Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability
Huixing Wu, … , Kwang Sik Kim, Francis X. McCormack
Huixing Wu, … , Kwang Sik Kim, Francis X. McCormack
Published May 15, 2003
Citation Information: J Clin Invest. 2003;111(10):1589-1602. https://doi.org/10.1172/JCI16889.
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Article Pulmonology

Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability

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.

Authors

Huixing Wu, Alexander Kuzmenko, Sijue Wan, Lyndsay Schaffer, Alison Weiss, James H. Fisher, Kwang Sik Kim, Francis X. McCormack

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Antimicrobial activity of calcium-bound surfactant-associated proteins. ...
Antimicrobial activity of calcium-bound surfactant-associated proteins. Human surfactant was repeatedly washed by sedimentation in the presence of calcium, eluted with EDTA, purified by mannose-Sepharose affinity chromatography, and size-fractionated by fast protein liquid chromatography on a Superose 6 column. (a) Elution of proteins from the column was monitored by UV absorbance at a wavelength of 280 nm. (b) The SP-A and SP-D content of individual fractions was determined by Western analysis. (c) The antimicrobial activity of the original sample and selected fractions, including 8, 12, and the tenfold concentrate of fraction 21, was assessed by measurement of 3H-uridine incorporation in E. coli K12. Controls shown included protein-free ultrafiltrates (molecular weight cutoff of 10,000) of the most concentrated sample used in each set (designated F), and 2 mM EDTA, the highest possible concentration of EDTA in any sample. Data are mean ± SEM; n = 3; *P < 0.01.