Degradation of Outer Membrane Protein A in Escherichia coli Killing by Neutrophil Elastase

See allHide authors and affiliations

Science  18 Aug 2000:
Vol. 289, Issue 5482, pp. 1185-1187
DOI: 10.1126/science.289.5482.1185


In determining the mechanism of neutrophil elastase (NE)–mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient,E. coli. Also, whereas NE-deficient mice had impaired survival in response to E. coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E. coli. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.

After bacterial infection, neutrophils engulf and kill bacteria by oxidative and nonoxidative pathways. Nonoxidative mechanisms are less well defined, but they predominantly relate to the ability of peptides to alter the bacterial membrane permeability (1). NE has long been regarded as an antibacterial protein, but its mechanism of bacterial killing remains unclear (2). NE is a potent serine proteinase whose catalytic activity relies on the His-Asp-Ser triad (3). Recently, we have demonstrated that NE is required for host defense against Gram-negative, but not Gram-positive, bacteria (4). These findings prompted us to delineate the mechanism by which NE kills E. coli, a common and virulent pathogen.

Using immuno–electron microscopy (immuno-EM), we confirmed that NE was localized within the phagolysosomes following ingestion of E. coli by neutrophils (Fig. 1A) (5, 6). To determine whether the antibacterial activity of NE was related to its catalytic activity, we cultured E. coli [106colony-forming units (CFUs)] with or without human NE (2 μM), and bacterial viability was monitored over time by plating serial dilutions. The addition of NE markedly decreased E. coligrowth (Fig. 1B). This decrease in bacterial growth was dependent on the time of incubation (4) and NE concentration but was independent of the growth phase and salt concentration. Upon heat inactivation or active site inhibition of NE with secretory leukocyte protease inhibitor (SLPI) (7), the bacteria cultured with NE grew similarly to the bacteria cultured without it. Thus, NE catalytic activity was required for bactericidal activity. This was further confirmed by using fluorescent staining to distinguish live versus dead bacteria (8). In the presence of NE, DNA of dead bacteria with damaged membranes fluoresce bright green, whereas DNA of live bacteria with intact membranes stains blue (Fig. 1C). Neither cathepsin G (CG) (9) nor gelatinase B (GB) (10) was able to kill this strain, suggesting selective antibacterial activity of neutrophil granule proteinases.

Figure 1

NE localization and effect on bacteria. (A) Immuno-EM localization of NE in neutrophils. Human neutrophils were incubated with freshly grown bacteria at a 1:100 ratio, and the reactions were processed for immuno-EM with antibody to NE. A phagolysosome containing an E. colibacterium is shown. There is an increased number of gold particles inside the phagolysosome. The estimated size of (A) is 3.5 μm. Ph, phagolysosome; E, E. coli. (B) NE kills E. coli through its catalytic activity. E. coli were incubated with or without NE. Similar experiments were repeated where NE was inactivated and viable bacterial counts were determined. SLPI (at 0.05 μM) inhibited NE activity (>95%) but exhibited unsubstantial antibacterial activity against E. coli. Results represent the mean of four experiments; error bars indicate the standard error of the mean. (C) Fluorescence microscopy (FM) of bacteria. E. coli were incubated without (live) or with NE (+NE) as described above, and the reactions were stained with a mixture of DAPI and SYTOX. DNA of live (intact cell membranes) and dead (disrupted membranes) bacteria fluoresce blue with DAPI and bright green with SYTOX (magnification, ×2000). (D andE) Electron micrographs of bacteria incubated with (+) or without (−) NE. E. coli and S. aureus were cultured with or without NE, and the reactions were processed for SEM and TEM. Single cells and aggregates of bacteria are shown. (E) In the presence of NE, the coccus morphology (S. aureus) remained intact, and the bacillus morphology (E. coli) was distorted (determined by SEM) (scale bar, 1.5 μm). (D) In the absence of NE, the outer and inner membranes are intact, but the addition of NE resulted in a distorted structure (determined by TEM) (scale bar, 0.5 μm). Insets represent cross sections of bacteria.

Unlike Gram-positive bacteria, Gram-negative bacteria typically have an outer membrane (Om). We hypothesized that Om proteins were susceptible to cleavage by NE, resulting in cell death. To address this, we cultured E. coli with or without NE, and the reactions were processed for both scanning and transmission electron microscopy (SEM and TEM) (5, 11). Upon addition of NE, the rodlike morphology of E. coli was perturbed, with the appearance of “nodules” and collapsed architecture (Fig. 1E). Loss of membrane integrity was further confirmed with TEM. In the presence of NE, E. coli lost its discernable inner and outer membranes, resulting in a disrupted and frayed appearance (Fig. 1D). In contrast, the coccus morphology of Staphylococcus aureus was intact after incubation with NE, consistent with the inability of NE to kill this Gram-positive bacterium (Fig. 1E).

To determine the bacterial target(s) of NE, we grew E. coliand subjected it to fractionation (12, 13). As previously shown (14) and because of its potent activity, NE degraded several proteins in both cytoplasmic (Cm) and outer membranes. The Om fraction, although complex, comprised two major bands identified as Om protein C and A (Omp C and OmpA) (Fig. 2A). Strikingly, OmpA, but not OmpC, was completely and rapidly degraded by NE (Fig. 2, A, C, and D). Similar findings were observed with Klebsiella pneumoniae (Fig. 2B). CG and GB (15) did not cleave OmpA (Fig. 2, E and F).

