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Projects arrow Killer Proteins

G-595

Analysis of Phage Specific Potentially Lethal Genes and Investigation of Antibacterial Activity of" Killer" Proteins

Project Status: 8 Project completed
Commencement Date: 01.10.2001
Duration in months: 42 months

Objective

Studying of any new means of struggle against infectious diseases, especially antibiotic-resistant bacterial infections is of great importance for many scientists worldwide. Rapid spread of multiresistant bacterial strains limits the effectiveness of antibiotic treatment. Phage therapy appears to be an alternative way of treatment. During the years bacteriophages have been used for diagnostics, prophylaxis and treatment of infectious diseases in the entire former Soviet Union. However, in some cases the usage of phage therapy is connected with some difficulties. Sometimes, baseless fear to treat infectious diseases by bacterial virus limits it application. Investigation of general mechanisms of interaction between phage and host bacterial cell exploited phage specific "killer" proteins. Identification of such proteins, their isolation and purification could provide an efficient strategy against various infections.

A huge collection of pathogenic and conditionally-pathogenic bacterial strains and virulent phages active against them exists in the Institute. Morphology, biological, serological, physical and chemical characteristics of several therapeutic phages were studied. General mechanisms of phage-host bacterial cell interaction were investigated in some phages, such as IRA (Salmonella typhimurium), PhI-1 and PhI-5 (E.coli. Shigella), Sb-1 (Staphylococcus aureus), fST1 (Pseudomonas aeruginosa).

Many phage and bacterial-coded proteins are involved in the process of phage infection. These include restriction-modification enzymes and phage-specific proteins protecting phage genome from cellular restriction, as well as many specific mechanisms to redirect cellular machinery to stop making host DNA, RNA and protein and become an efficient factory for making phage. Investigation of mechanisms of either host cell defense or the ones used by phages to inhibit bacterial cells could be important for development of new antibacterial phage preparations, as well as improvement of quality and effectiveness of existing ones.

The goal of our research is determination of virus-specific host takeover mechanisms, identification of “killer” proteins and elaboration of the methods for their identification and isolation. The objects of our investigation are IRA and Sb-1 phages. Phage-specific "killer" activities in infected bacterial cells will be studied. Potentially lethal genes from Sb-1 phage genome will be cloned in special vector system. Nucleotide sequence of the "host-takeover" modules in IRA and Sb-1 phage genomes will be analyzed. Sequences for efficient protein expression will be designed. Alterations occurred in transformed bacterial cells caused by the expression of cloned phage genes will be investigated. The effect of expressed cloned phage genes on host replication, RNA and protein synthesis and DNA integrity will be studied. The methods of "killer" proteins' identification, isolation and purification will be elaborated. Antibacterial activity of expressed proteins against various pathogenic and conditionally pathogenic bacterial cells will be investigated.

Standard, as well as modern genetic and molecular biological methodology such as cloning, transformation, sequencing etc. will be used for fulfillment of the project.

Determination of new phage gene product toxic to bacteria, will give the possibility to construct new bactericidal substances. Identification of new targets for such "antibiotics" and definition of the mechanisms by which these targets can be attacked will be conducted to create new remedies against various infectious diseases.

This work is planned to carry out in collaboration with Dr. Elizabeth Kutter (Evergreen State College, Olympia, Washington) and Dr.Charles Stewart (Department of Biochemistry and Cell Biology, Rice University, Houston, Texas). Dr. John Drake from National Institute of Environmental Health Sciences is US counterpart scientist.