BACKGROUND: Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown. METHODS: Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters. RESULTS: Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters. CONCLUSIONS: The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.
BACKGROUND: Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown. METHODS: Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters. RESULTS: Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters. CONCLUSIONS: The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.
Authors: Derrick E Fouts; Michael A Matthias; Haritha Adhikarla; Ben Adler; Luciane Amorim-Santos; Douglas E Berg; Dieter Bulach; Alejandro Buschiazzo; Yung-Fu Chang; Renee L Galloway; David A Haake; Daniel H Haft; Rudy Hartskeerl; Albert I Ko; Paul N Levett; James Matsunaga; Ariel E Mechaly; Jonathan M Monk; Ana L T Nascimento; Karen E Nelson; Bernhard Palsson; Sharon J Peacock; Mathieu Picardeau; Jessica N Ricaldi; Janjira Thaipandungpanit; Elsio A Wunder; X Frank Yang; Jun-Jie Zhang; Joseph M Vinetz Journal: PLoS Negl Trop Dis Date: 2016-02-18