Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4（Rv2820c） on Intracellular Viability of Mycobacterium smegmatis
Abstract
To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis(MS). Methods Csm 4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid. The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS_Csm4 in vitro and the influence of reactive N, O species on the growth of MS_Csm4were observed. The intracellular survival of MS_Csm4 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_Csm4, which did not affect the growth of the recombinant MS. Reactive N, O species decreased MS_Csm4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS_Csm4. Conclusion Recombinant MS_Csm4 is susceptible to reactive N, O species in vitro. THP-1 promotes NO release and thus discourages intracellular survival of MS.
Keywords: Mycobacterium tuberculosis, Csm4 gene, Recombinant, Mycobacterium smegmatis, Reactive N, O environment, Intracellular survival
Full Text:
PDFReferences
WHO. Global tuberculosis report 2016 [ 2017-05-10 ]. http://www. who. int/tb/ publications/global_report/en/. 2016.
MAKAROVA KS, KOONIN EV. Annotation and classification of CRISPR-Cas systems. Methods Mol Biol« 2015,1311: 47-75[2017-05-10], https;//link. springer, com/ protocol/10. 1007% 2F978-1-4939-2687-9 _ 4. doi: 10. 1007/978-1-4939-2687-9 4.
WESTRA ER, BUCKLING A. FINER AN PC. CRISPR-Cas systems;beyond adaptive immunity. Nat Rev Microbio, 2014 . 12(5):317-326.
HE L. FAN X, XIE J. Comparative genomic structures of mycobacterium CRISPR-Cas. J Cellular Biochem, 2012. 113 (7):2464-2473.
LAM JT, YUEN KY, HO PL, et al. Truncated Rv2820c enhances mycobacterial virulence ex vivo and in vivo. Microb Patbog.2011,50(6) :331-335.
PIETERS J. Mycobacterium tuberculosis and the macrophage:maintaining a balance. Cell Host Microbe,2008. 3(6);399-407.
NUMATA T, INANAGA H, SATO C, et al. Crystal structure of the Csm3-Csm4 subcomplex in the type Щ-A CRISPR-Cas interference complex. J Mol Biol,2015,427(2): 259-273.
RENGARAJANJ. BLOOM BR, RUBIN EJ. Genome-wide requirements for mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A, 2005, 102(23);8327-8332.
CHAN ЕD. CHAN J. SCHLUGER NW. What is the role of nitric oxide in murine and human host defense against tuberculosis? Current knowledge. Am J Respir Cell Mol Biol,2001,25(5) :606-612.
NATHAN CF, HIBBS JR JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol, 1991,3(1); 65-70.
PAHARI S. KHAN N. AQDAS M. et al. Infergen stimulated macrophages restrict mycobacterium tuberculosis growth by autophagy and release of nitric oxide. Sci Rep. 2016, 6: 39492 [2017-05-10]. https;//www. ncbi. nlm. nib. gov/pmc/articles/PMC5175149/. doi: 10. 38/srep39492.
YUK JM. SHIN DM. LEE HM,et al. Vitamin D3 induces autophagy in human monocytes, macrophages via cathelicidin. Cell Host Microbe,2009,6(3) :231-243.
TSOLAKI AG, GAGNEUX S, PYM AS, et al. Genomic deletions classify the Beijing/W strains as a distinct genetic lineage of mycobacterium tuberculosis. J Clin Microbiol, 2005,43(7):3185-3191.
Refbacks
- There are currently no refbacks.
