Asymmetric cell division in Mycobacterium tuberculosis and its unique features

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed

Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute similar to 10-20% of the population, and the high-buoyantdensity normal/long-sized cells (NCs), which form similar to 80-90% of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H2O2 and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H2O2 -exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria