No abstract

The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A–null mice and was increased in SP-D–overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D–mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane

Lung surfactant is a mixture of phospholipids, neutral lipids, and surfactant protein A (SP-A) 1 SP-B, SP-C, and SP-D, which are secreted into the air spaces by alveolar type II cells and Clara cells of the distal pulmonary epithelium (1). Although the primary function of surfactant is to reduce surface tension, the contribution of each molecular component to surface activity is not completely understood. Surfactant phospholipids form a film at the air-liquid interface that maintains air space patency by resisting compression as the alveolar radius decreases during expiration. Data from in vitro experiments, gene-targeted animals, and naturally occurring mutations in humans indicate that the hydrophobic surfactant proteins, SP-B and SP-C, participate in the assembly and biophysical properties of the surfactant film (2). The hydrophilic surfactant proteins, SP-A and SP-D, have a complex functional profile. The recognition that SP-A and SP-D are structurally homologous to mannosebinding protein has identified them as members of the collectin family of innate opsonins and directed attention to their host defense properties (3). Like mannose-binding protein, SP-A and SP-D bind to a wide range of microorganisms and enhance microbial phagocytosis and killing by alveolar macrophages. These in vitro activities appear to be physiologically relevant, since gene-targeted SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice clear microbial infections less effectively than pulmonary collectin-sufficient mice (4 -7). However, SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice also exhibit abnormalities of surfactant structure, metabolism and function (8 -10). Surfactant isolated from SP-A Ϫ/Ϫ mice does not contain the large aggregate tubular myelin and has impaired surface activity in the presence of plasma inhibitors (11). SP-D Ϫ/Ϫ mice develop progressive alveolar phospholipidosis and air space dilation (9, 10), associated with increased macrophage production of metalloproteinases and oxidant species (12). All of these defects are corrected by lung-specific expression of the cognate collectin in the SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice (13, 14). The structural basis of SP-A and SP-D surfactant functions has been explored by mutagenesis using in vitro and in vivo analyses. The primary structure of both proteins includes an N-terminal segment containing interchain linkages formed by Cys residues, a collagen-like region of Gly-X-Y repeats, a hydrophobic "neck" domain, and a carbohydrate recognition domain (CRD) (15,16). Trimeric association of subunits occurs by the folding of the collagen-like domains into triple helices (17) and coiled-coil bundling of ␣-helices in the neck (18). In the fully assembled molecules, the N-terminal sequences and di- ϩ/ϩ mice disrupted oligomeric assembly of the endogenous SP-D and produced air space dilation and foamy macrophage formation without phospholipidosis (24). These data suggested that the in vivo activity of SP-D is dependent on its oligomeric structure. The purpose of this study was to examine the role of the N-terminal segment-dependent olig...

We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A؉/؉ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A؊/؊ animals. Similarly, calciumdependent surfactant-binding proteins of SP-A؉/؉ mice increased membrane permeability more than those from SP-A؊/؊ mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O 2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A؉/؉ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.