Acquired resistance of Escherichia coli to complement lysis by binding of glycophosphoinositol-anchored protectin (CD59)

Abstract

Protectin (CD59) is a glycophosphoinsitol (GPI)-anchored defender of human cells against lysis by the membrane attack complex of complement. In this study, we examined whether protectin released from human cell membranes can incorporate into the surface of gram-negative bacteria. Analysis by using radiolabeled protectin, immunofluorescence, flow cytometry, and whole-cell enzyme-linked immunosorbent assay demonstrated that protectin bound to nonencapsulated Escherichia coli EH237 (Re) and EH234 (Ra) in a calcium-dependent manner. The incorporation required the GPI-phospholipid moiety since no binding of a phospholipid-free soluble form of protectin was observed. Mg2+ did not enhance the binding, and a polysialic acid capsule prevented it (strain IH3080 [O18:K1:H8]). Bound protectin inhibited the C5b-9 neoantigen expression on complement-treated bacteria. Protection against complement lysis was observed in both a colony counting assay and a bioluminescence assay, where viable EH234 bacteria expressing the luciferase gene emitted green light in the presence of the luciferine substrate. In general, two- to four-times-higher serum concentrations were needed to obtain 50% lysis of protectin-coated versus noncoated bacteria. The results indicate that protectin can incorporate in a functionally active form into the cell membranes of the two nonencapsulated deep rough E. coli strains studied.

FIG. 1

Effects of Ca²⁺ and Mg²⁺ on binding of protectin to E. coli EH234, EH237, and IH3080. Three strains of E. coli were incubated (30 min at 37°C) with ¹²⁵I-CD59_E (0.4 μg/10⁹ bacteria) in VBS in the presence of the indicated concentrations of Ca²⁺ (A) or Mg²⁺ (B). Note a dose-dependent enhancing effect of Ca²⁺ on the binding of CD59_E to the nonencapsulated strains EH234 (Ra) and EH237 (Re) but not to the encapsulated strain IH3080 (O18:K1:H8). Mg²⁺ has no effect on the binding of lipid-tailed protectin to the bacteria examined. (C) Comparison of binding of ¹²⁵I-CD59_E and soluble ¹²⁵I-CD59_U to E. coli EH237 in the presence of 2.5 mM Ca²⁺. Unlike CD59_E, soluble CD59_U fails to bind to the rough, nonencapsulated strain EH237. bd, background.

FIG. 2

Demonstration of binding of the glycolipid-tailed CD59_E to E. coli EH237 by indirect immunofluorescence microscopy. In this assay, 0.4 μg of CD59_E was incubated with 10⁹ bacteria in the presence (A) or absence (B) of 2.5 mM of Ca²⁺ for 30 min at 37°C. Washed bacteria were incubated with a mouse MAb (BRIC229) against CD59 and further with FITC-conjugated secondary antibody. Control stainings were performed by omitting CD59 (C) or the primary antibody (D) or by incubating the bacteria with an irrelevant primary antibody (E). Washed bacteria were mixed with the mounting medium, spread on microscope slides, and covered with a coverslip. A membranous staining of EH237 for CD59 can be seen in panel A. The staining for CD59 is weak in the absence of Ca²⁺ (B) and negative in the controls (C to E). Bar = 5 μm.

FIG. 3

Analysis of CD59 binding to E. coli EH237 by flow cytometry. A 0.4-μg aliquot of CD59_E was incubated with 10⁹ bacteria (EH237) in the presence of 2.5 mM of Ca²⁺ at 37°C for 30 min. The washed bacteria were incubated with an antibody against CD59 (mouse MAb BRIC229) and exposed to FITC-conjugated secondary antibody (rabbit anti-mouse IgG). A total of 10,000 cells were counted, and histograms showing counts per channel (y axis) relative to fluorescence intensity (x axis) are shown. Bacteria incubated with CD59 show a mean intensity of 8.0, whereas the native bacteria show a mean intensity of 3.8 for green fluorescence.

FIG. 4

Protection of E. coli EH237 against complement lysis by CD59_E. Human erythrocyte protectin was incorporated into E. coli EH237 as for Fig. 2, whereafter the bacteria were incubated (30 min, 37°C) with indicated concentrations of NHS. The ability of bacterium-bound CD59_E to prevent complement-mediated bacteriolysis was studied by counting the CFU of serial 10-fold dilutions of the bacterial suspensions. The survival of serum-treated bacteria is expressed as percentage of surviving bacteria exposed to NHSi. After incorporation of CD59_E, the survival (at 1.7% of NHS) rose from 4 to 45%. Mean (±SD) values of duplicates are shown in this representative example of two similar experiments.

FIG. 5

Analysis of the ability of bacterium-bound CD59_E to prevent cell death by using the bioluminescent recombinant E. coli strain EH234. A gene (lucGR) encoding the luciferase enzyme was cloned and expressed in E. coli EH234 (Ra). The recombinant bacteria were incubated with CD59_E or VBS as for Fig. 2 and treated with increasing concentrations of NHS. d-Luciferin, the substrate for luciferase, was added, and the luminescence of the bacteria was measured after 60 min with a luminometer. In the presence of the luciferase enzyme and luciferine substrate, the bacteria emit green light but cell death leads to the loss of enzyme activity and light emission. Mean (±SD) values of quadruplicates are shown in this representative example of four similar experiments.

FIG. 6

(A) Inhibition of MAC assembly (as measured by C5b-9 neoepitope expression) by incorporation of CD59_E into E. coli EH237 studied by whole-cell ELISA. The glycolipid-anchored protectin was incorporated into E. coli EH237, and the bacteria were incubated with the indicated dilutions of NHS. Washed bacteria were incubated with a mouse MAb against a C5b-9 neoepitope (or CD59, in panel B) and further with a peroxidase-conjugated secondary antibody (rabbit anti-mouse). Results from control incubations performed by omitting the primary antibody have been subtracted as background (optical density [OD] of <0.4). Results are shown as mean ± SD (n = 6). The significances of differences in MAb binding were examined by the two-tailed paired Student’s t test. ∗∗∗, P < 0.001; ∗∗, P < 0.01. (B) Correlation between CD59_E binding and C5b-9 assembly on E. coli EH237 analyzed by simple linear regression analysis. The binding of CD59_E and C5b-9 epitope expression on the bacteria were found to be inversely proportional.