The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection

Abstract

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number (n = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.

Keywords: enteroaggregative Escherichia coli, complement; ficolin-2; lectin pathway; serum resistance.

Figure 1

Consumption of recombinant MBL (rMBL), rficolin-1, -2, and -3 by prototypic EAEC strains. EAEC prototype strains JM221, 042, C1010-00, and 55989 were incubated with (A) rMBL (0.5 µg/ml), (B) rficolin-1 (2.0 µg/ml), (C) rficolin-2 (0.5 µg/ml), or (D) rficolin-3 (0.5 µg/ml) and the unbound protein fraction was transferred to wells coated with mannan or acetylated bovine serum albumin and detected by specific mAb against mannose-binding lectin (MBL), ficolin-1, -2, or -3.

Figure 2

Consumption of serum ficolin-2 by 56 clinical Enteroaggregative Escherichia coli (EAEC) isolates. 56 clinical EAEC isolates and a control containing GlcNAc beads were incubated with 10% normal human serum and the unbound protein fraction was transferred to ELISA plates coated with acetylated bovine serum albumin, in twofold serial dilutions. Specific mAb were used to detect ficolin-2. Boxes show isolates binding ficolin-2 and * marks isolate C322-17 showing high binding of ficolin-2.

Figure 3

Western blot detection of bound serum ficolin-2 to isolate C322-17. Isolates C322-17 (high binding), E3-1065539 (low binding), and H57553 (low binding) was incubated with 10% normal human serum and the eluates were separated by SDS PAGE and detected by western blot. 0.25 µg rficolin-2 was added as a positive control and specific mAb was used to detect ficolin-2.

Figure 4

Ficolin-2 binding is independent of calcium. Isolate C322-17 was incubated with 10% normal human serum (NHS) in Barb-fetal calf serum (FCS), and 10% NHS in Barb-FCS + 10 mM ethylene diamine tetraacetic acid (EDTA). Binding of ficolin-2 was detected with specific biotinylated mAb against ficolin-2 and FITC-conjugated Streptavidin. Complement deposition was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Results represent the means of three independent experiments ± SD.

Figure 5

Inhibitory effects of ficolin-2 inhibitor on ficolin-2 binding. 10% normal human serum (NHS) was combined with ficolin-2 inhibitor FCN212 (5 µg/ml) and incubated with isolate C322-17. Detection was performed with specific biotinylated monoclonal antibodies against ficolin-2, followed by FITC-conjugated Streptavidin. (A) Ficolin-2 deposition was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). (B) Residual EAEC cells from flow cytometry was placed on slides by cytospin and examined by bright field (BF) and fluorescent microscopy (FITC). Results represent the means of three independent experiments ± SD, ****p ≤ 0.0001 (unpaired Student’s t-test).

Figure 6

Inhibitory effects of a ficolin-2 inhibitor on C4b, C3b, and membrane attack complex (MAC) deposition. 10% normal human serum (NHS) was combined with ficolin-2 inhibitor FCN212 (5 µg/ml) and incubated with isolate C322-17. Detection was performed with specific biotinylated monoclonal antibodies against (A) C4b, (B) C3b, and (C) MAC, followed by FITC-conjugated Streptavidin. Complement deposition was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Results represent the means of three independent experiments ± SD, ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired Student’s t-test).

Figure 7

Inhibitory effects of a C1q inhibitor on C4b, C3b, and membrane attack complex (MAC) deposition. 10% normal human serum (NHS) was combined with C1q inhibitor C1q-85 (5 µg/ml) and incubated with isolate C322-17. Detection was done with specific biotinylated monoclonal antibodies against (A) C4b, (B) C3b, and (C) MAC, followed by FITC-conjugated Streptavidin. Complement deposition was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Results represent the means of three independent experiments ± SD. No significant differences were detected between the C1q inhibitor and the mock inhibitor, ns p > 0.05 (unpaired Student’s t-test).

Figure 8

Inhibitory effects of a factor D inhibitor on C3b, and membrane attack complex (MAC) deposition. 10% normal human serum (NHS) was combined with a factor D inhibitor (5 µg/ml) and incubated with isolate C322-17. Detection was done with specific biotinylated monoclonal antibodies against (A) C3b and (B) MAC, followed by FITC-conjugated Streptavidin. Complement deposition was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Results represent the means of three independent experiments ± SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired Student’s t-test).

Figure 9

Inhibitors of ficolin-2 and factor D reduce serum-mediated enteroaggregative Escherichia coli (EAEC) killing. Bacterial growth in normal human serum (NHS) measured by colony-forming unit (CFU)/ml. EAEC isolate C322-17 was incubated with 10% NHS in combination with complement inhibitors of ficolin-2, factor D, C1q, C3 (compstatin), and C5 (eculizumab). A baseline (No NHS) was established from an overnight culture under normal growth conditions without the presence of NHS or inhibitors. Results represent the means of three independent experiments ± SD. All conditions are compared to 10% NHS, ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01 (unpaired Student’s t-test).