Page 34 - Biennial Report 2018-20 Jun 2021

BLOOD BASED SIGNATURES OF TUBERCULOSIS FOR DIFFERENTIAL DIAGNOSIS
IN EXTRAPULMONARY TB

Differential diagnosis of Tuberculosis in cases of extrapulmonary tissue involvement is clinically
challenging, especially in deep tissues such as bone, brain, and lymph nodes as these often
present as paucibacillary disease yet result in debilitating conditions. Blood based transcriptional
signatures have paved the way for novel molecular diagnosis of pulmonary TB. In a study funded
by DBT, Sheetal Gandotra and Vivek Rao, in collaboration with Rajesh Gokhale at National
Institute of Immunology and Joy Sarojini Michael at Christian Medical College began a study to
identify blood-based signatures in extrapulmonary TB. In this study, the first step was to test if
the blood-based signature of pulmonary TB would work in differential diagnosis of
extrapulmonary TB from other diseases involving the same tissue site. Patient recruitment took
place at CMC Vellore where blood samples were collected and stored. The blood was stored at -
80 up till processing. RNA was extracted from whole blood samples of patients at IGIB and NII.
qRT-PCR was used to quantify gene expression using a set of genes 16 experimental and 4
housekeeping control extracted from literature. Initial optimization involved screening for the
most stable housekeeping gene among the 4 controls that similar studies had previously used.
The most stable housekeeping gene was further used for analysis. A total of167 blood samples,
69 cases and 58 control samples (TB and non-TB samples of pulmonary, bone, lymph node, and
CNS diseases) have been collected and analyzed for the 16-gene signature previously described
in literature. While several of the 16 gene set classifier facilitate distinction between pulmonary
TB and non-TB cases, these signature genes do not show distinction between EPTB cases and
controls. It is therefore important perform transcriptome wide analysis on these and similar
samples in the future.

ROLE of CALCIUM HOMEOSTASIS OF Mycobacterium tuberculosis IN
PATHOGENESIS

Under this project funded by the Department of Science and Technology, a group of scientists
led by Rakesh Sharma and Bhupesh Taneja aim to functionally characterize the putative calcium
ion transporters (CtpE and CtpF) of Mycobacterium tuberculosis. By evaluating the role of
calcium in cellular physiology and morphology of Mycobacterium and the importance of calcium
transporters in pathogenesis of Mycobacterium tuberculosis, a clear picture of calcium
homeostasis in this pathogen can be drawn.

Gene knockout of putative calcium ion transporter protein ctpE was constructed in M.
tuberculosis using the recombineering methodology. M. tuberculosis Erdman strain mutants for
ctpE gene were selected on hygromycin containing growth media. The knockout of ctpE gene
was confirmed by Southern hybridization and PCR based strategies. Phenotypic characterization
of the mutant strain is in progress. Knockout of another putative calcium transporter gene, ctpF,
was constructed based on homologous recombination in Mycobacterium smegmatis and
confirmed by PCR-based methodology and Southern hybridization. The mutant showed slower
growth in Sauton's medium. Further phenotypic characterization is in progress. Knockout work

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