Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5

The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated 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 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 three 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 biomarker.

Introduction release of C5b6 from the bacterial surface (Fig. 3b), as was already observed previously for 137 MAC-sensitive MG1655 [27]. Binding of C7 also appeared to increase C5 conversion on 138 MAC-resistant 552059.1 (Fig. 3b). However, binding of C8 to the nascent MAC triggered 139 release of C5b from MAC-resistant 552059.1, even in the presence of final MAC component 140 C9 (Fig. 3b). Binding of C8 also triggered some release of C5b from the surface of MAC-141 sensitive MG1655, but this was prevented when C9 was also present (Fig. 3b). Quantification 142 of C5b6 by ELISA revealed that binding of C8 and C9 released 4-fold more C5b6 from the 143 surface of MAC-resistant E. coli (Fig. 3d). These data suggest that binding of C8 and C9 to 144 C5b-7 triggers release of the nascent MAC from MAC-resistant E. coli. 145 Release of MAC precursors from E. coli triggers lysis of bystander human erythrocytes, 146 but is prevented by serum regulators vitronectin and clusterin 147 Although we measured the release of C5b (Fig. 2), the composition of the released complexes 148 and their capacity to lyse cells remained unclear. Previous reports showed that complement 149 activation on erythrocytes can cause MAC-dependent lysis of bystander cells that are not 150 recognized by the complement system, so-called bystander lysis [28][29][30]. We next tested 151 whether complement activation and subsequent release of MAC precursors from MAC-152 resistant E. coli can also cause bystander lysis. Therefore, E. coli were labelled with 153 convertases in C5-depleted serum as done in Fig. 2. Next, these convertase-labelled bacteria 154 were incubated with purified MAC components and unlabelled human erythrocytes to measure 155 bystander lysis (Fig. 4a). This resulted in lysis of bystander erythrocytes for all MAC-resistant 156 E. coli strains and MAC-sensitive 547563.1 (Fig. 4b), corresponding with the production of 157 sMAC (S4). Lysis was prevented in the presence of C5 conversion inhibitor OmCI (Fig. 4b), 158 suggesting that lysis was MAC-dependent. These data show that release of MAC precursors 159 from E. coli can result in bystander lysis of human cells. However, when we studied bystander 160 lysis in a human serum environment, we observed that the 552059.1 did not trigger bystander 161 lysis of erythrocytes (Fig. 4c). Serum regulators Vn and Clu are known to scavenge and 162 inactivate sMAC [31,32]. Indeed, both Vn and Clu inhibited bystander lysis of erythrocytes 163 when MAC assembled on convertase-labelled E. coli 552059.1 (as described in Fig. 4a) at 164 concentrations representative for 10% serum (Fig. 4c). SDS-PAGE revealed that Clu, but not 165 Vn, prevents the formation of polymeric-C9 in the supernatant (S5a). This suggests that Vn 166 and Clu interfere at different stages in the assembly of sMAC. Vn and Clu both specifically 167 prevent lysis of bystander cells by MAC, since Vn and Clu did not inhibit binding of C9 (S5b), 168 or MAC-dependent killing (S5c) when MAC was assembled by local conversion of C5 on 169 convertase-labelled MAC-sensitive MG1655. Altogether, our data suggest that release of MAC 170 precursors from E. coli can trigger bystander lysis of human erythrocytes, but that serum 171 regulators Vn and Clu can inhibit this bystander lysis. 173 So far, our data indicate that on Gram-negative bacteria, sMAC is mainly released from MAC-174 resistant bacteria. Since sMAC is also increased in patient plasma that suffer from bacterial 175 infections with Gram-positive bacteria [15,16], we also wanted to measure release of sMAC 176 was released in serum by Gram-positive bacteria. Three Staphylococcus aureus strains 177 (SH1000, Wood46 and Newman), one Staphylococcus epidermidis strain (KV103) and one 178 Streptococcus agalactiae strain (COH-1) were incubated in serum. sMAC was formed in all 179 five strains and was 3-to 5-fold higher compared to MAC-sensitive E. coli MG1655 (Fig. 5a), 180 corresponding with MAC-resistant E. coli (Fig. 1d). Release of sMAC also coincided with 181 generation of more C5a (Fig. 5b), although the increase compared to MAC-sensitive MG1655 182 was smaller for Gram-positive bacteria than for MAC-resistant E. coli (Fig. 3a). Altogether, 183 these data indicate that Gram-positive bacteria also form sMAC in serum. Next, we aimed to define the molecular composition of sMAC generated when complement is 187 activated on bacteria. sMAC was generated by incubating MAC-resistant E. coli 552059.1 in 188 C6-depleted serum with His-tagged C6 and captured and isolated with HisTrap beads (S6).

