The Role Of Rv0394c In Adherence Of Mycobacterium Tuberculosis To Alveolar Epithelial Cells

Tuberculosis(TB) is one of the major infectious diseases endangering human and animal. Especially in recent years, with the emergence of multi-resistant mycobacteria and the mixed infection with the virus, the treatment of TB is becoming more and more difficult. Therefore, study on the pathogenesis of tuberculosis and the development of new effective TB vaccines has become urgent. The secretory protein of tuberculosis is the important virulence factors to cause the diseases and to evade the body’s immune system. It has been proved that some secretory proteins of mycobacterium tuberculosis(Ag85b, ESAT- 6, etc.) were important to its virulence, but the molecular mechanism of pathogenesis and escaping from the body’s immune system remains unclear.Our previous study found that Rv0394c(SEC) protein, encoded by M. tuberculosis rv0394 c gene, contains transmembrane area, thus we presumed that the SEC protein may be a secretory protein. Moreover, rv0394 c gene exists only in pathogenic mycobacteria such as Mycobacterium tuberculosis, M. africanum, M. bovis, therefore the protein is supposed to be associated with mycobacterial pathogenesis; The SEC protein exhibits hyaluronidase and chondrosulfatase activities, whose substrates constitute the main components of the extracellular matrix and belong to the glycosaminoglycans macromolecular substances. Furthermore hyaluronic acid widely exists on the surface of various cells, so we speculated that the SEC protein may play an important role in the adhesion process of mycobacteria to the host cells. In order to verify this presumption, we performed the following studies.Firstly, we obtained the recombinant SEC protein using E. coli expression system and immune mice using the purification product by affinity chromatography. Then, we prepared monoclonal antibodies of the protein using the cell fusion, cloning and screening technology, which as experimental tool in the following study. Indirect Enzymelinked immunosorbent assays revealed that the SEC protein displayed capability of binding to DNA, HA and chondroitin sulfate. Furthermore, confocal laser microscopic analysis showed that SEC protein adhere to Human Lung Epithelial Cells(A549 cells). To furtherly confirm the specific adherence of SEC to human cells, rv0394 c gene was cloned to E. coli-mycobacterium shuttle vector pMV262, and the resultant recombinant plasmid was transformed into Mycobacterium smegmatis strain MC2155 for constitutive expression by electroporation. The results showed that the capability of the recombinant bacteria in adhesion to A549 cell surface was dramaticly improved compared with the parent strain. In addition, the adhesion was blocked by SEC protein and monoclonal antibodies of the protein. The mice were challenged intranasally with recombinant bacteria and detection of bacterial colonization was performed at different time points past challenging. The results showed that colonization ability in mice’s lungs of the recombinant bacteria was considerablyenhanced compared with the parent strain and the recombinant bacteria caused pathological changes in mice’s lung tissue as well. However, adding the exogenous HA during the challenging could promote the removal of the recombinant bacteria in mice’s lungs.To investigate the localization of SEC protein in mycobacteria, subcellular components were prepared and the existence of the protein in these components was tested by Western blot. The results indicated that SEC localizes on the cell wall as a surface anchoring protein, providing additional evidence supporting the conclusion that SEC protein plays a role in mycobacterial adherence to host cells.In conclusion, these studies prepared monoclonal antibodies of SEC protein, providing a research tool for structural and functional study of SEC protein. We identified a novel adherence factor of mycobacteria-SEC protein, which displays hyaluronidase and chondrosulfatase activities, by adhesion and adhesion blocking experiments. The results showed that it may be involved in the pathogenic process of mycobacteria. Our findings contributed to a thorougher understanding of the pathogenesis of mycobacteria and lay a foundation for furtherly exploring the specific role of this hyaluronidase in pathogenic mechanism of mycobacteria.