Genetic manipulation of mycobacterial strains using different techniques to determine the role of specific mutations associated with drug resistance
Date
2021-09-06
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Abstract
Functional genomic studies are limited by difficulties surrounding the construction of in vitro Mycobacterium tuberculosis mutants using site-directed mutagenesis. We aimed to use different mutagenesis techniques to insert different point mutations into M. tuberculosis to determine their roles in drug-resistance, or for further physiological investigations in future studies.
In this study we showed proof-of-concept in M. smegmatis on the feasibility of constructing double point mutations using two-step homologous recombineering. Our results showed that it is possible to sequentially introduce two independent point mutations in different genes of the same genome by homologous recombineering. This was achieved in M. smegmatis with katG Pro347His and gyrA Ala288Asp point mutations, which was confirmed to be resistant to isoniazid (INH; ˃4 μg/ml) and ofloxacin (OFX; 0.5μg/ml).
Furthermore, homologous recombination and homologous recombineering were used to attempt to insert a point mutation in M. tuberculosis H37Rv for investigation of the alternative roles of the inhA promoter C-15T mutation. We achieved the construction of the inhA promoter mutant with homologous recombination. A Luria Delbrück assay was also employed to study whether a drug-resistant genetic background would preferentially result in the acquisition of inhA promoter mutations in an ofloxacin (OFX) resistant M. tuberculosis Beijing mutant strain (B01) with a gyrA Asp94Gly compared to its progenitor strain. The Luria Delbrück assay showed that it may be impossible to induce inhA promoter mutations by INH selection pressure in an in vitro system in the strain genetic backgrounds chosen for this study.
We also investigated the association of two different mutations, rv0678 Thr33Ala and atpE Ile66Val, with bedaquiline (BDQ) and clofazimine (CLZ) resistance. This was done by the insertion of each of these mutations into M. tuberculosis H37Rv by homologous recombineering and recombination, respectively in separate experiments. Whole genome sequencing was used to confirm the genetic background of all the genetically manipulated in vitro M. tuberculosis mutants, as well as the progenitor. BDQ, CFZ and INH MIC determination for the mutant isolates was achieved using the Mycobacterium Growth Indicator Tube (MGIT) system to show the association between the manipulated genotypes and their phenotypes. The MGIT MIC determination confirmed that the inhA promoter C-15T mutation confers low-level INH resistance (MIC of ≥0.1μg/ml), while rv0678 Thr33Ala mediates high-level BDQ and CFZ resistance (MIC of ≥1.0μg/ml) and atpE Ile66Val confers low-level BDQ (MIC of 0.25μg/ml) and CFZ resistance (MIC of 0.5μg/ml).
In conclusion, homologous recombination and homologous recombineering were both efficient in generating mycobacterial mutants. However, homologous recombination proved more efficient in M. tuberculosis than homologous recombineering. Proof-of-concept in M. smegmatis demonstrated that double katG (Pro347His) | gyrA (Ala288Asp) point mutations can be introduced in a single strain. While in vitro phenotypic drug susceptibility testing confirmed that katG (Pro347His) caused high-level INH resistance, and gyrA (Ala288Asp) caused low-level OFX resistance in M. smegmatis. The M. tuberculosis inhA C-15T mutation caused low-level INH resistance in vitro. However, C-15T mutation appears to not be acquired spontaneously during in vitro INH selection. While M. tuberculosis rv0678 Thr33Ala and atpE Ile66Val mutations caused an increase in the MIC values for BDQ and CFZ, compared to the wild-type progenitor.
Description
Biomedical Sciences: Molecular Biology and Human Genetics
Keywords
Mycobacterium smegmatis, Mycobacterium tuberculosis, Genetic manipulation, Point mutation, Homologous recombination, Homologous recombineering, Isoniazid, Clofazimine, Bedaquiline, MGIT MIC, Tuberculosis