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Antipneumococcal Properties of Killer Toxin

#G-1651


The Antipneumococcal Activity of K9 Killer Toxin and its Effect on the Alterations Provoked by S. pneumoniae in the Gene Expression, Brain-Blood Barrier Permeability, Immune System and Neuro-immune Interactions of Mice

Tech Area / Field

  • BIO-MIB/Microbiology/Biotechnology

Status
3 Approved without Funding

Registration date
29.05.2008

Leading Institute
Ministry of Education and Science of Georgia / Beritashvili Institute of Physiology, Georgia, Tbilisi

Collaborators

  • University of Abertay Dundee, UK, Scotland, Dundee

Project summary

The specific aims of this Project are: (i) to investigate the antipneumococcal mechanism of action of K9 killer toxin produced from Hansenula mrakii and (ii) to clarify, if this toxin has positive effect on the alterations provoked by Streptococcus pneumoniae on gene expression, brain barrier permeability, immune system and neuro-immune interactions in mice. These objectives are supported by our research of K9 killer toxin antibacterial properties demonstrated that this toxin is active against some Gram-positive bacteria. The aims are within the expertise of principal investigators.

The emergence and rapid dissemination of infectious agents resistant to antibiotic treatments have emphasized the need for alternative chemotherapies. Among the new antimicrobial molecules under investigation, yeast killer toxins represent promising candidates because of wide spectrum of activity. Some of them are capable to kill sensitive cells of their species and those of other species.

The European Antimicrobial Resistance Surveillance System database contains antimicrobial resistance data on approximately 260,000 invasive isolates of five species of indicator bacteria. Streptococcus pneumoniae is one of them.

Provoked by Streptococcus pneumoniae, streptococcal meningitis is one of the most frequent causes of community-acquired bacterial meningitis in adults. In spite of continuing development of new antibiotics, the mortality rate of S. pneumoniae remains high. Besides, streptococcal meningitis, as a severe inflammatory disease of the central nervous and immune systems, leads to various long-term neurological and immunological sequelae. The need for new preparations against streptococcal meningitis is actual.

In our previous study - together with scientists from Abertay University of Dundee (Scotland) - we have investigated antibacterial activity of K9 killer toxin from Hansenula mrakii, using flow cytometry with a highly sensitive cell viability fluorescent marker - DisBAC 2(3). It was demonstrated that the K9 killer toxin is active against Gram-positive bacteria (Bacillus subtilis; Streptococcus griseus, Streptococcus pyogenes, Staphylococcus epidermidis). Our brief study has highlighted the antimicrobial potential of K9 yeast killer toxin but further research is required to evaluate this potential. Now, in in vitro and in vivo level, we plan to study the K9 killer toxin pharmaco-kinetic and pharmaco-dynamic properties, mechanism of antipneumococcal action and its possible effect on some alterations provoked by S. pneumoinae on gene expression, brain blood barrier permeability, immune system and neuro-immune interactions in mice.

Our working hypothesis is: K9 killer toxin produced from Hansenula mrakii possesses antipneumococcal properties and it will have positive effect on the changes provoked by S. pneumoniae on gene expression, blood-brain barrier permeability, immune system and neuro-immune system of mice. As a control for K9 toxin, pneumococcal bacteriophage will be specially isolated and used.

For testing this hypothesis, the following tasks will be performed


Task 1. Clarification of mechanism of action of K9 killer toxin and pneumococcal bacteriophage. Especially: (i) Production, isolation and purification of K9 killer toxin, (ii) Isolation and propagation of pneumococcal bacteriophage, (iii) Study of antipneumococcal activity of K9 killer toxin and (iv) Study of mechanism of antipneumococcal action of K9 killer toxin and pneumococcal bacteriophage will be performed.
Task 2. (i) Determination of the dosage of (i) S. pneumoniae required for provoking streptococcal meningitis in mice. Determination of the dosages of (ii) K9 killer toxin and (iii) pneumococcal bacteriophage needed for the treatment of streptococcal meningitis in mice.
Task 3. Electron-microscopic study of the structure of BBB (microvascular endothelial cells, gap/tight-junctions, surrounding astrocytes) in mice: (i) infected with S. pneumoniae; (ii) infected with S. pneumoniae and treated with K9 killer toxin; (iii) infected with S. pneumoniae and treated with pneumococcal bacteriophage; (iv) two groups of intact mice, one treated with K9 killer toxin and second - with pneumococcal bacteriophage. Any quantitative and qualitative changes will be described.
Task 4. The study of immune system activation in (i) mice infected with S. pneumoniae; (ii) mice infected with S. pneumoniae and treated with K9 killer toxin; (iii) mice infected with S. pneumoniae and treated with pneumococcal bacteriophage; (iv)) two groups of intact mice, one treated with K9 killer toxin and second - with pneumococcal bacteriophage. Especially, in all animals: (1) the level of IL-1 and TNF-alpha in blood plasma, (2) the quantitative analysis of IL-I and TNF-alpha-containing cells in the hippocampus and prefrontal cortex – brain regions preferentially damaged after streptococcal meningitis, (3) immunohystochemical analysis of activated astrocytes (prostaglandin-expressed types) in the hippocampus and prefrontal cortex and (4) immunohystochemical analysis of activated microglia (Prostaglandine-E receptors expressing cells) in the hippocampus and prefrontal cortex will be performed.
Task 5. Analysis of differential gene expression by DNA microarray analysis in the hippocampus of mice i) infected with S. pneumoniae; (ii) infected with S. pneumonieae and treated with K9 killer toxin; (iii) infected with S. pneumonieae and treated with pneumococcal bacteriophage; (iv) two groups of intact mice, one treated with K9 killer toxin and second - with pneumococcal bacteriophage.

All participants of the project have high experience in the neuroscience, molecular biology, microbiology, phagotherapy. More than 50% of researchers are weapon scientists. All results of the research will be unique.

If antipneumococcal properties of K9 killer toxin will be well documented, it will be possible to consider this research as an important step for a new antibacterial treatment preparation: production of yeast killer toxin seems to be more quickly and economic method than isolation and purification of specific bacteriophages; moreover the raise of microbial resistance using the killer toxins is not yet documented.

In addition to the detailed analysis K9 killer toxin antibacterial properties, our research will extend existing knowledge about alterations provoked by streptococcal meningitis in the central nervous and immune systems.

The role of collaborators will be maximal. Especially, the part of investigation (tasks 2, 3) will be performed in the Abertay University of Dundee, Scotland, in the collaborators lab, with their materials, and other kind of non-financial support (it is indicated in the letter of collaborator).

The multidisciplinary approach and the most recent methods of research will be used.


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