Tuberculosis, a worldwide health problem, claims at least 3 million lives annually. The causative intracellular bacterium, Mycobacterium tuberculosis (M.tb), is transmitted by inhalation into the alveoli of uninfected individuals. The interaction between M.tb and the pulmonary lining represents the initial contact of the bacterium with the host immune system. Since this bacterium multiplies mainly within the mononuclear phagocyte (especially the alveolar macrophage), the factors which affect its phagocytosis by this host cell are important to our understanding of disease pathogenesis. Surfactant and its associated proteins, in conjunction with alveolar macrophages, are important components of the pulmonary alveoli. Two major surfactant associated proteins, surfactant protein A (SP-A) and surfactant protein D (SP-D), contain carbohydrate recognition domains (CRDs). Our preliminary studies indicate that human SP-A enhances the uptake of the virulent Erdman strain of M.tb by human monocyte-derived macrophages (MDM) and human alveolar macrophages (HAM) by a direct interaction with the macrophages. SP-A from patients with alveolar proteinosis (APP SP-A) enhances uptake of M.tb to a greater degree than SP-A from healthy individuals. This result may provide an explanation for the known association between alveolar proteinosis and mycobacterial disease. Rat native and recombinant SP-A also enhance uptake of Erdman M.tb. Removal of the carbohydrates from both rat and human SP-A eliminate the M.tb uptake enhancing activity of these proteins. The effects of SP-A on M.tb uptake are selective since SP-D reduces M.tb uptake by MDM. Our studies indicate that SP-A also binds directly to M.tb. More SP-A binds to Erdman M.tb than to the attenuated H37Ra strain. Thus, SP-A may affect uptake of virulent and attenuated strains of M.tb differently. Our hypothesis in this proposal is that SP-A may play a role in the pathogenesis of tuberculosis by enhancing the ability of M.tb to enter and survive within its host niche.
Our specific aims are to: determine the domain(s) of SP-A that bind to human macrophages to enhance M.tb phagocytosis; determine whether SP-A binding to macrophages upregulates surface expression and function of those receptors that mediate M.tb phagocytosis; determine the influence of SP-A on the viability of M.tb within macrophages; characterize the binding of SP-A to M.tb strains and determine whether the CRD of SP-A plays a role in this binding; and construct chimeric proteins to further define the function of specific domains of SP-A and SP-D in M.tb-macrophage biology. We will study M.tb phagocytosis by MDM and HAM in an in vitro assay in the presence and absence of soluble or matrix-bound SP-A. The specific domains of SP-A involved will be studied using variant forms of protein produced by enzyme digestion of native and recombinant SP-A and site directed mutagenesis of recombinant SP-A in baculovirus and mammalian expression systems. Macrophage surface receptor expression and function in the presence of intact or variant SP-A will be studied by flow cytometry. ELISA and in binding assays. M.tb intracellular multiplication will be studied by colony counts of macrophage lysates or metabolic labeling of M.tb. Binding of intact or variant SP-A to M.tb strains will be studied by ELISA and by [125I] SP-A binding. Finally, we will construct SP-A/SP-D chimeric proteins to further define the functional maps of these proteins in influencing M.tb phagocytosis and intracellular survival. With the re- emergence of tuberculosis as a significant health problem and the appearance of drug-resistant strains, enhanced effort to define the molecular events in early tuberculosis infection is necessary and may provide the basis for novel therapeutic strategies.
