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|Title:||MOLECULAR AND BIOCHEMICAL STUDIES ON ANTHRAX LETHAL TOXIN|
|Authors:||Swain, Prabodha Kumar|
|Keywords:||MOLECULAR;BIOCHEMICAL STUDIES;ANTHRAX;LETHAL TOXIN|
|Abstract:||Anthrax lethal toxin consists of two proteins, protective antigen (PA), and lethal factor (LF). These proteins are individually non-toxic. However, combination of PA and LF is toxic to animals and some macrophage cell lines and is known as lethal toxin (LT). In the process of cytotoxicity, PA (binding moiety) binds to the cell surface receptors and facilitates the internalization of LF (catalytic moiety) into the cytosol for its activity. In the present study, LF gene was subcloned, expressed and purified from E. coli. The biological activity of E. coli expressed LF was comparable to LF purified from B. anthracis. LF is the cytotoxic molecule of the toxin, because when PA and LF alone or in combination were internalized into the cytosol through virosomes, LF or PA + LF were toxic to the macrophage cells. Like other bacterial toxins, which require intracellular processing and activation for their biological activity, PA of LT is proteolytically cleaved for internalization and translocation of LF into the cytosol. No proteolytic activation or intracellular processing of LF was required for its biological activity. Intracellular distribution studies using 125I-LF indicated that large fraction of LF remains associated with lysosome and cytosol. Studies using brefeldin A suggested that golgi has no role to play in the activity of LT. Inhibition of protein synthesis was not the primary event in the toxicity of LT as reported for several bacterial protein toxins. However, inhibition of host cell protein synthesis by cycloheximide provided protection to the macrophages against LT challenge. These results suggest that host cell factors are essential for the cytotoxicity of LT. In the process of toxicity Ca++ influx precedes the cytolysis of the macrophages. LT enhances the inositol phosphate production in the cell, which could be responsible for the increased influx of Ca++ and cell lysis. Studies on the structure of the toxin proteins suggest that PA is predominantly a /?-class protein, whereas LF has both a-helix and /J-sheets in its secondary structure. Spectrofluorometric studies reveals that LF exposes hydrophobic residues at acidic pH, which may facilitate the translocation of the protein into the cytosol. Stoichiometric studies suggest that PA and LF combine in an equimolar ratio. These studies will be helpful in elucidating the mechanism of action of anthrax lethal toxin and could be useful in designing and developing new therapies against the disease.|
|Research Supervisor/ Guide:||Singh, Yogendra|
Singh, Rajesh P.
|Appears in Collections:||DOCTORAL THESES (Bio.)|
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