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

Impaired regeneration in LGMD2A supported by increased Pax7 positive satellite cell content and muscle specific microRNA dysregulation

Introduction—Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, upregulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods—We examined the histopathological stages, Pax7 SC content, and muscle specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis. Results—Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206. Conclusions—These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.This work was supported by NIH NIAMS U54 AR050733-05, Jesse’s Journey, and the muscular Dystrophy Associatio