Figure 2

NE, but not GB and CG, degrades OmpA. E. coli were grown and subjected to subfractionation. (A) Although complex, the Om fraction contains two major proteins that were identified by NH2-terminal amino acid (a.a.) sequencing (24). (B) Membrane fractions (Cm/Om and Om) were incubated with NE for 1 hour. (C and E) The Om fraction was incubated with NE, GB, and CG for varying times. These reactions were performed in duplicate. Reactions (C) and (E) were resolved with SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and were stained with Coomassie blue. Duplicate reactions (D) and (F) were processed for Western blotting by using antibody to OmpA. Arrow indicates NE; the asterisk shows GB.

OmpA is highly conserved in a wide range of Gram-negative bacteria, and multiple potential functions have been ascribed to it, including maintenance of virulence, structural integrity, and porin activity (16). To determine the importance of OmpA cleavage for NE-mediated killing of E. coli, we first verified that OmpA was evenly distributed on the cell surface by immuno-EM (17,18) (Fig. 3A). Incubation of intact bacteria with NE was accompanied by considerable degradation of OmpA, as demonstrated by immunoblotting (19) (Fig. 3B), coinciding with bacterial death (Fig. 3C). Next, to assess the contribution of OmpA to NE-mediated killing, we incubated NE with wild-type (WT)E. coli, isogenic mutants where OmpA expression was disrupted (OmpA), and OmpA E. coli that were rescued by complementation (OmpA+) (8, 11, 20). Exposure of the WT strain to NE for 4 hours resulted in altered morphology and a decrease in growth (Fig. 3C). However, in the absence of OmpA, NE had no effect on viability or the morphology of OmpA (Fig. 3D). Reconstitution of OmpA expression restored WT E. coli growth characteristics and NE-mediated killing for OmpA+ (Fig. 3E).

Figure 3

Characterization of E. coliand isogenic mutant strains after incubation with NE. (A) WTE. coli were processed for immuno-gold negative labeling using antibody to OmpA. Shown is an E. coli bacterium (2.5 μm) with OmpA evenly distributed on the Om. (B) E. coli (106 CFUs) were cultured with or without NE (2 μM) for 4 hours, boiled, and resolved directly with SDS-PAGE and processed for Western blotting using antibody to OmpA. There is a decreased level of OmpA in the presence of NE (arrowhead). (C) WT, (D) OmpA, and (E) OmpA+ E. coli were cultured with or without NE. Viability and morphology of bacteria were followed after 4 hours of incubation. Bacterial growth data represent the mean of four different experiments; error bars indicate the standard error of the mean {scale bar, 1.5 μm [applicable to SEM images in (C) through (E)]}.

Next, we compared the ability of WT (NE+/+) and NE-deficient (NE–/–) neutrophils to kill WT E. coli and its isogenic OmpA in vitro (4,21). Both neutrophils recognized and interacted with both E. coli strains equally (time 0 in Fig. 4, A and C). But, NE–/–exhibited less bactericidal activity than NE+/+ after incubation with WT E. coli (Fig. 4A) (4). In Fig. 4C, both NE+/+ and NE–/– killed OmpA. However, there was no difference in the killing when we compared NE–/– to NE+/+, suggesting that bacterial death was independent of NE and that the presence of OmpA was required for NE bactericidal killing.

Figure 4

(A and C) Capacity of NE+/+ and NE−/− neutrophils to kill WT and OmpA E. coli. Bacteria (107 CFUs) were incubated in the presence of 105 freshly isolated NE+/+ and NE–/– neutrophils. After 15 min of exposure (time 0), nonadherent bacteria were removed. At that time (time 0) and 60 min later, neutrophils were solubilized with 0.1% Triton, and the number of viable bacteria (intracellular and cell-associated bacteria) was determined. Data shown are expressed as the mean of four independent experiments; error bars indicate the standard error of the mean. (B) Survival curves for NE+/+ and NE–/– mice in response to being ip challenged with WT E. coli (P = 0.0018, Wilcoxon test). (D) Survival curves for NE+/+ and NE–/– mice in response to being ip challenged with OmpA E. coli(P = 0.6852, Wilcoxon test).

To confirm the relevance of this mechanism in vivo, we infected NE–/– mice and NE+/+ littermates intraperitoneally (ip) with either WT E. coli or OmpA, and their survival was monitored over time (22). Infection with 4 × 104 CFUs of WTE. coli resulted in the death of all NE–/–mice, whereas 35% of NE+/+ mice survived (Fig. 4B) (P = 0.04, Fisher's exact test) (4). OmpA were less virulent than WTE. coli, but a dose that caused 50% of NE+/+mice to die [median lethal dose (LD50)] was achieved with 3.5 × 105 CFUs. The same LD50 was observed with NE–/– mice (Fig. 4D) (P = 0.5, Fisher's exact test) (23). Thus, the absence of OmpA negates the role of NE in host defense against E. coli.

These data confirm the role of NE in host defense againstE. coli and demonstrate that NE-mediated killing requires the presence of OmpA. NE degradation of OmpA results in cell death either by a loss of bacterial integrity or by localized weakening of the cell wall followed by osmotic lysis. Alternatively, OmpA cleavage could allow NE access to internal protein(s), resulting in further proteolysis and bacterial death. These findings highlight the importance of NE as a host defense molecule and demonstrate bacterial killing through the proteolytic attack of a specific Om protein. Thus, the function of NE needs to be reconsidered if intracellular NE inhibition is pursued to treat destructive diseases, including cystic fibrosis. Also, OmpA may represent a target in the design of therapeutic strategies against Gram-negative bacteria.

  • * To whom correspondence should be addressed. Present address: Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8052, St. Louis, MO 63110, USA; E-mail: belaaouaja{at}


View Abstract

Stay Connected to Science

Navigate This Article