| Salmonella enterica serovar Enteritidis(S.Enteritidis),Salmonella enterica serovar Pullorum(S.Pullorum)and Salmonella enterica serovar Gallinarum(S.Gallinarum)are common Salmonella serovars in veterinary.They have close evolutionary genetic relationships and share similar genome structures.However,there are various in their host range,host age and pathogenic characteristics.S.Enteritidis is a kind of important zoonotic pathogen,which can causes food-borne poisoning and gastroenteritis.S.Pullorum and S.Gallinarum is mainly pathogenic to young birds,causing acute,and systemic septicemia.It is worth noting that,unlike S.Enteritidis and other Salmonella serovars,S.Pullorum and S.Gallinarum lack of flagella and motility.However,the reasons why S.Pullorum and S.Gallinarum absence of motility has not been well studied for a long time.Flagella is an important virulence factor of bacteria.In addition to imparting motility to bacteria,it also plays an important role in adhesion,invasion,biofilm formation and other aspects in the pathogenic process of bacteria.The synthesis and assembly of bacterial flagella is very complicated,and about 60 genes are involved in this process.By searching for a series of flagella-related genes in the genomic sequence of S.Pullorum and S.Gallinarum,we noticed that although chicken S.Pullorum and S.Gallinarum did not have flagella,they have a full set of flagella-related genes.Interestingly,we noticed that some flagella-related genes are pseudogenes in S.Pullorum and S.GallinarumPseudogenes are DNA sequences that resemble functional genes but are generally thought to have no function due to point mutation,deletion,insertion and other defects.Commonly denoted by the symbol "?".So,do all S.Pullorum and S.Gallinarum have flagella-related pseudogenes?What are the characteristics of pseudogene distribution?Is the non-flagellar phenotype of S.Pullorum and S.Gallinarum caused by the pseudogene?In addition,some studies have shown that the pseudogenization of some virulence-related genes of pathogenic microorganisms may increase their pathogenicity.Will the pseudogenization of flagella-related genes affect the pathogenicity?Based on the above questions,researches are carried out mainly on the following aspects:1.Sequencing and bioinformatics analysis of flagella related pseudogenes of S.Pullorum and S.GallinarumIn order to explore the distribution and mutation characteristics of flagella-related pseudogenes in S.Pullorum and S.Gallinarum.In this study,the published genomic sequences of S.Pullorum(5 strains)and S.Gallinarum(3 strains)were obtained from the NCBI genome database.Then the whole genome sequence(AM933172.1)of S.Enteritidis strain P125109,which is known to be flagellar positive,was used as the reference sequence.Specific primers were designed according to the reference sequence.The flgK,flhB,flhA,flgl,cheM,fliN,fliL,motB and ycgR genes of four domestic standard strains of S.Pullorum(CVCC523,CVCC526,CVCC535 and CVCC540)were amplified by PCR and then sequencing was performed.The sequence obtained by sequencing and sequence obtained from NCBI were compared with the reference sequence.The results showed that all of the tested S.Pullorum and S.Gallinarum strains contained 2-6 pseudogenes.The distribution of each pseudogene was different,among which flgK and flhB were pseudogenes in all S.Pullorum and S.Gallinarum strains.There were two types of mutations in the pseudogenes:(1)The single base mutation results in the premature of stop codons in the sequence.(2)The insertion or deletion of bases which is non-triple integer multiple leads to a frameshift mutation in downstream.The results in this chapter provide necessary information and basis for further study of flagella-related pseudogenes.2.Effect of flagella-associated pseudogenes deletion on flagella formation and motility of S.EnteritidisTo explore the effects of flagella-related pseudogenes on flagellum formation and motility,the domestic standard strain of S.Enteritidis strain 50336,a known flagellum positive reference strain,was used as the research model in this study.Nine flagellum gene deletion mutants were constructed using ?