Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous <it>Mycobacterium tuberculosis </it>genes are being characterized. In this study we report the dual functionality of <it>tlyA </it>gene product of <it>Mycobacterium tuberculosis </it>annotated as Rv1694, a 268 amino acid long basic protein.</p> <p>Results</p> <p>The recombinant purified Rv1694 protein was found to exhibit hemolytic activity <it>in vitro</it>. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated <it>de novo </it>by <it>in vitro </it>transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of <it>E. coli </it>as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of <it>E. coli </it>expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, <it>E. coli </it>expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed <it>E. coli</it>. The S30 extract of <it>E. coli </it>expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [³H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity.</p> <p>Conclusions</p> <p>The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by <it>Mycobacterium tuberculosis</it>.</p

Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway

BACKGROUND: Wild type Staphylococcal alpha-hemolysin (alpha-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by alpha-HL are unknown till date. RESULTS: Cells treated with H35N (a mutant of alpha-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of alpha-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of 'kiss and run' dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1. CONCLUSIONS: This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal alpha-hemolysin

In Vitro Activities of New 2-Substituted Quinolines against Leishmania donovani▿

A series of 9 quinolines and 18 styrylquinolines was evaluated for the drugs' in vitro antileishmanial activities and cytotoxicities. The 7-aroylstyrylquinoline scaffold appeared to be the most promising one, with the most interesting compound, no. 35, exhibiting a 50% inhibitory concentration (IC50) of 1.2 μM and a selectivity index value of 121.5. Compound 35 was 10-fold and 8-fold more active than miltefosine and sitamaquine, the reference compounds, with selectivity indexes 607-fold and 60-fold higher, respectively

Abstracts of International Conference on Innovations in Business Management

This book contains abstracts of the various research ideas of the academic community and practitioners of management presented at the International Conference on Innovations in Business Management (ICIBM 2020). The researchers have contributed toward various themes of the conference such as sustainable economy, supply chain, women-empowerment, export-import, microfinance, government policies, etc. We strongly believe that it will open up further scope for in-depth research in various disciplines of business management. Best wishes to the participants to have detailed discussions on the above-said wide range of areas. Conference Title: International Conference on Innovations in Business ManagementConference Acronym: ICIBM 2020Conference Date: 16-17 January 2020Conference Location: ICFAI University, Dehradun, IndiaConference Organizers: ICFAI Business School, ICFAI University, Dehradun, India &amp; University of Derby, United Kingdo