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Caf1M and V-antigen of Yersinia pestis in crystal and solution

#2352


Study on the Structure and Conformational Properties of Molecular Periplasmatic Chaperone Caf1M and V-antigen of Yersinia pestis in Crystal and Solution as a Way for Construction of Antibacterial Drugs of a New Generation

Tech Area / Field

  • MED-DRG/Drug Discovery/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-SFS/Biosafety and BioSecurity/Biotechnology

Status
3 Approved without Funding

Registration date
01.11.2001

Leading Institute
Institute of Bioorganic Chemistry, Russia, Moscow

Supporting institutes

  • Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany

Collaborators

  • Universidad de Gradada, Instituto Andaluz de Ciencias, Spain, Granada\nFreie Universität Berlin, Germany, Berlin\nHauptman-Woodward Medical Research Institute, Inc., USA, NY, Buffalo\nUniversitaet zu Luebeck, Germany, Luebeck

Project summary

The aim of the project: Conducting of crystallization in microgravitation conditions and study on conformational properties of genetically engineered periplasmatic molecular chaperone Саf1М and V-antigen of Y. pestis as the base for constructing of antimicrobial drugs of a new generation and components of vaccine against yersinioses.

Yersinia pestis is the agent of plague – one of the most severe and dangerous infectious diseases. This zoonotic disease taking shelter almost in every continent manifests impressive ability to conquer host defense in mammals. Y. pestis effectively evades the inborn host stability and prevents development of the adequate adaptive immune response. Realization of Y. pestis pathogenic properties in the infected host organism is provided via regulation systems of co-ordinated expression of a whole set of factors for virulence and pathology of a various functional purposefulness. Y. pestis is an optional intracellular pathogen now considered as one being inside the host cell only at early stages of infection with predominant extracellular growth at the following stages. Yop-virulon of pathogenic bacteria of the Yersinia genus takes a special part among the virulence factors affecting intracellular defense. Plague bacteria use Yop-virulon to inject toxic effector proteins paralyzing macrophage functions into the host cell. Y. pestis ability to prevent effectively host phagocyte attack results in an unchecked propagation of the plague bacteria at early stages of the infection and, hereinafter, in defects of the adequate immune response formation. Reproduction of Y. pestis inside the macrophages is an obligatory stage of plague pathogenesis.

More than a hundred years Y. pestis is known to form large capsules covering bacteria. F1 protein subunits are the main capsule component. F1 (Caf1) capsular antigen is considered to be one of the pathogenicity factors of the plague microbe firstly for antiphagocytic properties detected in polymer forms of the capsular substance. Lately, adhesive function has been found in Y. pestis F1 (Caf1) capsular antigen in its low molecular forms. Caf1 specific interaction with the host cells is performed via interleukin-1 receptors (IL-1R). Detected competition for IL-1R between Caf1 and interleukin-1 is of extreme importance for adhesion and holding of Y. pestis bacteria on the host cell surface. Formation of a tight contact between bacterial outer membrane and cytoplasmatic one of the host cell results in maximal induction of biosynthesis of yersiniae virulence factors - Yop proteins and in the following formation of a translocational channel providing direct communication between the cytoplasms of bacteria and the host cell. Low molecular forms, most possibly, Caf1 dimer anchored on the Y. pestis cell surface by Caf1-A usher-protein participates in the adhesive function realization. Apparently, in conditions of the polymer capsule loss, plague bacteria enhance their adhesive ability obtaining a view of a highly virulent cultures grown at 26-280 С and having minimal Caf1 protein quantities. The mechanism of Y. pestis capsule formation has two key details which are characteristic of other pathogenic gram-negative bacteria. Periplasmatic molecular chaperones play the leading part in these mechanisms. Y.pestis Caf1M protein is the most typical representative of the chaperone superfamily. Сaf1M performs the most important functions of polypeptide homeostasis periplasmatic system. It aids regular spatial packing of Caf1 capsular antingen polypeptide chain immediately after its translocation via cytoplasmic membrane into periplasm, protects capsular antigen from the cleavage by proteases, prevents its untimely polymerization, transports Саf1 to the outer membrane and transfers it to Саf1A usher-protein. Expression of Y.pestis capsular subunit Caf1 serves as a mediator to the production of caf1M gene.

Universal for all the gram-negative pathogens mechanism of the interaction of chaperone with an adhesion subunit (for Y.pestis, the interaction of Caf1M and Caf1) could be neutralized, for example, using synthetic peptides corresponding to the sequences of the secreted virulence factors, forming adhesion organelles (phili, capsules, fimbriae, filaments) on the surfaces of pathogenicbacteria.


Expected results.

Thus, a new class of antimicrobial preparations could be constructed, aimed only against pathogenic microflora. And the data on the thin structure and molecular organization of the chaperones (in the case of the plague agent, it is Caf1M) is needed for the construction of such preparations.

At the beginning, the studies on virulence determinants of Y. pestis Yop-virulon were considered to be of a great importance in connection with determination of virulon molecular elements critical for its function. V-antigen (LcrV) plays one of the key parts in Yop-virulon systems in three pathogenic yersinia including Y. pseudotuberculosis and Y. enterocoiltica. V-antigen is a multifunctional secreted protein participating in regulation of Yop-virulon components biosynthesis. LcrV- mutants are avirulent. Immunization with V-antigen provides an active defense in laboratory animals against plague virulent bacteria. LcrV suppresses natural immune response by lowering cytokine production (-interferon, TNF-) and by neutrophil chemotaxis inhibition.

In connection with LcrV key role in yersinia virulence and protective immunity induction, obtaining information about its spatial structure and localization of the active centers aimed for the search of substances able to block selectively V- antigen functions and to provide yersiniosis treatment is seemed to be of topical interest.

For these purposes crystallization technology is supposed to be used in microgravity conditions substantially lowering deformations while crystal formation. It makes it possible to obtain crystals of the needed size of such proteins crystallization of which on earth is inconvenient or impossible. Obtaining of highly purified preparations of bacterial proteins such as Сaf1М and LcrV in crystals would permit to conduct X-ray analysis, to collect important data on protein spatial structure, to decode molecular mechanisms of yersiniae resistance to the host immunity and to determine ways for constructing of antimicrobial drugs and molecular vaccines of a new generation.

Auhors have had an arrangement with the Russian Space Agency on protein cryslallization in conditions of microgravitaion at the International Space Station «Alpha».


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