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Projects / Programmes source: ARIS

Molecular Mechanisms of Listeriolysin Toxicity

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   

Code Science Field
P310  Natural sciences and mathematics  Proteins, enzymology 
P340  Natural sciences and mathematics  Lipids, steroids, membranes 
B110  Biomedical sciences  Bioinformatics, medical informatics, biomathematics biometrics 
B120  Biomedical sciences  Molecular biophysics 
Keywords
Listeria monocytogenes, Listeriosis, listeriolysin, lipid membranes, pH dependence, cholesterol, site-directed mutagenesis
Evaluation (rules)
source: COBISS
Researchers (5)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  15686  PhD Gregor Anderluh  Biochemistry and molecular biology  Head  2007 - 2009  980 
2.  27751  Andrej Bavdek  Biochemistry and molecular biology  Researcher  2007 - 2009  30 
3.  30203  Aleksander Kocuvan  Microbiology and immunology  Researcher  2009  28 
4.  06994  PhD Peter Maček  Biochemistry and molecular biology  Researcher  2007 - 2009  523 
5.  12278  PhD Maja Rupnik  Microbiology and immunology  Researcher  2007 - 2009  689 
Organisations (2)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  67,269 
2.  2334  University of Maribor, Faculty of Medicine  Maribor  5089638048  15,954 
Abstract
Natural toxins are extremely important molecules for the interaction of organisms with their environment. The understanding of how these molecules act at the molecular level is important in medicine, health, basic and applied science. In this project we plan to study certain aspects of the molecular mechanism of action of listeriolysin O (LLO), a bacterial toxins that affects the integrity of cell lipid membranes. LLO is the most important virulence factor of the intracellular Gram-positive bacteria Listeria monocytogenes, which is a causative agent of listeriosis, a serious food borne disease. Its main task is to enable escape of bacteria from the phagosomal vacuole to the cytoplasm. LLO belongs to a cholesterol-dependant cytolysin family, but is different from other members, as it needs low pH for optimal activity. We plan to address its cholesterol and pH dependence by a range of biochemical and biophysical techniques and by using wild-type LLO and its variants produced as recombinant proteins in Escherichia coli. The main methodological approach will be a surface plasmon resonance, which is important biochemical technique not yet fully explored in protein-membrane interactions. We expect to describe the cholesterol and pH dependence of LLO at the molecular level, i.e. which parts of the molecule and which amino acid residues promote these two important features of LLO. This knowledge will have important implications for understanding of listeriosis pathogenicity, protein recognition of particular membrane lipids, in general, and will provide a firm basis for the development of therapeutics and molecular tools used in cell biology.
Significance for science
Listeriolysin is the main virulence factor of Listeria monocytogenes, a pathogenic bacteria, which causes listeriosis. Listeriolysin belongs to a big protein family of cholesterol dependant cytolysins. These are powerfull toxins that are able to generate large pores in the membranes of target cells. Listeriolysin is different from other cholesterol dependant cytolysins as it works the best at low pH, which enable bacteria to escape from phagolysosome to the cytosol of the cell, where it can multiply and escape to other cells. This research project enabled us to describe pH dependence of listeriolysin and how is its activity regulated in the cytosol of the host cell. We show that pH is not affecting much the ability to form transmembrane pores, since hemolytic activity and pore formation in planar lipid membranes is retained at phyiological pH of 7.4. Listeriolysin inactivates only when it is incubated at higher temperatures, above 37 °C, while at low pH it does not aggregate. Aggregation of listeriolysin as described in this project is the main mechanism for inactivation in the cytosol of the host cell. With structural methods we show that listeriolysin is a dimer at low pH, which has probably an important role in preventing aggregation at this pH. We did not observe dimers at high pH. We also developed a screening test for inhibitors of listeriolysin aggregation, which will be used in future studies. We plan to search for small molecules that will be able to block listeriolysin aggregation specifically. The research project also enabled us to describe parts of the listeriolysin molecule important for the protein function, particularly some amino acids that participare in membrane binding and hemolytic activity were determined. This study also shows that it is possible to use members of cholesterol dependant cytolysins as molecular tools in cell biology. By the surface plasmon resonance approach we show that they can be used to sense the amount of cholesterol in membranes. It will be possible to study the distribution of proteins that participate in raft binding and how the lipid composition of membranes affect the exposure of cholesterol and its binding by proteins.
Significance for the country
The applicative value of this research project for Republic of Slovenia was multiple. It enabled us to educate and train researchers, which employed modern biophysical and biochemical methodology. Both undergraduate and postgraduate students participate in the project, which employed the technology that is available in the laboratory of the prinicipal investigator and collaborators from other laboratories. We were also able to develop some new approaches in the biochemistry of the lipid membranes. Particularly, we have developed the surface plasmon resonance as a method for monitoring interaction of proteins with lipid membranes, which render Slovenian science more competitive. The approach is relatively new to the science and not many groups explore it fully. The group of principal investigator is becoming very experienced in protein-lipid interaction studies by using SPR, which is also highlighted by invitations to meetings and preparation of review papers and book chapters. With publications in highly cited international scientific jornals and with lectures at academic institutions and research conferences the research group definitely participate in making slovanian science more visible, particularly in the field of biophysical approaches in basic sciences. This was a basic research projects, but it has some practical applications. It is possible that knowledge obtained within this project will be used to design and develop novel therapeutic approaches, as LLO is a major virulence factor of Listeria monocytogenes. The description of the membrane binding site will enable to design markers for cholesterol that may be used in cell biology to label lipid rafts. Traditionally cholera toxin is used, but it has certain deficiencies that may be overcomed by engineered cholesterol binding domain. The methodology and procedures that were developed within the project may be immediately used by other researchers in academia and pharmaceutical companies. Particularly, surface plasmon resonance methodology is interesting for a wider scientific community in Slovenia and in other neighbouring countries.
Most important scientific results Annual report 2008, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2008, final report, complete report on dLib.si
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