Biochemical Characterization of VgrG-1 Toxin of Vibrio Cholerae and Leishmania Major Formins to Study Their Role in Actin Cytoskeleton Dynamics

Ali, Jijumon S. (2015) Biochemical Characterization of VgrG-1 Toxin of Vibrio Cholerae and Leishmania Major Formins to Study Their Role in Actin Cytoskeleton Dynamics. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Jjumon A.S. (10MS77)) JIJUMON_A.S_10_MS_77_MS_THESIS_2015.pdf - Submitted Version Restricted to Repository staff only Download (18MB)

Abstract

Actin is a 42.5 kDa protein which is highly conserved in all eukaryotes and has the ability to transit between monomeric G-actin and filamentous F-actin states under the control of nucleotide hydrolysis and large number of actin binding proteins. Actin participates in more protein-protein interactions than any other known protein with a wide range of functions including cytokinesis, cell motility, cell shape and polarity. With time many pathogens have acquired a capacity to exploit the cellular systems by altering normal actin cytoskeleton dynamics. From literature, we know Vibrio Cholerae induces rapid cell rounding in infected intestinal epithelial cells. This is due to destruction of intrinsic actin structure by crosslinking of cellular monomeric G-actin into dimers, trimers and higher multimers. Two toxins have been identified in V. cholerae that shows actin crosslinking activity – MARTX and VgrG-1 toxin with an Actin Crosslinking Domain (ACD). First aim of my thesis is to figure out the molecular mechanism behind the VgrG-1 toxin mediated actin crosslinking. The preliminary results of crosslinking and co sedimentation assays of wild VgrG-1 ACD shows for actin crosslinking VgrG-1 should bind to actin and we found VgrG-1 has a putative WH2 like domain (WASP-Homology 2, or Wiskott- Aldrich homology 2) an actin monomer-binding motif. In vitro co-sedimentation assay shows that putative WH2 fragment of wild VgrG-1 is able to bind the actin and Mutation of specific amino acid residues from the proposed WH2 like domain makes VgrG-1 fails to bind and crosslink the actin in vitro and in vivo. So our results conclude neither the G actin nor F actin can be the substrate for ACD and VgrG-1 crosslink actin by binding the substrates with the help of a WH2 like domain. Characterization of ACDs provides opportunities to design compounds targeting their active sites. Second aim of my thesis is to characterize two putative formins-Formin A and Formin B in Leishmania major. Formin proteins are potent actin nucleators binds to the actin filament's barbed end, modulating its elongation and protecting it from capping proteins and act as a processive cap that moves with the barbed end as it elongates. Our results confirm that L. major have two formins - formin A and Formin B, which is going to be the pioneer formins reporting in protozoans. Leishmania major formins are slow actin nucleator protein and capable to nucleate filamentous actins in a concentration dependent manner, able to elongate actin filaments but fails to replace the CapZ protein from barbed end.

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