Protein Interaction between Pathogens and Host Cells.
Structural and functional Properties of Proteins Involved in the Interactions between Pathogenic Yersinia and Hst Cells, and Development of Approaches for their Application in Medical and Veterinary Practices
Tech Area / Field
- BIO-SFS/Biosafety and BioSecurity/Biotechnology
8 Project completed
Senior Project Manager
Melnikov V G
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- London School of Hygiene&Tropical Medicine, UK, London\nUniversity of California / Department of Chemistry and Biochemistry, USA, CA, Santa Cruz
Project summaryLately, cardinal changes have been observed in the understanding of molecular mechanisms of Gram-negative pathogenic bacteria virulence. These changes resulted, above all, from the discovery of the type III secretion system specialized in the secretion of virulence factors delivered into an eukaryotic cell. The system operates within Yersinia spp., Salmonella spp., Shigella spp. and many other animal and plant pathogens. A paradigm of the family of these related systems is the Yop virulon of Yersinia genus, which ensures direct communication between the bacterial cytoplasm and that of the target host cell during their close contact. The virulon is used by pathogenic Yersinia strains to inject into the host cells toxic effector proteins inhibiting phagocytosis and interfering with the normal function of the target cells. Yersinia virulence factors paralyze macrophage activities critical for an appropriate immune response of the cells. Determination of the virulon molecular elements essential for its functioning would facilitate purposeful search for inhibitors of this system, and potentially novel therapeutic remedies to treat a number of infections caused, in particular, by Yersinia spp.
One of the key functions in Yersinia Yop-virulon is performed by V antigen (LcrV). V antigen induces both active and passive immunity in mice against challenge with virulent Y. pestis strains. LcrV mutants have been shown to be avirulent for mice.
V antigen is a multifunctional secreted protein (molecular weight, about 39 kDa) involved in the regulation of biosynthesis of Yop-virulon components and essential for Yersinia virulence. It suppresses the natural immune response by lowering the production of cytokines, particularly, g- interferon and tumour necrosis factor-alpha. Besides, Lcr V inhibits neutrophil chemotaxis. The virulence of attenuated Y. pestis bacterial strains in mice increases as a result of purified V antigen inoculation. V antigen is also being necessary for the secretion of translocation proteins, YopB and YopD, which conclude the channel for virulon secretion forming a pore in the membrane of the target host cell. While carrying out this function, LcrV is likely to interact with YopB and YopD proteins in the bacterial cytoplasm, because the latter are not excreted from the bacterial cell without LcrV.
The role of V antigen in yersiniosis pathogenesis is not quite clear yet. A certain amount of secreted LcrV is supposed to be able of binding to the host cell prior to the establishment of a close contact between the bacterium and the target cell. This interaction may be imperative for the initiation of the secretion channel development. Lcr V specific antibody injection into laboratory mice protects them against plague. These antibodies seem to block those sites of the extracellular V antigen molecule that are functionally very important for Yop virulon function.
The key part of LcrV in Yersinia virulence and in the induction of protective immunity impart actuality both to a further improvement of specific preventive agents created on its basis and to the search for substances capable of selective blocking of V antigen (Yop virulon) activity in Yersinia cells thus making it possible to ensure urgent therapy of yersinioses. The procedure of construction and selective choice of randomized single-chain oligonucleotides (aptamers) originating from DNA and RNA (SELEX - Systematic Evolution of Ligands by EXponential enrichment) will be first used in search of Yersinia Yop virulon inhibitors.
LcrV-protein together with Caf1 capsular antigen are planned to be used for development of a new plague vaccine being extrecorporally cultured dendritic cells (Dcs), activated in vitro by cytokines (GM-CSF, G-CSF, IL-4, TNF-a), and loaded afterwards with the mentioned Y.pestis antigens. Dcs using as natural vectors for specific vaccination is based on undoubtedly paramount role of antigen-presenting cell in the process of initial T-cell activation. Such vaccine is supposed to have increased ability to protect against human pneumonic plague as immune defects against Y.pestis aerogenic challenge greatly depends on efficacy of antigen presentation by antigen-presenting lung cells resulted in inducing of specific cell response. In this supposed Project we are going to study in experiment the conditions for maturation, activation and loading with plague antigens in vitro of Dcs obtained from inbred line mice. Then we are going to study immunity formation following the vaccination with such vaccines based on dendrite cells (Y. pestis antigen + Dcs) of syngenic recipients. After proving the fact that immunization with vaccines based on dendrite cells (Y. pestis antigen + Dcs) induce T-cell immune response specific to the used antigen in recipient mice and evaluating of such response characteristics, the immunized mice would be challenged with different doses of Y. Pestis vaccine strain. Animals vaccinated with antigen unloaded Dcs (negative) and animals Lcr V and Caf1 proteins (positive) would serve as the control.
The aim of the present investigation is to study structural and functional properties of LcrV Yersinia Yop-virulon proteins: to develop approaches to construction of plague vaccines based on Dcs expressing Y.pestis protective antigens LcrV and Caf1; to study efficacy of the post-vaccinal immunity; search for substances able to block selectively functions of LcrV protein and prevent yersiniosis development.