We have established that the virulent H37Rv strain of Mycobacterium tuberculosis fragments and disrupts the phagosomal membranes of normal rabbit alveolar macrophages (AM) following phagocytosis of the organisms in vitro. In contrast, the avirulent H37Ra lacks this putative virulence characteristic. It is of special interest that BCG-immune rabbits are remarkably resistant to the membrane destroying potential of the H37Rv strain. We propose to expand this study significantly as follows: 1) We will extend our in vitro and in vivo observations by examining several additional strains of virulent and avirulent mycobacteria and compare their potential phagosome-membrane disrupting properties following interactions with normal and BCG-immune AM. 2) We also plan a comparative study using normal mouse peritoneal macrophages (PM) and AM with selected virulent and avirulent strains of mycobacteria. Our main objective is to determine if rabbit AM respond differently than mouse PM because of previously reported results. Investigators reported that the virulent H37Rv strain caused inhibition of lysosome-phagosome fusion but did not disrupt the phagosomal membrane. 3) Since BCG-immune AM are resistant to the membrane disrupting potential of virulent mycobacteria, we plan to determine and compare the lipid composition of normal and BCG-immune plasma membranes. Protein/lipid, saturated/unsaturated lipids, cholesterol/protein and cholesterol/phospholipid ratios as well as individual lipid analysis will be carried out. 4) In addition, purified plasma membranes form normal rabbit AM and BCG-immune AM will be analyzed for fluidity properties. Fluidity will be analyzed by fluorescence anisotropy and with electron spin resonance spectroscopy. The data obtained will be compared with data obtained form lipid analysis of normal and BCG-immune AM membranes. 5) We will initiate a collaborative investigation in a search for potential phagosomal membrane disrupting moieties associated with virulent mycobacterial strains. Initial studies will examine the sulfatides of virulent mycobacteria. Ancillary supportive studies will examine a) the role of BCG-immune sera in protecting normal AM from phagosome disruption, b) potential of virulent low sulfatide strains to disrupt phagosomes and c) effects of virulent mycobacteria on purified plasma membranes as determined by lipid analysis of supernates and affected membranes.
| Chida, K; Myrvik, Q N; Leake, E S et al. (1987) Chemiluminescent responses of alveolar macrophages from normal and Mycobacterium bovis BCG-vaccinated rabbits as a function of age. Infect Immun 55:1476-83 |
