Page 35 - Biennial Report 2018-20 Jun 2021

for ctpF gene in M. tuberculosis is in progress and the phage based knockout constructs are
ready.

GTP-BINDING PROTEINS: COMPLEX REGULATORS OF SURVIVAL AND
PATHOGENESIS IN Mycobacterium tuberculosis H37Rv

Whole genome sequencing of M. tuberculosis found various GTPases (Era, Obg, LepA, engA
phoH1 (Rv2368c), Rv3362c, ppa, ftsY, Rv2923c, hflX (Rv2725c) phoH2, and Rv1112c) that are
important in various cell signaling processes. At IGIB, Laxman Singh Meena initiated a project to
understand the significance of the GTP-binding family genes in the survival and pathogenic
mechanism of M. tuberculosis H37Rv. Previous work describes the importance of Rv3906c, which
is also a predicted GTPase. Rv3906c is a conserved hypothetical gene of M. tuberculosis and
contains many GTP binding protein motifs DXXG demonstrating that this protein might exhibit
GTPase activity. Aspartate is thought to be essential at this site; therefore, to further confirm
this essential role of aspartate residue in whole GTP binding and hydrolyzing activity, this residue
was mutated to other amino acids one by one. The impact of stress conditions like pH and
temperature on the stability of the protein were studied. Alteration in pH did not affect stability
of the protein while decreasing the temperature reduced its stability.

FtsY shows strong binding with Ffh and Rv3362c. All these proteins are confirmed to contain
intrinsic GTP hydrolyzing activity. FtsY also contains consensus GTP binding motif DXXG (D45,
D71, D312 and D367) and GXXXXGK at G230 position. Phosphorylation site prediction shows that
this protein is phosphorylated at serine, threonine and tyrosine. A 3D model of this protein has
been made by I-TASSER server and validated by SAVES server. Mutational analysis has been done
by I-Mutant 3.0 server and it shows that mutating D45 to alanine showed a large decrease in
stability at 25°C and pH 7. Molecular docking results of these proteins, both before and after
mutation, prove that interaction between these two proteins decreases maximally at D312.

The essential role of the ftsY gene in the secretion process was seen and that seems to have a
role in the pathogenic mechanism of this bacterium. ftsY mutations were characterized and top
ten mutations that had the maximum decrease in stability were selected. Rv1907c was found to
be a proficient thioredoxin protein. Thioredoxin (Trx) proteins are redox proteins that can exist
in either oxidised (disulfide) or reduced (dithiol) forms and are an important component of many
cellular redox reactions. The Trx protein of this study contains a conserved motif CXXC that is
essential for its enzymatic activity. Mutational analysis of Rv1907c confirmed the essential role
of this conserved motif for maintaining the stability of this protein.

This protein might be essential in cellular processes of M. tuberculosis and thus has a potential
of being a novel drug target for tuberculosis treatment. Rv1514c gene of M. tuberculosis is an
essential glycosyl transferase. Functional partner prediction was made using String database
server that predicted its interaction with an fcl (epiA), Rv1508A, gmdA, Rv1516c, Rv3264c,
Rv1515c, Rv3032, Pima, Rv2188c and glgA. Rv1514c was modelled using I-TASSER server and
further evaluated by ProSA, Rampage, Verify 3D and ERRAT. Binding sites were predicted by
COACH that confirmed UDP (Uridine diphosphate) might be bound to it for transferring glycosyl
groups.

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