Page 132 - CSIR-IGIB Annual Report 2020-21

macrophages abrogates the innate immune genes involved in remodeling of lipids, which
response to infection, we are interested in involves steps of degradation and transfer of acyl
understanding the role of LD-localized ARL8B in chains is not well studied in Mtb. The patatins
macrophage lipid mobilization and infection family of enzymes is particularly adept at such
induced innate immune responses. Key to this is to functions in mammals. Pathogenic bacteria exhibit
understand what regulates localization of ARL8B expansion of this family of enzymes compared to
to the LD. By generating several mutant variants of non-pathogenic species of the same genus. We are
ARL8B we are evaluating features of ARL8B that studying this family of enzymes in mycobacteria.
enable localization to the LD. We initiated a Of the eight patatin genes present in Mtb,
collaborative project with Dr Abdou Rachid Thiam orthologues of two are present in the fast-growing
at ENS Paris to understand ARL8B-LD interaction. non-pathogenic organism M. smegmatis. These
Complementary approaches of cell and infection are Ms5284-Ms5285, corresponding to the
biology in our lab and biophysical tools developed Rv1062-Rv1063c locus in M. smegmatis. We
in his lab are being used for this. As ARL8B is a generated an M. smegmatis Ms5284-5285 double
lysosomal localized protein, we wanted to address deletion mutant (DKO) and performed total lipid
if ARL8B is involved in LD-lysosome interaction. In profiling using thin layer chromatography. An
ARL8B silenced THP1 macrophages we found that unknown polar lipid species was found to be
LD-lysosome interaction was decreased. ARL8B significantly reduced in abundance in the DKO
silencing is known to prevent anterograde motility strain and this phenotype could be rescued by the
of lysosomes resulting in lysosome restriction to expression of Ms5284. We used multiple reaction
the perinuclear region. To understand if the monitoring to monitor abundance of
reduced interaction events were due to this phospholipids and triglycerides and found that
differential lysosomal localization, we made use of several species of PE and PS were increased in the
ARL8B mutants. We are currently evaluating the DKO and most features could be rescued by the
role of the ARL8B variants in mediating LD- expression of Ms5284 alone while others were
lysosome interaction and the downstream effects also contributed by expression of Ms5285. The
of these inter-organelle interactions in lipid identity of the unknown lipid species is currently
mobilization. In the future we will be studying the being investigated. To address if the Mtb
role of LD-localized ARL8B in lipid mobilization orthologues are indeed functional orthologues,
during Mtb infection and the innate immune we expressed Rv1062 and Rv1063c in the DKO
response to infection. strain and found that Rv1063c is the functional
orthologue of Ms5284. Deletion of Rv1062 and
Mycobacterial patatins: regulators of phospholipid Rv1063c in Mtb was carried out leading to the
homeostasis same loss of the unknown polar lipid species. This
Mtb is endowed with an unusually large repertoire phenotype despite additional six patatins being
of genes involved in lipid metabolism. This feature expressed in Mtb indicates that Mtb patatins play
is evolutionarily fitting in the light of its encounter non-redundant roles in phospholipid homeostasis.
with a lipid rich pathology generated by the We plan to investigate the role of these changes in
pathogens. While genes involved in synthesis of Mtb virulence using cellular and animal models of
complex lipids has garnered interest in the past, Mtb infection

Preprints

Inhibition of granuloma triglyceride synthesis imparts control of Mycobacterium tuberculosis through curtailed
inflammatory responses. Stanzin Dawa, Dilip Menon, Prabhakar Arumugam, Akash Kumar Bhaskar, Moumita Mondal,
Vivek Rao, Sheetal Gandotra bioRxiv 2021.05.10.443218

Annual Report 2020-21 129