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1.
Microb Drug Resist ; 28(12): 1057-1064, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36534487

ABSTRACT

Background: The virulent ATP-binding cassette (ABC) importers from Mycobacterium abscessus, the most common native multidrug resistant and emerging opportunistic pathogen in rapidly growing NTM, were explored by comparative genomic study, in view of the fact that the ABC importers of Mycobacterium tuberculosis, responsible for uptaking metals, anions, amino acids, peptides, sugars, and other crucial substances from the host, had been proved to be closely related with the bacillus's virulence, survival in the host macrophages, antibiotic resistance, modulation of host immune system, and so on, although detailed mechanism was unclear. Methods: For virulent ABC importers from M. abscessus predicted by orthology and phylogeny analysis of nucleotide-binding domains (NBDs) of Mycobacterium smegmatis, M. abscessus, and M. tuberculosis, the antibiotic susceptibility of overexpression transformant and knockout mutant was assayed after confirmation by in vitro experiment. Results: Three-domain importers were dominant ones in M. abscessus (60.0%), four-domain ones dominant in M. tuberculosis (87.5%), whereas both types were same in M. smegmatis (41.9%). In the phylogenetic tree, the importers of M. abscessus (53.3%) and M. tuberculosis (62.5%) were mainly distributed in clay A, whereas the clay E was exclusively composed of M. smegmatis NBDs, which hinted possible reprogramming of the transporter system during the pathogen evolution. In clay A, MAB_2178 and others were predicted virulence-associated because of high sequence similarity to M. tuberculosis virulence importers. Conclusions: The importance and complexity of antibiotics resistance mechanisms of MAB_2176-2177-2178 were pointed out by its overexpression enhancing bacterial resistance to ciprofloxacin, clarithromycin, cefoxitin, and sensitivity to amikacin, and knockout having opposite phenotypes.


Subject(s)
Mycobacterium Infections, Nontuberculous , Mycobacterium abscessus , Mycobacterium tuberculosis , Tuberculosis , Humans , Anti-Bacterial Agents/pharmacology , Mycobacterium abscessus/genetics , Clay , Phylogeny , Microbial Sensitivity Tests , Clarithromycin , Mycobacterium tuberculosis/genetics , Genomics , Adenosine Triphosphate , Mycobacterium Infections, Nontuberculous/microbiology
2.
Sci Adv ; 8(47): eadd5907, 2022 11 25.
Article in English | MEDLINE | ID: mdl-36417506

ABSTRACT

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Mycobacterium tuberculosis , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Mycobacterium tuberculosis/genetics , CRISPR-Cas Systems , Genomics/methods , Genome
3.
Front Genome Ed ; 3: 734436, 2021.
Article in English | MEDLINE | ID: mdl-34957465

ABSTRACT

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

4.
mBio ; 11(1)2020 01 28.
Article in English | MEDLINE | ID: mdl-31992616

ABSTRACT

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coliIMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , DNA End-Joining Repair , Gene Editing , Genome, Bacterial , Mycobacterium tuberculosis/genetics , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , CRISPR-Cas Systems , Models, Biological , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/metabolism , Protein Binding , RNA, Guide, Kinetoplastida , Rec A Recombinases/metabolism
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