Fig 1.
Step-wise assembly of the Membrane Attack Complex.
(A) Illustration depicting how a C5 convertase bound to a bacterial surface triggers the assembly of the Membrane Attack Complex (MAC). The C5 convertase converts C5 into C5a and C5b. C5b immediately binds C6 which forms the bimolecular C5b6 complex. C5b6 binds to C7, which triggers conformational changes that exposes a lipophilic domain in C7 that anchors to membranes. C5b-7 then recruits C8, which causes initial membrane protrusion. C5b-8 then recruits multiple copies of C9 which forms a membranolytic pore called the MAC. (B) Local C5 conversion and MAC assembly by a C5 convertase are essential to form a MAC pore that kills bacteria. (C) Isolated C5b6 can still trigger the assembly of MAC pores, but these MAC pores fail to kill bacteria.
Fig 2.
Killing of E. coli by MAC pores requires rapid interaction between C7 and C5b6.
(A) Schematic overview of the assay to determine the effect of the presence of C7 during C5b6 formation on bacterial killing by MAC pores. Bacteria were labelled with convertases in C5-depleted serum and washed (@) to get rid of remaining serum components. MAC pores were then either directly formed by adding all MAC components (C5-9 at t = 0), by forming C5b6 in the presence of C7 at and adding C8 and C9 after 60 minutes (C7 at t = 0) or by forming C5b6 in the absence of C7 and adding C7, C8 and C9 after 60 minutes (C7 at t = 60). Sytox is added to all bacteria after 60 minutes to analyze IM damage after 90 minutes by flow cytometry. (B) The percentage of Sytox positive bacteria of four different complement-sensitive E. coli strains (MG1655, BW25113, MC1061 and 547563.1) which were treated as described in (A). Samples with all MAC components at t = 0 are represented in black triangles, samples with C5b6 formed in the presence of C7 in blue squares and samples with C5b6 formed in the absence of C7 in red circles. (C) Schematic overview of the assay in (D) to determine the time-sensitivity of the interaction between C5b6 and C7 on bacterial killing by MAC pores. Convertase-labelled bacteria were incubated with C5 and C6 at t = 0 and C7 was added at different points in time. At t = 15, C5b6 formation was stopped by adding 25 μg/ml C5 conversion inhibitor OmCI. After 60 minutes, C8, C9 and Sytox were added to measure the percentage of Sytox positive cells by flow cytometry. (D) The percentage of Sytox positive bacteria of MG1655 bacteria which were treated as described in (C). The solid line represents samples where C7 was added in time without stopping C5b6 formation, the dashed line represents samples where C5b6 formation was stopped with OmCI at t = 15. (E) MG1655 was incubated with 1% C7-depleted serum followed by addition of C7 in time (solid line). 25 μg/ml OmCI (dashed line) was added after 30 minutes to block C5b6 formation. IM damage was determined by measuring the percentage of Sytox positive cells by flow cytometry after 90 minutes. Data represent mean +- SD (B, D and E) of at least 3 independent experiments. Statistical analysis was done using a paired 2-way ANOVA with Tukey’s multiple comparisons’ test in which all samples from one strain were compared with each other (B). Significance was shown as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 or **** p ≤ 0.0001.
Fig 3.
C7 prevents release of C5b6 from the surface.
(A) Representative Western blot for the detection of C5 and C5b in cell pellets and the supernatants taken from convertase-labelled E. coli MG1655 incubated with 100 nM C5, 100 nM C6 in the absence (red arrows) or presence (blue arrows) of 100 nM C7 for 60 minutes. As control, 50 pmol of C5, C5b6 or C5 + C5b6 was added to determine if the polyclonal anti-C5 binds both C5 and C5b to an equal extent. C5 is characterized by containing an uncleaved α-chain and β-chain, C5b by containing a cleaved α-chain (α’) and β-chain. (B) C5b6 ELISA of a titration of the supernatants used in (A). C5b6 formed on bacteria in the absence of C7 is depicted in red circles and C5b6 formed in the presence of C7 in blue squares. A mixture of 10 nM C5 and 10 nM C6 was taken as control for specificity of the ELISA for C5b6. (C) Deposition of C6-Cy5 on convertase-labelled E. coli MG1655 was shown after 60 minutes with 10 nM C5, 10 nM C6-Cy5 in the absence or presence of 10 nM of C7. C6 deposition was measured by flow cytometry and plotted as the geometric mean of fluorescence intensity (geoMFI) of Cy5 of the bacterial population. (D) Relative deposition of C6-Cy5 in time for convertase-labelled bacteria incubated with C5, C6-Cy5 in the absence or presence of C7. At t = 15, C5b6 formation was stopped by adding 25 μg/ml C5 conversion inhibitor OmCI (represented by the vertical dotted line). Relative C6 deposition was calculated by dividing the geoMFI at t = x by the geoMFI at t = 15. Data represent mean +- SD (B, C and D) of at least 3 independent experiments (A-D). Statistical analysis was done using a paired two-tailed t-test (C). Significance was shown as * p ≤ 0.05.
Fig 4.
