Identification, Structural and Functional Characterisation of the Molecule that Induces Asymmetric Cell Division in Mycobacteria

Abstract

Phenotypic heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions, is known in mycobacterial populations. Such population heterogeneity had been observed in in vitro cultures, TB patients, and in animal models. Our laboratory had earlier shown that about 20-30% of the 15% septating cells of Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium xenopi mid-log phase cultures divide by highly deviated asymmetric cell division (ACD), generating subpopulations of short cells and normal-sized/long cells. The remaining 70-80% of the septating cells divide by symmetric cell division (SCD) with 5-10% deviation of the division site constriction from the median. The proportion of short cells amounted to about 3-5% of the total population, while the remaining 97-98% of the population was constituted by normal-sized/long cells. This proportion of short cells has been found to be consistent and reproducible irrespective of culture media. Comparable proportion of short cells of tubercle bacilli has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum also. It indicated that such processes must be occurring in the tubercle bacillary population in the TB patients too and that the presence of short cells has some physiological relevance. Thus, ACD has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, there has not been any study on the mechanism that generate such subpopulation of short cells through ACD. Therefore, in the present study, we investigated the mechanism behind the ACD that generates short cells in the Mycobacterium smegmatis and Mycobacterium tuberculosis populations. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different areas of research in bacterial physiology that are linked by the present study. These areas of research include bacterial cell division process per se, the proteins involved in bacterial cell division, cell division in general in mycobacteria, different modes of cell division in mycobacteria, generation of cell size heterogeneity through symmetric and asymmetric cell division in mycobacteria, cell-cell communication in bacterial systems, and the characteristics and physiological significance of diadenosine polyphosphates from bacteria to humans. The complete account of the research in these areas that are linked in the present study thus justifies the introduction to the results that are given in the ensuing chapters. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the cell division bioassay used to score for the proportion of cells undergoing symmetric and asymmetric divisions, the biochemical assays performed to find out the biochemical identity of the molecule, the isolation and fractionation methods for the ACD-IM from the concentrated culture supernatants (CS), and finally the mass spectrometric methods used for the elucidation of the structure of the molecule are given The Chapter 3 forms the first data chapter that presents results on the detection of the presence of the asymmetric cell division inducing molecule (ACD-IM) from the concentrated culture supernatant (CS) of Mycobacterium smegmatis and Mycobacterium tuberculosis, as inducing ACD in higher proportions of cells. Further, it shows that the levels of ACD-IM increase at late growth phases of 0.8 and 1.0 OD600 nm. The chapter is concluded with a discussion of the results. The Chapter 4 describes the biochemical analyses of the CS to find out the chemical nature of the ACD-IM. DNase I, snake venom phosphodiesterase (SVP), RNase A, lipase, and proteinase K were used to find whether exposure of CS of M. smegmatis and M. tuberculosis cells to these enzymes could abolish the ACD inducing activity of the molecule in the CS on the respective cells. These experiments showed that the ACD-IM was susceptible to DNase I and SVP, but not to RNase A, lipase, or proteinase K. However, proteinase K showed direct effect on the cells by decreasing the proportion of cells dividing by ACD, indicating the probable presence of the receptor to the molecule on the external cell surface. The data also shows the conservation of the molecule in M. smegmatis and M. tuberculosis by demonstrating that the CS of M. smegmatis could induce ACD in higher proportions of M. tuberculosis cells and vice versa. The structural identification of ACD-IM present in the concentrated CS of M. smegmatis and M. tuberculosis using LC-ESI-MS and MS-MS analyses as diadenosine hexaphosphate (Ap6A). The structure of the natural Ap6A molecule was confirmed using synthetic Ap6A molecule subjected to LC-ESI-MS and MS-MS analyses. Further, the ACD inducing activity of the synthetic Ap6A molecule, its susceptibility to DNase I and SVP, but not to RNase A, lipase and proteinase K were verified to establish that the structural, biochemical, and functional properties of the naturally occurring Ap6A in the CS of M. smegmatis and M. tuberculosis were identical to those of synthetic Ap6A. The chapter is concluded with a discussion of the results.

The Chapter 5 presents the data on the genes involved in the degradation and synthesis of Ap6A in M. smegmatis and M. tuberculosis. The knockout of MSMEG_2936 gene of M. smegmatis resulted with significant increase in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found significantly higher concentration than wild type. These results showed that MSMEG_2936 protein might catalyse the degradation of Ap6A in mycobacteria. The knockout of MSMEG_2932 gene of M. smegmatis resulted with significant reduction in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found comparable to wild type. These results showed that MSMEG_2932 protein might not involve in the synthesis of Ap6A in mycobacteria. The chapter is concluded with a discussion of the results. Thus, the present study, for the first time, establishes the identity, structure, and function of Ap6A as the molecule that induces asymmetric cell division in mycobacteria.