-Red homologous recombination system,including flgK,flhB,flhA,flgl,cheM fliL,fliN motB and ycgR,respectively.The ability of flagellum formation and motility of each deletion mutant and complementary strains were investigated by semi-solid motility tests,slide agglutination tests and electron transmission microscope tests.The results showed that the flgK,flhB,flhA,flgI,fliL and fliN genes affected the formation of flagellum and motility.The gene motB did not affected bacterial flagella formation,but affect the motility.The cheM and ycgR genes had no effect on either flagellum formation or bacterial motility.3.Effect of deletion of flgK and flhB gene on pathogenicity of Salmonella EnteritidisPrevious studies have shown that the deletion of six genes,including flgK,flhB,flhA,flgI,fliL and fliN,could lead to non-flagellar phenotype of mutants.This study further discussed whether the non-flagellar phenotype affects the pathogenicity of the mutants.We chose gene flgK and flhB as target,to explore the relationship between the non-flagellar phenotype and the pathogenicity of S.Enteritidis strain 50336.The results showed that the adhesion ability of mutants 50336?flgK and 50336?flhB to IPEC-J2 and Caco-2 cells were significantly(p<0.05)impaired when compared to the wild-type strain 50336.In addition,the ability mutants 50336?flgK and 50336?flhB invasion into IPEC-J2,Caco-2 cells,HD-11 and RAW264.7 macrophages were also significantly(p<0.05)decreased.However,the survival ability of mutants 50336AflgK and 50336AflhB within macrophage HD-11 and RAW264.7 cells were significantly(p<0.05)increased.These results suggest that the non-flagelled phenotype caused by flgK and flhB gene deletion can promote the viability of S.Enteritidis CMCC(B)50336 in macrophages and increase pathogenicity to some extent.4.Effect of flgK and flhB pseudogenazation of S.Enteritidis on flagella formation and motilityPrevious studies have shown that the deletion of flgK and flhB genes directly affects the formation of flagella of S.Enteritidis.However,the effects of pseudogenization of flgK and flhB on flagella and motility are unknown.The replacement mutants 50336?flhBSP and 50336?flgKSP were constructed by using homologous recombination mediated by temperature-sensitive plasmid pHSG415,the genes flhB and flgK of S.Enteritidis CMCC(B)50336 were replaced by pseudogenes ?flhBSP and ?flgKSP,which were derived from S.Pullorum.Pseudogenized mutants were proved to be non-flagelled and non-motile,by semi-solid motility tests,slide agglutination tests and electron transmission microscope tests.The results indicate that pseudogenization offlgK and flhB affects flagella formation and motility.5.Effect of pseudogenization of flgK and flhB on pathogenicity of Salmonella EnteritidisIn this study,the pathogenicity of 50336?flhBSP and 50336?flgKSP strians were analyzed.The results showed that the adhesion ability of these two strains to IPEC-J2 and Caco-2 cells were significantly(p<0.05)impaired when compared to the 50336 wild-type strain.In addition,the ability of pseudogenized mutants invading into IPEC-J2,Caco-2 cells,HD-11 and RAW264.7 macrophages were also significantly(p<0.05)impaired.However,when compared to the wild-type strain,the survival ability of replacement mutants within macrophage HD-11 and RAW264.7 macrophages were significantly(p<0.05)increased.The results showed that the pseudogenization of flgK and flhB decreased the ability of adhesion and invasion to host cells.On the contrary,the pseudogenization of flgK and flhB enhanced their viability in macrophages of S.Enteritidis CMCC(B)50336 strain.6.Reparation of pseudogenes flgK and flhB of S.Pullorum and the analysis of flagella-related functionAccording to the distribution of pseudogenes in S.Pullorum strains in chapter one,two pseudogenes(flhB and flgK)in S.Pullorum CVCC535 and CVCC540 were repaired.Firstly,the pseudogenes ?flhBSP and ?flgKsp of S.Pullorum CVCC535 and CVCC540 were replaced by functional genes flhBsE and flgKsE,which were derived from S.Enteritidis 50336;and the replacement mutants CVCC535flhBsE and CVCC540flhBsE were constructed.The complementary plasmids carrying the functional flgKSE gene deriving from S.Enteritidis were transformed into the above replacement mutants above;the complementation mutants CVCC535flhBSE/pBRflgKSE and CVCC540flhBSE/pBRflgKSE were constructed.Semi-solid motility tests,slide agglutination tests and electron-transmission microscope tests were conducted.The results showed that the mutants still non-flagelled and non-motile.The results suggest that,there may be other pseudogenes in flagellum coding system of S.Pullorum,therefore more research are needed to reveal the mechanism of non-flagellar phenotype of S.Pullorum.This study shows that the deletion or pseudogenization of flhB and flgK genes affected the formation of flagellum and motility of S.Enteritidis CMCC(B)50336.Pseudogenization of flhB and flgK genes affected the pathogenicity of S.Enteritidis CMCC(B)50336,that is,contributes to the survival of macrophages. |
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