Persistence and Pathogenicity of Mycoplasmas
Molecular Genetic Mechanisms of Mycoplasmic Persistence and Pathogenicity
Tech Area / Field
3 Approved without Funding
Gamalei Institute of Epidemiology and Microbiology, Russia, Moscow
- Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- University of Amsterdam / Academic Medical Center, The Netherlands, Amsterdam\nThe Cleveland Clinic Foundation / Lerner Research Institute, USA, OH, Cleveland
Project summaryThe aim of the Project: study of molecular mechanisms providing persistence of pathogenic mycoplasmas.
The subject of this project is stated in frames of a new and urgent trend of medical microbiology dealing with the understanding of pathogenicity nature. This trend offers investigation of molecular genetic mechanisms responsible for differential gene activity of pathogens in the course of infectious process. In this connection researches of the above problem are of importance for the development of effective approaches to precautions and treatment of infectious diseases.
Mycoplasmas, representatives of Mollicutes class, are the smallest selfreplicating prokaryotic human and animal pathogens, which infectious role is undoubtedly, underestimated, the fact is connected with the absence of fundamental knowledge about their virulent properties. There are accumulating evidence supporting mycoplasmas’ role in pathogenesis of acute and chronic diseases of respiratory and urogenital tract, in newborns and immunocompromised patients with wide–ranging complications (Prozorovsky et. al., 1995; Baseman, Tully, 1997; Taylor-Robinson D., 1996, 2002). The main feature of mycoplasmal infections is the establishment of chronic forms due to a persistence of a pathogen, which is difficult to eradicate even after prolonged drug therapy and in patients with competent immune system. At present microbiologists and clinicians began to realize the changing image of mycoplasmas from innocent bystanders to emerging and reemerging pathogens in human and animal diseases (Blanchard, Bebear, 2002, Lo, 2002).
However, it remains unanswered how mycoplasmas colonize and persist in the human host and cause acute or chronic disease. There are evidences that these “simple” microorganisms possess multiple mechanisms by which they adapt to the privileged and hostile environment of the human host, at the same time mycoplasmas can either avoid host defense factors or influence on macroorganism’s cells and it’s immune system.
Pathogenic mycoplasmas used the main molecular-genetic mechanisms enabling the microbe to change antigenic structure and to regulate the expression of pathogenicity factors. (Himmelreich et al., 1996, 1997, Herrmann, 2002, Krause D.C., 1996, 1998, Laderfoged S.A., 2000, Razin, 1998, 1999). But, there is no enough data on the participation of the global regulatory systems in mycoplasma adaptation to the existence in the macroorganism environment. The less studied field in that respect is the understanding of which precisely mechanisms function effectively in vivo.
The recent findings that some microbial pathogens modulate apoptosis to accommodate the organism’s life cycle and facilitate infection shed an important and exciting new light on the study of pathogenesis. Mycoplasmas are among the few procaryotes that have a close interaction with host cells for long periods without causing acute cytopathic effects and they evolved strategies to modulate the apoptotic program of the host cell to their favour. Identification of bacterial proteins blocking cellular death and study of the mechanism of their action are of topical importance in the frames of the given problem. This will open a possibility to approach to the development of a new generation of therapeutic drugs aiming at prevention of infectious process to become chronic.
The main goals of the study:
Analysis of differentially expressed genes of mycoplasmas in host environments at different stages of infection by using the RNA microarray technology. Development of in vitro and in vivo models for the study on differential gene expression of M.pneumoniae in acute and persistent infections.
– Identification of mycoplasmal differentially expressed genes at acute and chronic infections and their functional analysis.
– Study of the genetic control of mycoplasmal gene expression in persistent forms modulated at the level of DNA topology. Mass spectrometric identification of DNA-binding proteins participating in downregulation of gene expression in persistent mycoplasmas.
– Characteristics of the identified DNA-binding proteins: definition of their physical-chemical properties, nature and specificity of the binding to DNA.
– Study of mycoplasma influence on the host cell apoptosis in acute and chronic infection models in the cell culture.
– Development of approaches to the identification of the mycoplasma factors having a modulating effect on apoptosis.
Laboratory of mycoplasmas and bacterial L-forms of The Gamaleya Institute of Epidemiology and Microbiology Russian Academy of Medical Sciences possesses a collection of M.pneumoniae isogenic strains with various virulence and both laboratory and clinical M.fermentans, M.hominis, U.urealyticum strains, the biological and virulent properties of which were characterized by the experiments in vitro and in vivo.
Recently in our studies it has been demonstrated that during the establishment of a persistence of M.pneumoniae the downregulation of genes encoding antigenic determinants and pathogenicity factors takes place. The regulation makes it possible for parasite to avoid immune response and coexist durably with the host. It has been shown due to development and application of a molecular tool for identifying and isolating differentially expressed genes, so called RNA arbitrarily primed PCR or RNA fingerprinting. It has been shown that expression of many genes in an avirulent strain are switched off comparing with a virulent strain. Interaction with host organism induced gene expression which led to restoration of virulence. We managed to identify several differentially expressed genes of M.pneumoniae. Among them are genes encoding antigenic determinants, pathogenicity factors, DNA methylating enzymes, enzymes of phosphotransferase system and so on.
Our preliminary data let us advance an original hypothesis concerning the mechanisms of mycoplasmal persistence establishment. We proposed to investigate a role of DNA topology changing and DNA-binding proteins in global modulation of gene expression under environmental signals. Identification of these DNA-binding proteins may facilitate investigation of unknown mechanism of the pathogen adaptation and survival within host organism.
Apoptosis plays an active role in the control of viral and bacterial infections. To establish a successful infection, pathogens developed a variety of strategies for modulating apoptosis of the host cell. In the frame of the project we plan to study whether mycoplasmal infection interferes with apoptotic machinery and which mechanisms mycoplasmas evolved to ensure their own survival in the infected cells.
Investigation of gene regulation in persistent forms of mycoplasmas and identification of mycoplasmal antiapoptotic factors may allow to search of specific inhibitors and development of medicines for chronic infections treatment.