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Expression Profiling of Yersinia Pestis

#2528


Expression Profiling and Study of Molecular Evolution of Hazardous Microbial Pathogen Yersinia Pestis Using High-Density Oligonucleotide Microarrays

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-INF/Bioinformatics/Biotechnology

Status
3 Approved without Funding

Registration date
04.06.2002

Leading Institute
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk

Supporting institutes

  • Institute of Bioorganic Chemistry, Russia, Moscow

Collaborators

  • University of Kentucky / Department of Microbiology and Immunology, USA, KY, Lexington

Project summary

Yersinia pestis, etiologic agent of plague is, in many senses, remarkable pathogen. First, the plague, one of the most lethiferous bacterial diseases caused devastating pandemics, which took more that 200 millions lives. According to the WHO, despite the availability of a number of highly effective therapeutic agents, mortality due to plague still remains unacceptably high. Second, Y. pestis is now believed to be extremely young species emerged as a clone of Y. pseudotuberculosis less than 20 thousands years ago [1, 2]. Actual reasons of Y. pestis evolution are unknown, but it is believed that it accompanied transition of human population to the settled lifestyle. It is likely, that the pathogen continues to evolve rapidly since comparison with Y. pseudotuberculosis reveals much of recently altered or silenced genes [3]. Finally, during very short evolution, Y. pestis acquired the ability to infect fleas, the insect vector, which transmits the pathogen between rodents in the natural plague foci.

Globalization changes the world continuously. While overall number of plague cases declined dramatically since the first half of XX century, now clear trend of re-emergence of plague in Africa has been established according to the WHO data. Changes in geographic distribution of plague are accompanied by genetic changes in Y. pestis and subsequent emergence of new hazardous biovars [4]. An instructive example of evolutionary plasticity of Y. pestis is provided by isolation of multidrug-resistant strain in Madagascar [5]. It has been further noted that Y. pestis continued to acquire antibiotic resistance [6]. Acquisition of new plasmids is easily detectable, but is known about new genomic variants of the pathogen. Overall, these data led to consider Y. pestis as the re-emerging pathogen. Given the evolutionary adolescence of plague, consequences of such re-emergence are difficult to predict.

It is still much puzzling in mechanisms of Y. pestis pathogenicity. In many cases genes thought to be responsible for pathogenicity exclusively in Y. pestis are present in Y. pseudotuberculosis, in other yersiniae, and even in yet another pathogens like enterohaemorrhagic E. coli strain O157:H7 [7] or Salmonella typhi [8]. The latter strongly suggests complex pattern of evolution of pathogenicity in Y. pestis, which includes both deviation from the ancestor, Y. pseudotuberculosis and horizontal transfer from other bacteria. Thus, no convincing evidence obtained regarding the unique pathogenicity determinants that orchestrate the dreadful pathological profile of the plague.

A sizable share of plague decline in XX century was due to development of vaccines. However, currently available vaccines do not confer protection against the pneumonic form of plague, and are reactogenic [9]. Vaccine strains of Y. pestis are not exhaustively characterized from molecular genetic point of view. This and the absence of precise information about unique virulence determinants of Y. pestis make impossible preparation of safe and efficient vaccine strains or recombinant subunit vaccines.

Study of Y. pestis variability and pathogenicity appeared to be challenging for conventional molecular genetic approaches. We suggest that the genome- and transcriptome-wide analyses of the pathogen will be helpful in solving of at least some of the mentioned problems. Sequencing of the whole genome of Y. pestis biovar Orientalis is already completed [10] and its annotated version is expected to be released in autumn of 2001. Microarray-based studies already yielded invaluable information about genome structure and expression in wide variety of organisms from bacteria to mammals [12, 13].

We propose that construction of a microarray containing oligonucleotides complementary to all genes of Y. pestis and subsequent expression profiling of the pathogen in various conditions will help to delineate major genetic traits determining high virulence of the bacteria. Alterations in transcription profiles after growing the Y. pestis in the presence of mammalian cells, increased concentration of iron, and elevated temperature will help to dissect the gene ensemble responsible for infection and growth of Y. pestis in the host mammal. Study of Y. pestis transcription in fleas will help to understand the mechanism of maintenance of the parasite in natural reservoirs. Comparative analysis of Y. pestis and ancestral Y. pseudotuberculosis genomes akin to that accomplished for M. tuberculosis and BCG [11] will provide detailed information about genetic differences in these bacteria and knowledge about the genetic traits acquired by Y. pestis from other bacteria via horizontal transfer. Differences between gene expression in virulent and vaccine strains can be also assessed, yielding the information about the insufficiencies in vaccine strains which lead to limited protection from infection of the wild-type bacteria.

The results of transcription profiling and genetic analysis of Y. pestis obtained with the aid of microarray technology will be summarized in annotated electronic database hosted at the public Web server. It will contain the information about changes in expression of the Y. pestis genes dependent on the changes in the environment. The database will be subpided into the appropriate regulons studied throughout the project. Whenever possible, the annotation of the genes within the database will include actual or proposed function, results of homology searches and fold prediction of the protein encoded by given mRNA. The database will have highly open architecture providing the option for rapid changes and additions of information arose from functional studies as well as from new array-based analyses. [A separate domain of the database will be devoted to description of genetic differences between various Yersiniae.] This domain is expected to be expanded and updated continuously basing on new sequencing data and on worldwide analysis of genetic persity and evolution of Y. pestis. This domain will serve as efficient tool for analysis of the ongoing evolution of Y. pestis.

Basing on the results of microarray analysis of Y. pestis, proposals regarding further directions of studying of the pathogen will be made with respect to development of new therapeutics and vaccination strategies. Completion of the Project and development of a Y. pestis microarray will permit setting up highly precise typing of Y. pestis strains throughout the world. On the other hand, analysis of Y.pestis interaction with the host cells would enable a reciprocal microarray-based analysis of the host cells response to the pathogen invasion. Overall, the data obtained in the course of the proposed research would be extremely helpful in prevention of the plague re-emergence and provide new valuable insights in the mechanisms of rapid evolution of pathogenic bacteria.

Microarray-aided analysis of Y. pestis genome and transcription profiles would have significant medical importance. The data obtained by microarray analysis of Y. pestis can be further used in a number of biomedical investigations:

1. Knowledge about unique virulence determinants can be obtained;


2. Study of the evolutionary pathway from mild (Y. pseudotuberculosis) to hazardous (Y. pestis) can be substantiated;
3. New vaccine strains or subunit vaccines of high efficiency can be constructed;
4. A global survey of current Y. pestis biovars can be initiated which would yield new information about variability and spreading of the pathogen, and potential hazard of newly emerging variants of the microorganism.


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