Mechanisms of Mycobacterium leprae pathogenesis: Analysis of the extracellular proteins and effect on the host lipid metabolism.
The complete sequence of the Mycobacterium leprae genome, together with recent advances in the definition of its proteome, are contributing to our understanding of leprosy pathogenesis, so far a difficult task due to the unique properties of this bacterium. The major goal of the present study was toidentify and functionally characterize extracellular M. leprae proteins. Since these proteins are localized at the host-pathogen interface, they may play a relevant role in infection. A second objective was to analyze the potentialeffects of M. leprae on host lipid metabolism. Firstly, the M. leprae histonelike protein (Hlp), an adhesin potentially involved in the interaction with Schwann and epithelial cells, was better characterized. Hlp was shown to be expressed by M. leprae isolated from human lesions. Moreover, the capacityof the recombinant protein to bind to different extracellular matrix components was demonstrated. Also, the binding sites of Hlp to laminin and heparan sulfate were defined by using truncated recombinant proteins and a panel ofsynthetic peptides covering the entire protein. Our results indicate that the Ala/Lys/Pro-rich repeats present in the C-terminal region constitute a binding site for Heparin and Heparan sulfate. Another site located at the N- terminalregion (residues 45 to 65) was defined as a second interaction site with Heparan sulfate. A second adhesin, hemaglutinin-binding-heparin (HBHA), factor in M. tuberculosis by promoting its binding to epithelial cells of the respiratory tract and subsequent extrapulmonary dissemination, was analyzed in M. leprae. Proteomic analysis of armadillo-derived bacterium showed that HBHA is expressed in vivo by M. leprae. Subsequently, the recombinant protein (rHBHA) was shown toincrease the association of M. leprae to epithelial cells, suggesting that this protein could also contribute to M. leprae pathogenesis by promoting bacterial adherence to the respiratory tract. We also evaluated the immune response torHBHA and to its methylated form in individuals infected by M.leprae and in uninfected controls. Our findings suggest that, in contrast to what has been seen in tuberculosis, HBHA is not an immunogenic protein during M.leprae infection. Five additional M. leprae extracellular proteins were identified inarmadillo infected tissue by a proteomic approach. Two of them were new in the context of the M. leprae proteome: ML1273, a tryptophan synthase alpha subunit, and ML1029, a conserved hypothetical protein. Finally, a comparative analysis of the profile of neutral lipids was performed. We observed areduction of approximately 5 times in the percentage of cholesterol esther in infected tissue. Altogether, the results described in this work contribute for a better understanding of M.leprae pathogenesis.
Advisor:Maria Cristina Vidal Pessolani
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Mycobacterium leprae Leprosy Adhesins Bacterial Lipids Extracellular Matrix Proteins
Date of Publication:01/21/2008