Abstract
Tuberculosis remains one of the leading causes of death worldwide. Even if new antitubercular drugs are currently being developed, the rapid emergence and spread of drug-resistant strain remain a severe challenge. The CRISPR associated proteins 1 (Cas1), a most conserved endonuclease which is responsible for spacer integration into CRISPR arrays, was found deleted in many specific drug-resistant strains. The function of Cas1 is still unknown in Mycobacterium type III-A CRISPR family. In this study, the Cas1 (Rv2817c) defect was found in 57.14% of clinical isolates. To investigate the function of Cas1 in new spacer acquisition, we challenged Bacillus Calmette–Guérin (BCG) with a mycobacteriophage D29. Newly acquired spacer sequence matches D29 genome was not found by spacer deep-sequencing. We further expressed Cas1 in recombinant Mycobacterium smegmatis. We found that Cas1 increased the sensitivity to multiple anti-tuberculosis drugs by reducing the persistence during drug treatment. We also showed that Cas1 impaired the repair of DNA damage and changed the stress response of Mycobacterium smegmatis. This study provides a further understanding of Cas1 in Mycobacterium tuberculosis complex (MTBC) drug-resistance evolution and a new sight for the tuberculosis treatment.
Highlights
Tuberculosis is caused by Mycobacterium tuberculosis (MTB)
First we explored the function of type III-A clustered regularly interspaced short palindromic repeats (CRISPR) in the acquisition of phage genome by using mycobacteriophage D29 and Bacillus Calmette–Guerin (BCG) which has a similar type III-A CRISPR system with M. tuberculosis [27]
Type III-A CRISPR-CRISPR associated proteins (Cas) system in Mycobacterium tuberculosis complex (MTBC) is predicted to play a similar role with other type CRISPR-Cas systems in phage resistance, the spacer adaptation has not been observed in MTBC so far
Summary
Tuberculosis is caused by Mycobacterium tuberculosis (MTB). It remains one of the top ten causes of death worldwide and the leading cause from a single infectious agent [1]. Foreign DNA derived from phage genome or invasive plasmid is stored in unique spacer sequences and inserted into CRISPR arrays, which endow the CRISPR-Cas system with the acquired immunity of the past encounters [9]. Mycobacterium tuberculosis, a Gram-positive genus bacteria belonging to Actinobacteria, contains a type III-A CRISPR family [9, 13]. The spacer adaptation has not been observed in type III-A systems [14], and none of the BioMed Research International spacer elements in M. tuberculosis CRISPR array can perfectly match the known mycobacteriophage sequences [15]. CRISPR associated proteins 1 (Cas1), the most conserved protein in Cas family, has been proved to be a metaldependent DNA-specific endonuclease in many genera of bacteria and responsible for spacer integration into CRISPR arrays [16,17,18,19]. The expression of Cas in M. smegmatis impaired DNA damage repairing and increased susceptibility to multiple antibiotics and environmental stresses
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