Conditional Silencing by CRISPRi Reveals the Role of DNA Gyrase in Formation of Drug-Tolerant Persister Population in Mycobacterium tuberculosis

Abstract

Drug tolerance in mycobacterial pathogens is a global concern. Fluoroquinolone (FQ) treatment is widely used for induction of persisters in bacteria. Although FQs that target DNA gyrase are currently used as second-line anti-tuberculosis (TB) drugs, little is known about their impact on Mycobacterium tuberculosis (Mtb) persister formation. Here we explored the CRISPRi-based genetic repression for better understanding the effect of DNA gyrase depletion on Mtb physiology and response to anti-TB drugs. We find that suppression of DNA gyrase drastically affects intra- and extracellular growth of Mtb. Interestingly, gyrase depletion in Mtb leads to activation of RecA/LexA-mediated SOS response and drug tolerance via induction of persister subpopulation. Chemical inhibition of RecA in gyrase-depleted bacteria results in reversion of persister phenotype and better killing by antibiotics. This study provides evidence that inhibition of SOS response can be advantageous in improving the efficacy of anti-TB drugs and shortening the duration of current TB treatment.

An Update on Antimicrobial Peptides (AMPs) and Their Delivery Strategies for Wound Infections

“…Choudhary et al 2019 [210] and Wei et al 2019 [211] have used the endogenous CRISPR-Cas system as an efficient tool against Mycobacterium tuberculosis, which has developed multidrug-resistance, even against the most potent antibiotic rifampicin. In other studies, CRISPR-Cas9 was successfully delivered to selectively kill MDR bacteria E. coli [212], and S. aureus [213] using a phagemid (plasmid packed in a phage capsid)-mediated system by removing plasmids carrying AMR genes and, in turn, effectively re-sensitizing bacteria to antibiotics.…”

Impact of CRISPR interference on strain development in biotechnology

“…1). CRISPRi has been developed in a diverse group of bacteria, including E. coli [5] , Corynebacterium glutamicum [7] , Lactococcus lactis [18] , Rhodococcus opacus [19] , Burkholderia [20] , Clostridia [21–25] , Bacillus subtilis [26–29] , Bacillus methanolicus [30] , Streptomyces [31,32], and Mycobacteria [33–36], and applied for gene repression to interrogate their physiology or to identify gene targets for biotech applications (see Sections 3 and 4).…”

“…It turned out that the lipoprotein transport system represents an ideal drug target because its depletion resulted in cells prone to plasmolysis and reorganizing membrane in an elaborate manner [81]. Similarly, CRISPRi in Mycobacterium tuberculosis was used to screen and validate new genetic targets to develop novel therapeutics against this pathogen [33,35,36,82]. CRISPRi was further used to study the function of essential genes in two bacteria with reduced genomes that are actively used in systems biology studies, that is, Mycoplasma pneumoniae and Mycoplasma mycoides [83].…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Zuberi et al also used the same strategy to inhibit the expression of FimH, the key component for type I fimbriae in E. coli and responsible for the bacterial adhesion and biofilm formation, also alleviating its virulence. Furthermore, CRISPRi was used in other pathogenic bacterial species, such as S. aureus and M. tuberculosis , to reduce their virulence and antimicrobial resistance …”

“…Furthermore, CRISPRi was used in other pathogenic bacterial species, such Small Methods 2020, 4,1900560 www.advancedsciencenews.com www.small-methods.com as S. aureus and M. tuberculosis, to reduce their virulence and antimicrobial resistance. [12,[183][184][185][186][187]…”