Page 56 - Biennial Report 2018-20 Jun 2021

domain. The structure was obtained in complex with sinefungin, that helps identify the binding
pocket.

In order to gain insights into substrate binding, a 3-mer oligonucleotide was docked to the ligand-
bound structure of Rv3919c. The substrate docks at the positively charged surface near the
ligand-bound site. A slight increase in the radius of gyration suggests opening of the structure to
accommodate its substrate. Mutations in Rv3919c in the clinical strains of M. tuberculosis were
mapped on the structure to highlight roles of these residues in modulation of ligand binding. The
structure of Rv2966c (RsmD-like methyltransferase) in complex with sinefungin was determined
to 2.3Å resolution and revealed the molecular interactions involved in ligand binding. Role of key
residues was confirmed by site-directed mutagenesis resulting in loss of activity. The loss in
activity was further confirmed by monitoring the changes in the structure in respective mutants
by MD simulations. In order to identify the key methyltransferases that could be associated with
alteration of antibiotic response, genomics data of 9745 clinical strains of M. tuberculosis were
analyzed from the PATRIC database. Mapping of all the non-synonymous mutations in the 16S
rRNA methyltransferases indicated that Rv3919c (RsmG) had the highest number of mutations
(3991), followed by Rv2372c (RsmE) with 1820 mutations and 1357 for Rv1407 (RsmB). Rv3919c
(RsmG) and Rv2372c (RsmE) were hence selected for the study. For validation of in vivo role of
RsmG in m7G527 methylation, direct RNA sequencing of wildtype, RsmG knockout and
complemented strains using nanopore sequencing technology was employed. An analysis
pipeline was developed for the first time, to utilize direct RNA sequencing to identify methylated
nucleotides in 16S rRNA in wild type and complemented strains. The growth of RsmG-deleted
M. smegmatis was checked under increasing concentrations of osmotic agents, oxidising agents
and NaCl. RsmG-deleted M. smegmatis showed increased sensitivity at higher concentration of
hydrogen peroxide compared to the wildtype. The antibiotic response of wild type, RsmG-
knockout and complemented strains, tested with ribosomal and non ribosomal drugs, showed
nearly two-fold increased resistance towards streptomycin that was restored in the
complemented strain. No difference was observed for other tested ribosomal and non-
ribosomal drugs. Deletion of RsmE-homolog was similarly generated in M. smegmatis. Primer
extension assay of wildtype, RsmE-knockout and complemented strains confirmed its role in
highly specific methylation of m3U1498 in 16S rRNA. RsmE knockout strain of M. smegmatis
showed increased sensitivity to growth at low pH, as compared to the wildtype strain. However,
under other conditions of stress, only a marginal difference was observed. This suggests that
these methyltransferases play important roles in mycobacterial cells under certain stress
conditions. The RsmE-knockout exhibited nearly two-fold increased resistance towards several
aminoglycosides (kanamycin, amikacin, gentamicin, neomycin and paromomycin) while no
difference was observed for other tested ribosomal and non-ribosomal drugs. In summary, RsmG
and RsmE methyltransferases were identified to have an association with drug resistance as the
deletion strains exhibited altered response to aminoglycosides. These results are a step-in
further understanding drug resistance mechanisms in mycobacteria.

EVALUATION OF A SIMULATION-BASED SCORING SYSTEM IN INHIBITION OF
MOLECULAR TARGETS

Tuberculosis is the ninth leading cause of death worldwide by a single infectious agent [WHO TB
report 2017]. There is a requirement to develop and update new strategies for identification of

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