Regularities in Burkholderia Genus
Extended Experimental Search for Genomic Differences between B. Pseudomallei and B.mallei Species and Their Variants for Investigation of Pathogenicity Mechanisms and Development of Efficient Typing Tools in Cases of Melioidosis and Glanders
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Institute of Bioorganic Chemistry, Russia, Moscow
- Midwest Research Institute, USA, FL, Palm Bay
Project summaryBurkholderia pseudomallei, Burkholderia mallei, and Burkholderia thailandensis are three closely related species of Gram-negative bacteria. The first two are causative agents of melioidosis and glanders, respectively, severe diseases of human and animals, whereas the third, being a close relative of the first two, exhibits a low virulence towards sensitive laboratory animals. B. pseudomallei and B. mallei are members of categories of most severe human pathogens in national lists of biological agents of Russia, USA, Canada and Great Britain. In particular, both organisms have been considered as potential agents of bioterrorism.
Despite the danger, carried by these microorganisms, which even increases due to their relative resistance to antibiotics, up to now there are no concepts concerning molecular mechanisms responsible for their pathogenicity. In particular, the question is still open, why closely related genetically and immunologically B. pseudomallei and B. mallei are highly different both from each other and from non-pathogenic B. thailandensis from the epidemiological and zoonotic considerations.
Moreover, despite numerous investigations, the question, concerning the extent, causes, and physiological consequences of natural variability of these species, up to an uncertainty in taxonomic differentiation of B. pseudomallei and B. mallei. While it is generally accepted that although B. thailandensis is a very close, but nevertheless a separate species, close genetic and antigenic relationship of B. pseudomallei and B. mallei encourage some authors to consider B. mallei as a variant of B. pseudomallei. To give just one example, multilocus sequence typing (MLST) of 128 isolates of B. pseudomallei the authors grouped them into 71 sequence types thus demonstrating the extreme genetic variability of the B. pseudomallei species. The B. pseudomallei isolates were clearly distinct from the cluster of B. thailandensis isolates. However, the B. mallei isolates formed cluster within the B. pseudomallei group thus providing evidence, on population genetics ground, that B. mallei is a genetic variant of B. pseudomallei. Nevertheless, with the account of morphological, ecological, and epidemiological differences between the causative agents of melioidosis and glanders, the distinguishing of B. mallei as an independent species is generally accepted.
As to the variability of B. mallei, at least two different groups can be distinguished on the basis of antigenic properties. A high degree of the B. pseudomallei variability and overlapping of genetic and antigenic properties of B. pseudomallei and B. mallei raise serious problems in identification of infectious agents. In addition variability of pathogenic characteristics of B. pseudomallei strains within the species leads to strong difficulties in identification of the pathogen virulence factors.
In practice, the above-mentioned unsolved fundamental problems result in the fact that leading factors of pathogenicity of B. pseudomallei and B. mallei remain elusive, vaccines for active prophylaxis of glanders and melioidosis are absent, and identification of these infections is still a labor- and time-consuming task. The most rapid solution of both the above-mentioned fundamental and applied problems requires systematic investigations aimed at solution of the following problems:
· objective systematization of organisms under discussion on the basis of similarity and differences in genomic sequences and expression products, as well as revealing evolutionary and population regularities in Burkholderia genus;
· correlation of genome and expression profiles variability with phenotypic characteristics, in particular, with peculiarities of pathogenesis;
· identification of genetic factors responsible for different biological properties of Burkholderia strains by a comparison of genomic and expression product differences of B. pseudomallei, B. mallei and B. thailandensis strains;
· deciphering molecular mechanisms of pathogenesis by combination of genetic, immunological, and biochemical data with results of investigations of pathogenic properties on model animals.
An important stimulus of such systematic work is expected from deciphering and comparison of full-genome sequences, which has been already finished for B. pseudomallei K96243 and is close to completion for B. mallei ATCC 23344. However, the sequences obtained will give information only about two strains out of a great variety of natural populations, thus being unable to produce the comprehensive picture of correlation between variability in genome structure and phenotype features of B. pseudomallei and B. mallei species. It is evident that to reach this last goal one needs efficient cost and time saving nonsequencing technologies which would use the full genome sequences as necessary standard, suitable for comparison of rest strains and isolates, to draw a conclusion concerning the genome variability, which then could be correlated with the variability of pathogenic and other phenotypic properties.
The objective of this project is identification and analysis of genomic differences between B. pseudomallei and B. mallei that might be responsible for species characteristics of the causative agent of melioidosis, as well as of intraspecies differences between B. pseudomallei variants for objective systematization of the groups belonging to this species.
