Pulmonary mycobacterial infections affect approximately one third of the world's population and claim millions of lives annually. Infectious organisms that are inhaled into the airspaces encounter alveolar macrophages in the context of immune proteins in the alveolar lining fluid. Specifically, surfactant protein A (SP-A), a lung- specific collectin, orchestrates macrophage activation, phagocytosis and killing of mycobacteria via a 210 kDa (SP-8210) receptor. The long term objective of this application is to elucidate the mechanisms by which SP-A and its receptor direct alveolar macrophage host defense functions in the lung fn vivo. This proposal is based on these new findings: 1) the SP-A receptor is a heterooligomer of 210 kDa (SP-8210) and 240 kDa (SP-8240) cell- surface, and 78 kDa (SP-R78) intracellular proteins. Both SP-8210 and SP-R78 have been sequenced by mass spectrometry.2) The composition of the SP-A receptor heterooligomer may vary based on the state of macrophage differentiation. The central hypothesis of this proposal is that the interaction of SP-A with its receptor coordinates macrophage activation and mycobacterial clearance via distinct SP-A receptor- directed mechanisms. To test this hypothesis we will study SP-A receptor structure and function in the context of mycobacterial infection.
The Specific Aims are: 1) reconstitute a functional SP-A receptor in COS .cells and use a panel of recombinant wild type and mutant SP-A proteins to determine mechanisms of SP-A-binding and function in the phagocytosis of mycobacteria, 2) determine the role of the intracellular SP-R78 in the activation of an SP-A-specific pathway for intracellular targeting of mycobacteria, and 3) study the relative expression of the SP- A receptor components during macrophage differentiation to understand how the structure of the SP-A receptor directs SP-A-mediated mycobacterial clearance and activation of macrophages. To facilitate these studies we will utilize immature alveolar macrophages that we have isolated, and Mycobacterium bovis BCG and Mycobacterium tuberculosis Ra reporter strains that express green fluorescent protein.
