[Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm 4 ( Rv 2820 c ) on Intracellular Viability of Mycobacterium smegmatis ].

OBJECTIVE: To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis (MS).

METHODS: Csm 4 gene was amplified by PCR to construct pMV261- Csm 4 shuttle expression plasmid. The Csm4 protein expression in MS _Csm 4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS _Csm 4 in vitro and the influence of reactive N,O species on the growth of MS _Csm 4were observed. The intracellular survival of MS _Csm 4 and expressions of inducible nitric oxide synthase gene ( iNOS ) and nitric oxide production (NO) were detected after infection with THP-1 macrophages.

RESULTS: Csm4 protein was successfully expressed in MS _Csm 4,which did not affect the growth of the recombinant MS. Reactive N,O species decreased MS _Csm 4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS _Csm 4.

CONCLUSION: Recombinant MS _Csm 4 is susceptible to reactive N,O species in vitro . THP-1 promotes NO release and thus discourages intracellular survival of MS.