Characterization of Vacuolating Cytotoxin A of Helicobacter pylori

George, Meekha (2017) Characterization of Vacuolating Cytotoxin A of Helicobacter pylori. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Meekha George (12MS054)) Meekha_George.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Helicobacter pylori is a gram-negative, microaerophilic bacterium usually found attached to the lining of the stomach and small intestine of humans. The bacterium was identified in 1982 by Australian scientists Barry Marshall and Robin Warren. H. pylori infection occurs when the bacteria start to release several toxins that can damage mucosal lining of the stomach and gastric epithelial cells which ultimately leads to the occurrence of diseases such as peptic ulcer, duodenal ulcer, and even gastric cancer if left untreated for a long period of time. One of the key proteins identified which are responsible for H. pylori pathogenesis is Vacuolating cytotoxin A (VacA). VacA is synthesized as 140 kDa precursor protein with an N-terminal signaling sequence, 88 kDa passenger domain, and an autotransporter domain. The 88 kDa passenger domain that comprises of p33 and p55 subunits function as mature cytotoxin. It has been already found that treatment of gastric epithelial cells with VacA induces significant loss of actin stress fiber density but the exact molecular mechanism by which VacA disrupts actin cytoskeleton architecture is not known yet. Sequence alignment studies showed the presence of probable actin binding site in p33, p55 and autotransporter domain as well. So in this study, we tried to characterize p33 and p55 subunits separately to investigate the effects these subunits on actin cytoskeleton dynamics in vitro. The results of our experiments showed that both the subunits can bind to actin cytoskeleton purified from rabbit muscle (F-actin) in vitro. Fluorimetric or pyrene fluorescence assay (monomer sequestration/actin polymerization assay) with pyrene-labeled actin showed that both the subunits are capable of sequestering G-actin monomer and inhibits actin polymerization. Even at a very low concentration of p55, actin polymerization was found to have inhibited. This might be due to the presence of multiple actin binding domains in the protein or it might cap the ends F-actin filaments and inhibits further addition of monomeric G-actin. The results of this study provide a new insight into the mechanism by which vacuolating cytotoxin disrupts actin cytoskeleton integrity in H. pyloi infected gastric epithelial cells.

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