Biological activity and pathogenicity of M23 superfamily metalloendopeptidases of Leptospira interro-gans / 中华微生物学和免疫学杂志

ABSTRACT

Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR(qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells(HUVEC) with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190. 25 μmol/L, Kcat 4. 86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai(P<0.05). Conclusion The prod-ucts of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+-dependent M23 metalloendopeptidas-es, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness.