The Mycobacterium Tuberculosis Cas2 (Rv2816c) Mediates Susceptibility Of Mycobacterium Smegmatis To In Vitro Stressors And Macrophage Killing

Tuberculosis remains a major health problem worldwide. About a third of the global population has been latently infected by M. tuberculosis and around 1.3 million deaths can be attributed to TB annually[1]. The main reasons why TB is so difficult to overcome are the evolution of multidrug-resistant Mycobacterium tuberculosis, the persistence of Mycobacterium tuberculosis and the co-infection with HIV. Therefore, the further basic research on the Mycobacterium tuberculosis which is the pathogen of TB is very urgent.Mycobacterium tuberculosis will encounter the phages which are ubiquitous on the earth when survival in vitro. A series of ways have been evolved by bacteria to defense phages, such as restriction endonuclease, abortive infection[2] and clustered regularly interspaced short palindromic repeat （CRISPR） system. CRISPR system have been found in the genome of 90% archaea and 40% bacteria and been used to defense phages and other mobile genetic elements by these procaryotes[3,4]. CRISPR systems provide an adaptive defense mechanism against phages for archaea and bacteria. CRISPR system consist of a series of repeat sequences which are separated by non-repetitive sequences, a leader sequence and CRISPR-associated genes around it[5]. The functions of Cas proteins are diverse, such as ribonuclease, helicase, polymerase. Recently, CRISPR loci have been shown to be able to modulate gene expression[6]. The Cas9 in Francisella novicida regulate the expression of FIN₁103 which encode a lipoprotein[7]. The CRISPR spacers found homologous to the DNA sequences within their own genomes are involved in autoimmune and the regulation of gene expression[8, 9].Cas2 is a conserved RNA endonuclease crucial for the acquisition of spacers[10-12] widespread among microbial genomes containing CRISPRs[9,13,14]. Cas2 is very important for L. pneumophila to infect its hosts Hartmannella and Acanthamoeba and evade the macrophage immunity[15]. M. tuberculosis genome has two consecutive long CRISPRs and ensuing 9 Cas proteins encoding genes including Cas2[12]. The transcription of Cas2 encoding gene Rv2816c varied with treatment by inhibitors of metabolisms, such as antibiotics and agents causing stress[16]. Therefore, we speculate that Rv2816c might be involved in Mycobacterium tuberculosis stress response and antibiotic resistance.We alignmented Cas2 sequences of mycobacteria with Cas2 of Sulfolobus solfataricus and found the conserved active sites and secondary structure elements and found that Cas2 is conserved in pathogenic mycobacteria. We use Mycobacterium smegmatis as a modle to construct recombinant strains over-expressing Rv2816c （M. smegmatis-pALACE-Rv2816c）. We researched on the phenotype of M. smegmatis-pALACE-Rv2816c and the effect of Cas2 on the stress responses of M. smegmatis in vitro and the survival rate of M. smegmatis in macrophages. We found that the expression of Cas2 changed the growth rate, morphology, sliding motility and bio film formation of M. smegmatis. Those changes may due to the lipid changes of the cell envelope of the recombinant M. smegmatis caused by expression of Cas2. Otherwise, the expression of Cas2 led to the stress response changes and down-regulated transcriptions of SigB, SigE and SigH which are responsible to the stress response and virulence of mycobacteria. We also found that Cas2 decreased the survival rate of M. smegmatis-pALACE-Rv2816c within macrophages accompanying reduced production of host cytokines IL-6 and IL-10. This study provides new insights on the role of Cas2.