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.

Figure 1

Modulation of E. coli clearance from the lungs of pulmonary collectin mouse models. Bacterial CFUs in lung homogenates were quantified 6 hours after intratracheal inoculation with 5 × 10⁶ E. coli in age- and inbred strain–matched SP-A^–/– and SP-A^+/+ C3H/HeN mice, or Swiss Black SP-D–overexpressing mice (+/+, rSP-D) and littermate controls (+/+). Data are mean ± SEM; n = 10 per group; *P < 0.05.

Figure 2

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.

Figure 3

Time course of inhibition of E. coli K12 macromolecular synthesis by rat and human pulmonary collectins. E. coli K12 were incubated in the presence of ³H-uridine (a) or ¹⁴C-amino acids (b and c) and pulmonary collectins including hSP-A, rSP-A, or rSP-D for up to 2 hours. At the indicated time points, bacterial RNA or proteins were precipitated, collected on filters, and counted using a scintillation counter. Controls including no bacteria, no added protein, and heat-killed bacteria (heat) are shown. Data are mean ± SEM; n = 3.

Figure 4

Specificity of collectin-mediated growth inhibition of E. coli K12. (a) The purity of collectins used in the study, including the EDTA eluate of the surfactant pellet (lane 1), fast protein liquid chromatography fractions 8 (lane 2) and 12 (lane 3), rSP-A (lane 4), and rSP-D (lane 5), was assessed by silver staining after 8–16% SDS-PAGE electrophoresis. (b) ³H-uridine incorporation by E. coli K12 was assessed alone and in the presence of 2 mM EDTA; in the presence of hSP-A (100 μg/ml) or rSP-D (10 μg/ml) at pH 5.0, 6.0, and 7.4; in the presence of boiled hSP-A (100 μg/ml) or rSP-D (10 μg/ml); and in the presence of the ultrafiltrate (molecular weight cutoff of 10,000) of hSP-A (100 μg/ml) or rSP-D (10 μg/ml) before boiling (room temperature; RT) and after boiling (100°C). Data are mean ± SEM; n = 3; *P < 0.01.

Figure 5

Attenuation of E. coli K12–induced light scattering by SP-A and SP-D. (a) E. coli K12 were mixed with 25 μg/ml (open squares) or 250 μg/ml (open triangles) hSP-A or no protein (open circles) and incubated in LB at 37°C in a shaking water bath. Bacterial density was monitored by absorbance at 400 nm. (b) E. coli grown as above were incubated with hSP-A (filled circles), 300 μg/ml J5 LPS (open circles), 300 μg/ml J5 LPS plus 100 μg/ml hSP-A (open triangles), or no additives (x’s). (c) E. coli grown as above were incubated with 100 μg/ml rmSP-D (open squares), 300 μg/ml J5 LPS (open triangles), or 300 μg/ml J5 LPS plus 100 μg/ml rmSP-D (filled circles). Data are mean ± SEM; n = 3. (d) Bacterial aggregation was assessed under the light microscope after incubation of E. coli K12 with hSP-A, rSP-A, rrSP-A, a C-terminal fragment of SP-A containing residues A81–F228 (ΔSP-A), rSP-D, rmSP-D, a C-terminal fragment of SP-D containing residues D203–F355 (ΔSP-D), or BSA, and viewed with the light microscope.

Figure 6

Antibody-induced aggregation does not affect RNA synthesis in E. coli K12. (a) E. coli K12 were incubated in the presence of BSA, hSP-A (100 μg/ml), or E. coli–aggregating EK12 (100 μg/ml) or PM antibody (10 μg/ml) for 1 hour at 37°C and then visualized under the light microscope. (b) E. coli were incubated for 1.5 hours with ³H-uridine prior to assessment of RNA synthesis as above. Data are mean ± SEM; n = 3.

Figure 7

Inhibition of bacterial growth by SP-A and SP-D is independent of bacterial aggregation. Molten agarose was mixed with E. coli K12 (a and b) or a clinical E. coli isolate from a septic patient (d), plated in Petri dishes, and allowed to cool. Wells were bored in the agar, and proteins (0.5 or 5.0 μg/well) were added for overnight incubation. (a) BSA, lysozyme (Lys), rSP-A, or rSP-D was incubated with E. coli K12. (b) E. coli K12 was incubated with 5 μg rSP-D (first two wells) or the protein-free filtrate (molecular weight cutoff of 10,000) from the 1-mg/ml rSP-D reagent (last two wells). (c) Silver-stained SDS-PAGE gel of clinical E. coli isolate showing slow- and fast-migrating species, consistent with smooth LPS phenotype. (d) Clinical E. coli isolate incubated with the same proteins as E. coli K12, as well as hSP-A and ΔSP-D, a D203–F355 C-terminal fragment of recombinant mouse SP-D containing only the C-terminal domains.

