Collagenase activity and pathogenic mechanism of colA gene product of Leptospira interrogans
ZHAO Jin-fang1, TAN Pan-li1, WANG Zhe-jiong1, HU Wei-lin2, YAN Jie2, Kokouvi KASSEHNE2
1.Department of Laboratory Medicine, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China; 2.Department of Medical Microbiology and Parasitology,School of Medicine, Zhejiang University, Hangzhou 310058, China
Abstract:To investigate the distribution and sequence conservation of colA gene in different serogroups of L. interrogans, as well as to determine the collagenase activity, expression and secretion of colA gene product of the spirochete during infection of host cells, PCR and its product sequencing were performed in seven L. interrogans serogroups that prevailing in China and the colA gene product of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was expressed by a prokaryotic expression system and extracted by Ni-NTA affinity chromatography. By using spectrophotometry, the ability of the recombinant protein(rColA) to hydrolyze I-IV type natural collagen proteins and two synthetic substrates(Azocoll and Pz-peptide) were determined, and the Km and Kcat values of rColA were also measured. Real-time fluorescent quantitative RT-PCR and Western Blot assay were applied to detect the colA-mRNA level change and ColA secretion of L. interrogans strain Lai during infection of HUVEC, BEAS-2B, L-02 or HEK293 cells. The results showed that the entire colA gene segments were amplified from the different L. interrogans serogroups with high nucleotide and amino acid sequence identities(99.4%-100%). The rColA could hydrolyze the six substrates in varying extents, in which the hydrolytic ability on type-III collagen showed to be the strongest(P<0.05), with 2.16 mg/mL Km and 35.6 h-1 Kcat values. The colA-mRNA level of L. interrogans strain Lai was significantly increased(P<0.01) and the secreted ColA could be detectable in the supernatant of L. interrogans-cell co-cultures during infection of different host cells. These results indicated that the colA gene of L. interrogans is a sequence-conserved and extensively distributed collagenase-encoding gene. The up-regulated expression and external secretion of the gene product during infection of host cells imply its practical role in the process of L. interrogans infection.
赵金方, 谈潘莉, 汪浙炯, 胡玮琳, 严杰, Kokouvi KASSEHNE. 问号钩端螺旋体colA基因产物胶原酶活性及其致病机制研究[J]. 中国人兽共患病学报, 2016, 32(1): 7-12.
ZHAO Jin-fang, TAN Pan-li, WANG Zhe-jiong, HU Wei-lin, YAN Jie, Kokouvi KASSEHNE. Collagenase activity and pathogenic mechanism of colA gene product of Leptospira interrogans. Chinese Journal of Zoonoses, 2016, 32(1): 7-12.
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