| This experiment was based on the genome sequencing of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai type strain #56601 (virulent strain) by Chinese National Human Genome Center at Shanghai (CHGCS) in Oct. 2001. During genes annotation, a series of genes related to pathogenesis were found associated to adhesion and invasion, flagellum, mobility and chemotaxis, LPS synthesis, signal transference, hemolysis and haemorrhaging. The paper "Unique Physiological and Pathogenic Features of Leptospira interrogans Revealed by Whole Genome Sequencing" was published in Nature 2003, 422(6934): 888-893. Some results about identification and functional studies of genes related to hemolysis, haemorrhaging and bacterial apoptosis were come from this workL interrogans is a free-living, aerobic, motile, tightly coiled spirochete which is known worldwide to be responsible for the water-borne zoonosis, leptospirosis. It can not only proliferate in vivo but also be cultivated in vitro environment although many disadvantageous factors affect its growth. EMJH or Korthof is suitable medium for L.interrogans although its growth slowness (40-50 hours to reach exponential phase) and low strain concentration. Experiments have proved significant pathogenicity difference by infection guinea pig with virulent and aviruent strains Lai.Total ten hemolysin genes were predicted by bioinformatics encoded on L. interrogans chromosome, nine unknown genes were classified into two types: sphingomyelinase (LA1027, LA1029, LA3050, LA4004) and non-sphingomyelinase (LA0327, LA0378, LA1650, LA3937, LA0150). Cloned and expressed eight genes in prokaryotic system to get recombined hemolysin protein, and successfully detected their hemolysis. Some genes LA1027, LA1029, LA3050 and LA4004 were identified as sphingomyelinase by TLC and HPLC. It was the first time to discovered so many hemolysin genes encoded by one chromosome in microbial kingdom.RT-PCR results showed all hemolysin genes were transcripted in L. interrogans, Western-blot revealed all hemolysin genes except LAI 027 and LA3050 were translated into proteins whatever strain's pathogenicity in EMJH environment. ELISA showed all hemolysin except LAI027 and LA0378 were secreted outside of bacteria under Korthof environment.Studies on the cytotoxicity of L. interrogans hemolysins discovered both sphingomyelinase hemolysin LAI029 and non-sphingomyelinase hemolysin LA3937 have powerful damage to cells. For example, cellular floss disappeared, cellularmembrane burst or vanished completely, cytoplast leaked, cytoplasm dyed thinly, cellular organdies swelled and overflowed or even lost. Vacuole appeared in karyon and chromatin gathered, or even irregular brim of nuclear membrane with burst and serious cellular apoptosis. Some hepatocytes showed typical apoptosis such as mitochondria intumesce, nucleolus gathering aside and concentration with several nucleosome. About 14.73% to 57.92% hepatocytes L-02 cells became apoptosis after damaged by LA 1029 while no apoptosis of embryo kidney cells 293 in the same case. LA3937 can alter 293 cellular cycles. Using human 4,096 cDNA array technology to find hepatocytes L-02 gene expression profiles significantly changed after LA 1029 induction, total 90 genes (10 genes up and 80 genes down) changed compared to control group. These genes are associated with tumor, signal transduction, apoptosis, cellular cycles, metabolize and protein biosynthesis etc. Moreover, LA 1029 can stimulate lymphocytes and macrophages secrete inflammation factors IL-1 B and IL-6. Above all, Leptospira hemolysin can induce not only cellular necrosis but also apoptosis so as to be regarded as one of the most important factors related to inflammation, fever, organic dysfunctional.A series of genes related to Leptospirosis haemorrhage were also predicted containing one lysozyme gene {acm), one collagenase A gene (colA), seventeen YopM genes, one platelet activating factor acetylhydrolase gene (pafah) and two von Willebrand factor A domain genes (vwA 1 ,vwA2). RT-PCR...
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