Biomedical subjects

Puneet Khandelwal

Publications and source records attributed to Puneet Khandelwal.

6 recordsLinked to original sources

PubMed2005

Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis.

Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with l-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of l-arginine, N(omega)-hydroxy-l-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein from a pathogenic bacterium opens up avenues for further studies in understanding the importance of this protein in pathogenicity.

Amino Acid Sequence↗

PubMed2004

The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis.

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.

Animals↗

PubMed2004

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.

Agglutination Tests↗

PubMed2004

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

Agglutination↗

PubMed2003

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus.

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.

Animals↗

PubMed2002

Expression of anthrax lethal factor gene by osmolyte induction.

The anthrax toxin consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA mediates the entry of LF and EF to the cytosol where they exert their effects. Although PA is the major component of the vaccines against anthrax, LF has also been found to play an important role in enhancing protective immunity. We have developed an osmolyte-inducible LF expression system. The protein expression system contributed no additional amino acids to the recombinant LF making it suitable for the human vaccine trials.

Anthrax↗