Figure 8

Growth inhibition by rSP-A and rSP-D is blocked by J5 LPS. (a) E. coli K12 were grown in the presence of hSP-A (50 or 100 μg/ml) or rSP-D (5 μg/ml) and E. coli J5 LPS vesicles (300 μg/ml) for 90 minutes at 37°C. ³H-uridine incorporation was measured as above. Data are mean ± SEM; n = 3–5 per group; *P < 0.01 compared with E. coli alone. (b) E. coli–impregnated agar was prepared as above, and 0.5 μg of rSP-A or rSP-D was added to the well in the presence or absence of E. coli J5 LPS vesicles (100 μg/ml). Control wells with J5 LPS alone are also shown.

Figure 9

Growth inhibition of smooth and rough laboratory strains of E. coli and other Gram-negative isolates by pulmonary collectins. The effect of hSP-A (100 μg/ml) and rSP-D (10 μg/ml) on ³H-uridine uptake by laboratory strains and clinical isolates was assessed (a) and correlated with the LPS profile on SDS-PAGE analysis (b). The following rough E. coli strains are shown: an E. coli K12 strain containing an empty pGEM vector (F9+), and isogenic strains with pGEM-driven expression of glycosyltransferases that add two (OS4) and five (OS7) sugars to the core oligosaccharide; a K12 mutant with a defective lipid A missing an acyl chain (MLK217); E. coli J5; two luminescent E. coli HB101 strains (101a and 101b); and LCD25, an E. coli K12 acetate auxotroph. The following smooth E. coli strains are shown: 0111:K58 (B4) and 055:K59 (B5). Clinical isolates shown include four E. coli isolates, three smooth variants (Ec1, Ec2, and Ec3) and one rough variant (Ec4); two K. pneumoniae strains (Kp1 and Kp2); and two E. aerogenes strains (Ea1 and Ea2). Data shown are mean ± SEM; n = 3; *P < 0.01, ^#P < 0.05.

Figure 10

Deletion of OmpA enhances the susceptibility of smooth E. coli variant E98 to growth inhibition by SP-A and SP-D. (a) Silver-stained SDS-PAGE of E. coli strain E98. (b) A clinical isolate of E98 (parental), an isogenic OmpA-negative (ΔOmpA) mutant, and ΔOmpA bacteria containing either an empty vector (ΔOmpAv) or a plasmid directing overexpression of OmpA in the OmpA-negative background (ΔOmpA+) were embedded in agar and incubated overnight with albumin, lysozyme, rSP-A, rSP-D, rrSP-A, a C-terminal fragment of rrSP-A (ΔSP-A), hSP-A, and rrSP-D at 0.5 μg/well or 5.0 μg/well, as indicated.

Figure 11

Incubation of E. coli K12 with the pulmonary collectins results in release of protein into the extracellular space. E. coli were incubated with rSP-A, boiled rSP-A (⁁), a C-terminal fragment of SP-A (A81–F228, denoted ΔA), rSP-D, a C-terminal fragment of SP-D (D203–F355, denoted ΔD), or the permeabilizing antimicrobial peptide mellitin (mel) in HBSS for 15 minutes at 37°C and then sedimented by centrifugation. Thiol-containing proteins released into the extracellular space were measured using the fluorescent dye ThioGlo1. Controls in which bacteria or bacteria plus collectins were omitted are also shown. Data are mean ± SEM; n = 4–5. Statistical significance of differences in SH level in the presence and absence of collectins and controls is shown; ^#P < 0.05, *P < 0.01.

Figure 12

The pulmonary collectins enhance membrane permeability to Act D. (a) E. coli K12 were incubated with 1–10 μg/ml hSP-A, in the presence and absence of 10 μg/ml Act D, and incorporation of ³H-uridine was measured. RNA synthesis was also assessed in the presence of: 1, Act D (10 μg/ml) alone; 2, protein-free ultrafiltrate from 10 μg/ml rSP-A; 3, 0.05% DMSO vehicle; 4, ultrafiltrate plus Act D (10 μg/ml). (b and c) ³H-uridine incorporation by E. coli K12 incubated with a fixed SP-A, SP-D, or mellitin concentration and varying Act D concentrations. Data are mean ± SEM; n = 4–7.

Figure 13

Collectin-induced reduction in viability of E. coli. (a and b) E. coli K12 were incubated with rSP-A (a) or rSP-D (b) at various concentrations for 30 minutes at 37°C and then with green fluorescent nucleic acid stains STYO 9 and propidium iodide to assess viability. Data are mean ± SEM; n = 4–5. Statistical significance of differences in viability in the presence and absence of collectins is shown (^#P < 0.05, *P < 0.01). (c) Control experiments comparing viability of E. coli K12 grown in the absence (–) and presence (+) of collectins (100 μg/ml rSP-A, 10 μg/ml rSP-D), boiled collectins (100°C), or the ultrafiltrate of collectins (F) and boiled collectins (F100°C); *P<0.01.