Cloning, Expression And Preliminary Function Research Of Flagella-associated Genes Of Leptospira Interrogans

PartⅠ:Prokaryotic expression of Leptospira interrogans fliH/I/Y/N genes and determination of the protein locations in leptospiral envelopeBackground and Objective:Leptospirosis is one of the most widespread zoonosis that caused by pathogenic Leptospira species.The carriers of leptospires are mainly wild or domestic animals,especially rodents,small marsupials,cattle,pigs,and dogs. The clinical manifestations of human leptospirosis range from mild illness to multiorgan failure,characterized by jaundice,pulmonary haemorrhage and renal failure.Pulmonary diffuse hemorrhage,a serious clinical form of leptospirosis,is fatal in about 25%of patients.On the contrary,most of the infected mammalian reservoir animals,such as rodents,only present mild chronic disease or are asymptomatic,and shed infectious organisms in the urine for their lifetime.The reasons for this diversity of infection remain unclear.It's known that many pathogenic bacteria interact,invade and extract nutrients from their hosts successfully depending type three secretion system(T3SS),such as adhering cells,forming a structure of spiculiform to insert virulence factor into cells,interference and damaging cells.There're papers mentioned that flagella structure peoteins could be secreted by T3SS,some of which are supposed to be the members of the T3SS family.In the last few years,L.interrogans is proved to possessing a complete flagella formatting system and the flagella formatting related proteins are found lining up continuously on the macrochromosome forming Virulence-Island like structure.FliH,FliI,FliN and FliY,the flagella formatting related proteins of L.interrogans are highly homologous to Ysc proteins out of T3SS from Yersinia.If the flagella formatting related proteins mentioned have the functions in the L.interrogans' pathogenicity is up to be investigated.Methods:The segments of entire fliH,fliI,fliN and fliY genes were amplified by PCR using chloroform-phenol extracted genomic DNA as the templates and then were sequenced after T-A cloning.Prokaryotic expression systems of the target genes were subsequently constructed.Target amplification products were sequenced after T-A cloning and prokaryotic expression systerms of the genes were constructed and the expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzor were applied to examine the expressions of recombinant proteins rFliH,rFliI,rFliN and rFliY and Ni-NTA affinity chromatography was performed to extract the target expression products.Rabbits were subcutaneously immunized with each the four recombinant proteins to obtain antisera.ELISAs were performed to measure the titers of antisera and Western Blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH,FliI,FliN and FliY.Results:Segments amplified from fliH,fliI,fliN and fliY genes with 924,1365,318 and 1065 bp in size,respectively,were obtained by the PCRs.All the similarities of nucleotide and putative amino acid sequences of the segments from the four genes were 100%compared to the reported sequences.The constructed prokaryotic systems efficiently expressed rFliH,rFliI,rFliN and rFliY with the outputs of approximate 20% of the total bacterial proteins.The rFliH,rFliI,rFliN or rFliY immunized rabbits could produce antibody.The antisera had the titers of 1:100000 above and could recognize the corresponding recombinant proteins to display positive Western hybridization bands.FliH,FliI,FliN and FliY were found to distribute on the external surface of inner envelope,on the internal surface of outer envelope or the interspaces between the two layers of L.interrogans envelope.Conclusion:The prokaryotic expression systems to high efficiently express fagellum-associated proteins FliH,FliI,FliN and FliY of L.interrogans was successfully constructed in this study and the antisera with high titers to recognize their protein antigens were also obtained.Flagellum-associated proteins FliH,FliI,FliN and FliY are the inner envelope proteins and/or outer envelope proteins of L.interrogans.PartⅡ:Construction and preliminary study on the functions of fliH/I/N/Y genes mutants of Leptospira interrogansBackground and objective:Leptospirosis is one of the most widespread zoonosis that causes an acute febrile and systemic illness in humans caused by pathogenic Leptospira species.Although Leptospira virulence factors such as hemolysins, lipopolysaccharide,glycolipoprotein,peptidoglycan,heat shock proteins,flagellin,and others may contribute to the pathogenesis,their pathogenic mechanisms have not been clearly understood.FliN and FliY protein are both flagellar motor switch proteins in leptospires that hit YscQ of Yersinia pestis.YscQ is a T3SS protein with multiple functions,such as to be part of the switch/motor complex that controls the directional rotation of flagella,virulence-related and flagellar biosynthesis-related systems.FliH is a flagellar biosynthesis protein which presents a high sequence homology compared to YscL of Y.pestis,while FliI is a flagellum-specific ATP synthase just like YscN of Y. pestis.Therefor,this study is aimed to constructed the FliH~-,FliI~-,FliN~-,FliY~- mutants to study the function of these four proteins.Methods:The mutants of Leptospira interrogans serovar Lai strain Lai were constructed that fliH/I/N/Y genes were knocked out based on homologous recombination using suicide plasmid.The mutants were detected by ampicillin resistant,PCR and Western blot.Aimed at FliN~- and FliY~- mutant,the motility assay was used to detect the motility of the mutants.Fontana staining method was used to detect the adhesion of FliN~-,FliY~- mutant and wild strain.The Cell Apoptosis Detection Kit was used to staining FliN~-,FliY~- mutant and wild strain which infected murine macrophages,and then the apoptotic and necrotic cells were quantified by flow cytometry.Leptospiral challenge test in guinea pigs was used to test the lethal dose of FliN~-,FliY~- mutant and wild strain.Machite green assay was used to observe the relationship between FliH and the ATPase activity of FliI.Quantitative RT-PCR was used to detect fliH and fliI transcription of FliH~- mutant and wild strain.Results:The repeated PCRs demonstrated that the FliH~-,FliI~-,FliN~-,FliY~-mutants maintained the longer amplication fragments with 1878,2319,1272,2019bp in size,respectively.The Western Blot assay also revealed the negative expression of FliH,FliI,FliN,FliY in the FliH~-,FliI~-,FliN~-,FliY~- mutant.PCR and Western blot revealed the sucessful construction of the mutants.The colonies of FliN~-,FliY~-mutants were notably smaller than that of the wild strain,which showed the loss of motility of mutants.Lower adhesion to murine monocyte-macrophage-like cells (J774A.1)(25.5%in FliN~- mutant,22.9%in FliY~- mutant and 49.1%in wild strain for maximal adhesion rates) and weakened lethality to guinea pigs(100%death rate with 6×10~8 wild strain and 80%with 6×10~9 FliN~- mutant,60%with 6×10~9 FliY~-mutant),compared to the wild L.interrogans strain Lai.Additionally,the FliN~- and FliY~- mutant displayed an absolutely lower ability than the wild strain to induce the apoptosis/necrosis in the infected J774A.1 cells(29.7%in FliY~- mutant,27.4%in FliY~- mutant and 48.2%in wild strain for maximal apoptotic ratios).By studying the biochemical properties of FliH and FliI,we have confirmed ATPase activity of FliI and inhibitory effect of FliH.The expression of FliI was increased magnificently in FliH~-mutant compared to wild strain.Conclusion:The motility,adhesion and virulence of FliN~-,FliY~- mutants were all lower than wild strain,which suggested that fliN and fliY genes play the important roles in pathogenicity in vitro and in vivo of pathogenic Leptospira species.ATPase activity of FliI and inhibitory effect of FliH were detected.These studies laid a foundation for further research of the pathogenic potentials of the flagellar biosynthesis genes of L. interrogans.