The work under project will include four main steps:
1. Obtaining and analysis of differential libraries, containing genetic material present in genomes of different strains of B. pseudomallei, rather than B. mallei.
2. Classification of the various B. pseudomallei strains in the related genetic groups using hybridization techniques and PCR.
3. Hierarchic clustering of the B. pseudomallei strain collection using array technique.
4. Analysis of a correlation between the differences among revealed genetic groups of B. pseudomallei strains and their biological features in animal experiments.
At the first step comparative analysis of B. pseudomallei and B. mallei genomes will be carried out using the subtractive hybridization technique. A prerequisite for success of this project is the optimal selection of strains for subtraction. To do this, typing of the melioidosis pathogen strain collection will be performed using ribotyping and RAPD. The typing results will be used for selection of Asian and Australian B. pseudomallei strains that form different ribotypes and RAPD groups. The subtractive clone libraries will be designed which will contain the multitude of genetic differences between the B. pseudomallei and B. mallei genomes. Thus, the first step of the work will result in obtaining bank of base sequences, present in genomes of different groups of B. pseudomallei, but absent from genome of B. mallei.
At the second step the main task will be identification and analysis of B. pseudomallei-specific DNA regions. For this aim, specific subtractive clones, which are present in certain groups of B. pseudomallei strains, but absent from the others, will be analyzed.
Data on the subtractive clone group- and species-specificity will be obtained in the course of analysis of the B. pseudomallei, B. thailandensis and B. mallei strain collections by dot- and Southern-blotting. The comparison of the species- and group-specific sequences with known gene and protein databases will allow to put forward hypotheses on biochemical functions of differential clones. The difference in sets of functional components will make possible the evaluation of a possible role of cloned sequences in differences of phenotypic characteristics of B. pseudomallei and B. mallei. The group- and species-specific subtractive clones will be mapped on the B. pseudomallei genome. The sequence position on the map in the vicinity of certain genes will, in turn, allow one to draw additional conclusions on the functional role of differential clones.
In case clusters of group- and species-specific differences are detected the corresponding genomic regions of B. pseudomallei will be analyzed in more detail from point of view of their possible functional properties. A set of primers will be designed for determination of group- and species-specificity of B. mallei and B. pseudomallei strains.
At the third step the B. pseudomallei group-specific subtractive clones will be fixed on solid supports and hybridized with labeled genomic DNA of different strains of B. pseudomallei. The resulting hybridization profiles will be used for cluster analysis to show genetic relationship between B. pseudomallei strains. A dendrogram that shows phylogenetic structure of the species will be constructed. The obtained data will be eventually used for development of DNA microchips for rapid detection and identification of melioidosis pathogen.
At the fourth step, biological properties of B. pseudomallei strains, included in different gene groups, as described in preceding paragraphs, will be estimated in animals experiments, and the existence of correlation between gene groups of B. pseudomallei strains their phenotypic properties will be studied.
Main expected results of the project:
· differential clone libraries, containing the totality of genetic material, that differs the B. pseudomallei genome from that of B. mallei;
· characterization of the B. pseudomallei and B. mallei strain collections by ribotyping and RAPD techniques;
· identification of functional sites present in genomes of different groups of B. pseudomallei strains, rather than in B. mallei genome;
· hierarchic clustering of the B. pseudomallei strain collection using array hybridization technique.
· constructing of microchips for rapid detection and identification of genetic groups of B. pseudomallei strains.
Data, obtained under this project, will allow one to create a new system for genotyping the B. pseudomallei strains, which will be based on presence/absence of specific genetic structures in the genome of B. pseudomallei clone under investigation.
The identification of functional group-specific sites, inherent in specific genetic variants of B. pseudomallei, will form a basis for future investigation of molecular mechanisms of infection.
For realization of the project we shall use the results of the Partner ISTC Project #1178 “Cloning of Burkholderia pseudomallei specific nucleotide sequences useful for the development of test-system” that was successfully performed in laboratories of the State Research Center of Applied Microbiology (SRCAM) and Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (IBC).
Researchers of SRCAM have an essential experience in genetic investigations of bacterial pathogens of genus Burkholderia. The novel subtraction methods, developed and optimized for analysis of bacterial genomes by scientists of IBC, will be used in the work on this project.
The expected role of the foreign collaborator of the Project Dr. Hadfield (The Armed Forces Institute of Pathology, Washington, USA), who is an expert in the field of genetic investigations of B. pseudomallei and B. mallei will be the participation in joint working seminars, result discussion and participation in planning of experiments.