C5b6 that is released from the bacterial surface loses its bactericidal potential.
Convertase-labelled E. coli MG1655 was treated with a titration of supernatant from E. coli + C56 described in Fig 3A containing released C5b6 (red circles) or with 10 nM uncleaved C5 and 10 nM C6 (blue squares) in the presence of 10 nM C7, 10 nM C8, 100 nM C9-Cy5 and Sytox dye for 30 minutes. Supernatant of E. coli + C56 was supplemented with 25 μg/ml C5 conversion inhibitor OmCI to inhibit the conversion of the remaining uncleaved C5. (A) The percentage of bacteria with a damaged IM as determined by Sytox staining. (B) Deposition of C9-Cy5 on bacteria was plotted as geoMFI of the bacterial population by flow cytometry. Data represent mean +- SD of at least 3 independent experiments.
Fig 5.
The presence of C7 during the generation of C5b6 results in more efficient recruitment of C9.
Convertase-labelled E. coli MG1655 was treated as in Fig 3A in which C5b6 was formed for 60 minutes in the absence (red circles) or presence (blue squares) of C7 after which C8 and C9 were added and bacteria were analyzed by flow cytometry after 30 minutes. Deposition of C9-Cy5 (A) and C6-Cy5 (B) were both measured in parallel experiments and represented as geoMFI of the bacterial population. The fluorescence ratio between C9:C6 (C) was calculated by dividing the geoMFI of C9 by the geoMFI of C6. Data represent individual samples with mean +- SD of at least 3 independent experiments. Statistical analysis was done using a paired two-tailed t-test (A-C). Significance was shown as * p ≤ 0.05 or *** p ≤ 0.001.
Fig 6.
The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope.
(A) Schematic overview of trypsin shaving of E. coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 (black squares) or 10 nM pC5b6 with 10 nM C7 (black circles). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E. coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm2 scans of whole bacteria were shown on top and representative 500x500 nm2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm2 scans and 2 deg for 500x500 nm2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.
Fig 7.
Complement-resistant E. coli can prevent efficient anchoring of C5b-7 and insertion of MAC pores into the bacterial cell envelope.
(A) C3b deposition was measured for complement-sensitive (MG1655, filled squares, and 547563.1, filled circles) and complement-resistant (552059.1, open upward triangles, and 552060.1, open downward triangles) E. coli strains treated with 10% C5-depleted serum. C3b deposition was plotted as relative fluorescence by dividing the geoMFI of the bacterial population by the geoMFI of a control sample without antibody. Convertase-labelled bacteria were incubated with purified MAC components (C5-9) with Sytox blue to determine the percentage of Sytox positive bacteria (B) and the deposition of C9-Cy5 (C) by flow cytometry. C9-Cy5 deposition was plotted as relative fluorescence by dividing the geoMFI of the bacterial population by the geoMFI of bacteria incubated with C9-Cy5 only. Trypsin shaving experiments were performed on C5b-7 labelled bacteria by incubation in 10% C8-depleted serum. (D) C8 and C9-Cy5 were first added to C5b-7 labelled bacteria to assemble MAC pores, after which bacteria were treated either with buffer or 10 μg/ml trypsin. C9-Cy5 deposition was measured by flow cytometry. (E) C5b-7 labelled bacteria were first treated with 10 μg/ml trypsin after which 50 μg/ml soy-bean trypsin inhibitor was added together with C8 and C9-Cy5 to measure C9-Cy5 deposition by flow cytometry. For both (D) and (E), C9 deposition after trypsin treatment was plotted as the percentage of the C9 deposition compared to bacteria treated with buffer. This was done by dividing the geoMFI (Cy5) of the bacteria treated with trypsin by the geoMFI (Cy5) of bacteria treated with buffer. Data represent mean +- SD of at least 3 independent experiments (547563.1 in A was absent during one experiment, resulting in only two measurements for this strain). Statistical analysis was done using an ordinary one-way ANOVA (A) or paired one-way ANOVA (B, C, D and E) with Tukey’s multiple comparisons’ test. Significance was shown as * p ≤ 0.05, ** p ≤ 0.01 or **** p ≤ 0.0001.
Fig 8.
The relevance of direct assembly and anchoring of C5b-7 to the bacterial surface for bacterial killing by MAC pores.
(A) Illustration depicting the relevance of direct assembly and anchoring of C5b-7 to form a bactericidal MAC pore. A surface-bound C5 convertase converts C5 into C5b in the presence of C6 and C7 to directly assembly C5b-7 and anchor C5b-7 to the bacterial surface. Subsequent recruitment of C8 and multiple copies of C9 assembles a stably inserted MAC pore that kills bacteria. (B) Formation of C5b6 in the absence of C7 can lead to the release of C5b6 (I) and/or a delay in the interaction between C5b6 and C7 (II). Both result in inefficient anchoring of C5b-7 to the bacterial surface (III). Inefficiently anchored C5b-7 can recruit C8 and C9 to the bacterial surface, but this does not lead to bacterial killing and is therefore referred to as a ‘non-bactericidal MAC pore’.