Study On The Mechanism Of Hazard Forming Of Tdh/trh Negative Vibrio Parahaemolyticus From Aquatic Foods

Vibrio parahaemolyticus is an important and common food-borne pathogenic bacterium,and thermostable direct hemolysin and tdh-related hemolysin are the most important basis for the determination and food safety detection of the harmfulness of this bacterium.Recently,since we have detected the strains in clinical isolates which were both tdh/trh negative,while about 60%of strains isolated from aquatic foods and the environment are both tdh/trh negative and may still be pathogenic,leading to food safety incidents.Exploring and analyzing the function of new virulence factors and uncovering harm formation mechanisms has become an urgent problem.This thesis addresses the question of whether tdh/trh negative V.parahaemolyticus from current aquatic isolates are pathogenic.Firstly,based on our library of strains isolated in the early stage,PCR was used to genotype V.parahaemolyticus which were tdh~-/trh~-,and the virulence level difference strains were screened by cellular and animal experiments to identify the highly virulent strains.Then,to elucidate the cause of hazard formation of the highly virulent strains which were tdh~-/trh~-,the whole gene sequencing and comparative genomic analysis were performed to clarify their genetic characteristics and identify the significantly expressed genes by combining the transcriptome expression differences of the strains after the infection towards human cells.To reveal the hazard formation mechanism of the tdh~-/trh~-strains,the functions of the relevant virulence factors were analyzed by gene targeting knockout and complement and the regulation functions of the relevant virulence factors were investigated by weighted gene co-expression network analysis(WGCNA)method.To further elucidate the regulatory mechanism of the virulence factors,the proteomic analysis of significantly expressed proteins between the mutants and wild-type was performed to elucidate the mechanism of action of the virulence factors at the protein level.Finally,to investigate the pathogenic mechanism of the tdh~-/trh~-strains in vivo,the infection model of the tdh~-/trh~-strains was constructed and the pathogenesis was initially investigated.Meanwhile,the effect of Pediococcus pentosaceus selected by our laboratory,an active probiotic with an antagonistic effect against various foodborne pathogens,was evaluated for the prevention and control of tdh~-/trh~-strains.The main study contents and results are as follows:(1)Screening and omics features of the highly virulent tdh~-/trh~-strain VP353A tdh~-/trh~-high virulence strain VP353 was screened by cellular and animal experiments from a library of V.parahaemolyticus isolated from aquatic foods established by our laboratory earlier.The whole genome was sequenced and VP353 was found to be 5.28 Mb and consisted of 2 chromosomes,chromosome 1 consisted of 3.30 Mb with 2952 predicted ORFs and chromosome 2 consisted of 1.98 Mb with 1772 predicted ORFs.The VFDB database,VP353,a highly virulent tdh~-/trh~-strain of V.parahaemolyticus,was found to harbor both the new virulence genes hly A,hly B,hly C and hly D,and its protein affinity was most similar to that of Escherichia coli.Comparative genomic analysis of the T3SS gene cluster was performed for VP353,and it was found that the similarity of T3SS1 gene cluster of VP353 compared with the standard strain was over 99%.The similarity of the T3SS2 gene cluster was only 66%or more compared to the standard strain.Through transcriptome analysis before and after VP353infection towards Caco-2 cells,a total of 958 genes were found to be upregulated,mainly enriched in the biosynthesis and metabolic processes of organic nitrogen compounds,among which the virulence-related MSHA type IV pili,flagella,quorum sensing genes(Cqs A,Lux S),extracellular polysaccharide T2SS-related genes and hly A and hly C genes were significantly up-regulated.(2)Transcriptome-based regulatory mechanism of virulence Qrr5 from tdh~-/trh~-strain VP353Based on the screening of transcripts after VP353 infection towards the hosts,Qrr5 was confirmed as a novel virulence factor,and the△Qrr5 mutant and△Qrr5::Qrr5complementation strains of V.parahaemolyticus were successfully constructed by gene knockout and complement expression methods.The Qrr5 gene deletion significantly affected the expression of the T3SS1 gene cluster in the pre-infection and post-infection periods.Based on the transcript analysis of△Qrr5 mutant and wild-type infected hosts.The gene regulatory network of Qrr5 during infection towards the hosts was constructed with a total of 17 genes,among which VP2914,VP0485,VP2492,and VP1665 genes showed a strong correlation,and their expressions were verified by q RT-PCR,and the results were consistent with RNA-Seq results.(3)Proteome-based regulatory mechanism of virulence Qrr5 from tdh~-/trh~-strain VP353Quantitative proteomic analysis revealed that a total of 297 significantly expressed proteins were identified between the△Qrr5 mutant strain and the wild-type,of which 137 proteins were up-regulated and 160 proteins were down-regulated.Enrichment analysis of differentially expressed proteins by GO and KEGG revealed that the up-regulated proteins were mainly enriched in oxidoreductase activity,sulfur metabolism and ABC transporter protein pathway.Meanwhile,the down-regulated proteins were mainly enriched in intracellular signal transduction,two-component system and amino acid biosynthesis pathway.Enrichment analysis by prediction of protein domains revealed that the up-regulated proteins were mainly enriched in Na~+/H~+transporter protein,C-terminal Nha C-like and transcriptional regulator HTH and Lys R structural domains,and the main enriched structural domains of down-regulated proteins were winged helix-like DNA binding domain superfamily and Deo R-type HTH structural domain.Screening of the significantly expressed proteins and related phenotypic determination revealed that Qrr5 may be involved in the regulation of the regulatory factors Lys R family proteins,Omp A family proteins,and transfer protein Tra A to inhibit the ability of autoaggregation formation and competitiveness against E.coli.(4)Construction and prevention of a mouse model of highly virulent tdh~-/trh~-strain VP353infectionBy constructing a mouse model of a highly virulent tdh~-/trh~-strain V.parahaemolyticus353 infection,it was found that the survival rate of mice in the TDNH group was significantly lower than that of the control group,and the colonic tissues of mice in the TDNH group were significantly shortened,and significant atrophy and damage were observed.By measuring the indexes of the serum of mice in the infected group,it was found that the levels of IL-1βand IL-6 in the serum of mice in the TDNH group were significantly increased compared to the control group.Meanwhile,the levels of TNF-α,IL-1β,and IL-10 in the serum of mice in the TDPH group were significantly increased.By measuring the intestinal flora of mice in different infection groups,it was found that the TDNH infection group significantly reduced the abundance of Roseburia,Faecalibaculum and significantly increased the abundance of Eubacterium fissicatena,Erysipelatoclostridium.We applied the active probiotic bacterium P.pentosaceus IM96,which was selected from our laboratory and has a wide range of antagonisms against multiple foodborne pathogens,to intervene in TDNH infection to evaluate the prevention and control effect.It was found that the IM96 intervention group could significantly increase the survival rate of mice in the TDNH group as well as recovering the intestinal damage caused by the infection,and significantly reduce the serum IL-1βand IL-6inflammatory factor levels of mice in the TDNH group during P.pentosaceus IM96 intervention.IM96 also significantly reduced the abundance levels of Eubacterium coprostanoligenes,Clostridia vadin BB60,Marvinbryantia,while significantly improving the abundance levels of Romboutsia,Turicibacter,Parvibacter in TDNH group,suggesting that IM96 has a good protective effect against tdh~-/trh~-V.parahaemolyticus infection.In summary,this thesis systematically investigates the mechanism of hazard formation of tdh~-/trh~-type V.parahaemolyticus and provides new ideas and methods for detection and prevent research of V.parahaemolyticus.It is greatly significant to ensure food safety and promote the sustainable development of the aquatic food industry.