The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC is formed when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although the release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as a biomarker.

The file "Underlying data points graphs (Fig 1 until Fig 6 supplement 1a)" contain the underlying data for all graphs in Figure 1 up and until Figure 5 (including supplements), Figure 6ab, and Fig 6 supplement 1a. Descriptions of the experimental procedure for these data and what the data points represent are in the README file. Data underlying Figure 6c-f, Figure 6 - supplement 1b-e, and Figure 7 have been deposited to the ProteomeXchange partner MassIVE database and assigned the identifier MSV000088560 available at https://doi.org/10.25345/C5QW00.

The dataset "flow cytometry.zip" contains the supporting raw data (.fsc or .mqd files) used to analyze binding of complement components to bacteria by flow cytometry. Data have been organized in a folder underlying the figure for which they have been used and placed in a subfolder for each individual experiment (sorted by date, YYYY-MM-DD). FlowJo can be used to open and analyze the .fcs and .mqd. files. Also, .xlsx files have been added to indicate which sample corresponds with which .fsc or .mqd file.

The dataset "Gels.zip" contains complete images of all SDS-PAGE or BN-PAGE gels (after subsequent Western blotting, Silver staining or fluorescence imaging) used in this study. Lanes that were discussed in this study have been indicated in the image.