[The OmpA-like protein Loa22 from Leptospira interrogans serovar lai induces apoptosis in A549 via Ca2+ signal pathway].

OBJECTIVE: To study the effect of Loa22 mature peptide on the apoptosis of A549 to explore the mechanism of pulmonary impairment in severe forms of leptospirosis. METHODS: Loa22 mature peptide (100 microg/mL) was administered to culture with human lung adenocarcinoma cell line (A549). After 24 hours, the apoptosis, the concentration of calcium of the cells were evaluated. The F-actin cytoskeleton structure was observed and calmdulin (CaM) mRNA expression was also detected. At the same time, after the pretreatment of A549 with PLC specific inhibitor U73122, adding an appropriate amount of mature peptide of Loa22 to act on the cells for a period of time, then detection same index. RESULTS: Loa22 mature peptide could induce the increase of intracellular Ca2+ concentration ([Ca2+]i), CaM expression in mRNA level, the activity of LDH, and cytoskeleton rearrangement of F-actin. But after blocking the signal pathway of PLC, Loa22 mature peptide reduced the increase degree of [Ca2+]i, apoptosis rate, the expression of CaM mRNA, the activity of LDH compared with the unblocked group. CONCLUSION: These data suggest that Loa22 mature peptide involves in the pathological processes of L. interrogans invasion and increase apoptosis in A549 by increase of [Ca2+]i through signaling pathway of PLC.

[Construction and expression of recombinant Mycobacterium bovis BCG with the ompA-like membrane protein gene Loa22 of Leptospira interrogans serovar].

OBJECTIVE: To study the immunity of Loa22 from Leptospira interrogans serovar Lai strain 56601 by expressing its protein in BCG. METHODS: Amplified the mature peptide of Loa22 gene from the genome of of Leptospira interrogans serovar Lai strain 56601 and constructed recombinant plasmid rpMV36l-1oa22 with the E. coli-BCG integrating shuttle plasmid pMV361 and the Loa22 mature peptide gene. The rpMV36l-1oa22 plasmid was transformed into BCG by electroporation. The rBCG bearing rpMV36l-1oa22 was induced by high temperature of 45 degrees C and expressed protein was identified by SDS-PAGE and Western Blotting. Fifth 6-week-old BALB/c mice were randomly divided into five groups, which were inoculated intraperitoneally two times at 0-day and 21-day with BCG, rBCG-pMV361, rI3CG-1oa22, Loa22 and killed whole-leptospires respectively. All animals were dislocated from cervical vertebra on the 14Ih day after the last immunization. The proliferative reaction of splenic lymphocyte in tuitro were tested by XTT. RESULTS: The rpMV36l-1oa22 plasmid was constructed successfully and transformed into BCG. The rBCG expressed a 19 X io specifical protein identified by SDS-PAGE and Western Blotting. The splenic lymphocyte proliferate activity (SI) in rBCG-ioa22 group in intro was significantly higher than those in BCG group and rBCG-pMV361 group. CONCLUSION: We explored the expressing feasibility of Loa22 in Mycobacterium bovis BCG. may therefore make further researches on the induction of protective immunity against human and animal leptospirosis.