Himanshu Pandey

NMR elucidation of monomer-dimer transition and conformational heterogeneity in histone-like DNA binding protein of Helicobacter pylori

Helicobacter pylori (H. pylori) colonizes under harsh acidic/oxidative stress conditions of human gastrointestinal tract and can survive there for infinitely longer durations of host life. The bacterium expresses several harbinger proteins to facilitate its persistent colonization under such conditions. One such protein in H. pylori is histone‐like DNA binding protein (Hup), which in its homo‐dimeric form binds to DNA to perform various DNA dependent cellular activities. Further, it also plays an important role in protecting the genomic DNA from oxidative stress and acidic denaturation. Legitimately, if the binding of Hup to DNA is suppressed, it will directly impact on the survival of the bacterium, thus making Hup a potential therapeutic target for developing new anti‐H. pylori agents. However, to inhibit the binding of Hup to DNA, it is necessary to gain detailed insights into the molecular and structural basis of Hup‐dimerization and its binding mechanism to DNA. As a first step in this direction, we report here the nuclear magnetic resonance (NMR) assignments and structural features of Hup at pH 6.0. The study revealed the occurrence of dynamic equilibrium between its monomer and dimer conformations. The dynamic equilibrium was found to shifting towards dimer both at low temperature and low pH; whereas DNA binding studies evidenced that the protein binds to DNA in its dimeric form. These preliminary investigations correlate very well with the diverse functionality of protein and will form the basis for future studies aiming to develop novel anti‐H. pylori agents employing structure‐based‐rational drug discovery approach.

Biophysical and immunological characterization of the ESX-4 system ESAT-6 family proteins Rv3444c and Rv3445c from Mycobacterium tuberculosis H37Rv

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freunds incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patients sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.