The Molecular Basis for the Differential Antibiotic Susceptibility of Mycobacterial Subpopulations

Abstract

Mycobacterial populations have evolved various strategies to withstand diverse stress conditions in the environment. One among these strategies is maintenance of phenotypic heterogeneity, in terms of cell morphology, length, growth rate and division, metabolic status, and gene expression among others, in a genetically identical population. Phenotypically divergent but genetically identical subpopulations have been observed in mycobacterial populations in Tuberculosis patients, in animal model studies as well as in in vitro cultures. Phenotypic heterogeneity, in mycobacterial populations, based on cell length has been studied but the molecular level differences and the benefit of such variations remain unknown. In this regard, our laboratory had earlier reported the occurrence of highly deviated asymmetric cell division in a subpopulation of septating mycobacterial cultures giving rise to one short cell (SC) and one normal/long cell (NC). The proportion of cells undergoing highly deviated asymmetric cell division in a mid-log phase population remains consistent. The existence of short cells and normal cells was also observed in freshly diagnosed pulmonary TB patient’s sputum samples, indicating the clinical relevance of cell-length based heterogeneity in pathogenic Mycobacterium tuberculosis strains in TB patients. Our laboratory had also reported the physiological relevance of cell-length based phenotypic heterogeneity of mycobacterial SC and NC populations upon exposure to oxidative stress, nitrite stress and antibiotics stress. The SC subpopulation was found to be significantly more susceptible to all the stress conditions than the NC subpopulation. However, the molecular basis for the differential susceptibility between the SC and NC subpopulations needed investigation. Therefore, in the present study, we wanted to find out the molecular level differences between the SC and NC subpopulations that are responsible for their differential tolerance/susceptibility and the benefit such differences bestowed on the population upon antibiotic exposure. The data chapter that presents the benefit of the differential levels of hydroxyl radical generation in M. smegmatis SCs and NCs. Data showed that differential levels of hydroxyl radical generation was the reason for differential susceptibility of SCs and NCs, but with clear benefits to both the subpopulations that help their survival against anti-tuberculosis antibiotics. Hydroxyl radical is known to create genome wide nonspecific mutations which can give rise to resisters in a population. Differential levels of hydroxyl radical generation in SCs and NCs inflicted mutations in their genome, resulting in differential mutability or differential propensity for resister generation in SCs and NCs. This was demonstrated by plating unexposed SCs and NCs on various anti-tuberculosis antibiotics, selection for the resisters and confirming the presence of mutations on the hot spot regions of the target genes. Data showed a differential propensity of resister generation between SCs and NCs when selected on rifampicin, moxifloxacin and isoniazid. Mutation frequency of these cells against different antibiotics was also calculated and compared. ROS scavengers or inhibitors abolished the difference or reduced the mutation frequency in these cells. The overall experiments and the results therefrom have been discussed in this Chapter, which is concluded with a model depicting the overall data of the study