The rHis-Hcp and rGst-Hcp were used as the immunogen and detecting antigen,4 strains of hybridoma cells were screened.The titers of their ascitic fluid were over 2×107.Respectively,their immunoglobulin subclass was IgA,IgG1,IgG1,and IgG2a.The specificity experiment showed that both can recognize rGst-Hcp and the natural Hcp of C.jejuni specifically.The Western Blot detection method was established with antibody 4D6.There were 74 sequenced isolates used to compare the two detection methods.It was found that the two methods have high specificity,and the agreement is 100%;the multiplex PCR method is more rapid and convenient,providing technical support for the rapid detection of virulence factors of C.jejuni,while the Western Blot detection method is based on protein expression level is in the detection of whether the bacteria contain a functional T6SS.2.Analysis of distribution characteristics of C.jejuni T6SSBased on the Campylobacter strain library established in our laboratory,a total of 1430 C.jejuni isolates were obtained from different regions and host from 2007 to 2020.The T6SS multiplex PCR detection method established in this study was used to detect and analyze the carrying status of the T6SS gene cluster.The results showed that 25.80%(369/1430)of C.jejuni were T6SS positive.The T6SS-positive rate of C.jejuni isolated from humans(43.66%)was significantly higher than that from other animal sources(24.73%)(P<0.001).The C.jejuni isolated from the same poultry has significantly different positive rates in distinct farms,indicating that the positive rate has nothing to do with the host,and its distribution is polymorphic.The positive rate of C.jejuni isolated from diseased poultry is significantly higher than healthy.In addition,the proportion of serotypes associated with diarrhea in positive isolates(35.78%)was significantly higher than that in negative isolates(16.68%)(P<0.001).In the phylogenetic tree constructed based on the core genome,T6SS-positive isolates were clustered in partial branches.Up to 88.89%of isolates,their T6SS gene clusters were located in the genome of CJIE3,and 11.11%were located in plasmids.Furthermore,the 8 common virulence genes in C.jejuni are not related to T6SS,and it was found that the T6SS gene cluster can function on both the genome and the plasmid and can spread among C.jejuni by transferring the original.3.Preliminary analysis of the function of C.jejuni T6SSIn this study,the method of homologous recombination of the pMD-20T suicide plasmid was used to construct a deletion strain of the T6SS full gene cluster,and the biological characteristics of the WT and the T6SS were studied.First,the expression and secretion of Hcp were detected,then observed the effect of the T6SS gene cluster on the growth and motility of C.jejuni and studied its association with C.jejuni virulence.In addition,the sequenced 74 isolates were also tested to further determine the function of T6SS in C.jejuni.The results showed that after the deletion of T6SS,the Hcp could not be expressed and secreted normally.The growth rate of the T6SS same as WT,was no significant difference;WT and T6SS can spread and grow from the inoculation site to form a clear ring structure,and the diameter of the movement circle has no significant difference.In the adhesion and invasion experiments of Caco-2 cells,the adhesion rate of the T6SS was only 49.05%of WT,and the invasion rate was 79.87%.After deletion of the T6SS gene cluster,the lethality of G.mellonella larvae was reduced and resulting in a shorter duration of killing.The results of the study on the isolates showed that there was no significant difference in motility between T6SS positive isolates and negative isolates,but the adhesion and invasion ability of positive isolates and the killing effect on G.mellonella larvae were significantly higher than those of negative isolates.In general,T6SS enhanced the virulence of C.jejuni. |
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