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SEC was used to separate sMAC from monomeric-C6. The SEC profile of serum incubated 190 with MAC-resistant E. coli shifted to much shorter elution times compared to non-activated 191 serum (Fig. 6a, fractions B5-B12), indicating a mass shift. As a control, we analyzed 192 commercially available sMAC, which is generated with zymosan particles in serum.

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Commercial sMAC eluted from the SEC column in the same fractions (B5-B12), indicating 194 that these bacterial-eluate fractions contain sMAC. Blue-native PAGE (BN-PAGE) and 195 subsequent Western blotting for C6 and C9 confirmed that these fractions contain sMAC (Fig. 196 6b). Compared to commercial sMAC, bacterial sMAC eluted somewhat later from the SEC 197 column (Fig. 6b, most apparent in fractions B11 and B12). In addition, sMAC complexes 198 generated by E. coli seemed to run further into the gel. These data suggest that bacterial sMAC 199 complexes are have a different composition compared to commercial sMAC. 200 Menny et al. recently described that the commercially available sMAC used in our study 201 consists of C5b-8, 1-3 copies of C9 and several copies of Vn and Clu, using proteomics, cross-  Fig. 6c and S6b, c) corresponded with BN-PAGE ( Fig.   207 6b), suggesting that most bacterial sMAC eluted later from the SEC column than commercial 208 sMAC. For both bacterial and commercial sMAC, sMAC components C5, C7, and C8 were 209 present in equal amounts in a pooled sample of fractions B5-B12 (Fig. 6d). However, C6 and 210 Vn were both 2-fold more abundant for bacterial sMAC (Fig. 6d). The increased ratio of C6 is 211 likely explained by the fact that not all monomeric-C6 could be separated during SEC (as was 212 visible in fraction B9-B12 by BN-PAGE in Fig. 6b). Surprisingly, bacterial sMAC contained 213 on average less C9 per C5 (Fig. 6d, ratio of 2:1) than commercially available sMAC (Fig. 6d, 214 ratio of 3:1). The relative amount of C9 per C5 (Fig. 6e), but not other sMAC components 215 (S6d), decreased in fractions that eluted later from the SEC column for bacterial sMAC. This 216 was not observed for commercially available sMAC ( Fig. 6f and S6e). These data suggest 217 sMAC generated by bacteria contains more complexes with less C9 compared to commercial 218 sMAC.

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Finally, we assessed the relative abundance of sMAC components for sMAC that was 220 generated by Gram-positive bacteria. S. aureus Wood46 was incubated with human serum and 221 the supernatant was separated by SEC (without isolating sMAC via HisTrap beads), using non-222 activated serum as control. By profiling sMAC components in individual fractions by LC-223 MS/MS, we found that in non-activated serum these components elute later in the SEC profile, 224 corresponding to monomeric or low molecular weight complexes (Fig. 7). Upon activation, the 225 profiles of all sMAC components largely co-elute and are shifted to fractions that correspond 226 to the higher mass range. In fact, close to no monomeric MAC components were detected after 227 incubation with bacteria, suggesting that all available MAC components were incorporated into 228 hetero-oligomeric sMAC complexes. We did not observe any sMAC without Vn and Clu in 229 the bacterial activated sample, since these would be located around the 664 kDa molecular 230 weight marker, suggesting that all sMAC complexes were bound to multiple Vn and Clu 231 molecules (corresponding with the relative abundance in Fig. 6d). The complexes correlate 232 with a ratio of 1-3 C9 molecules and an average of 2 C9 molecules to C5b-8, with Clu as the 233 most abundant regulator bound to sMAC. Altogether, these data confirm that sMAC released 234 from bacteria is a heterogeneous assembly comprising a single copy of C5b-8 together with 235 multiple copies of C9, Clu and Vn in a mixture of stoichiometries.  Our study also provides molecular insight into how sMAC is formed when complement is

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The digested SEC fractions of isolated and commercial sMAC were analyzed using an Ultimate ionization was achieved using a coated fused silica emitter (New Objective) biased to 2 kV.

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The mass spectrometer was operated in positive ion mode, and the spectra were acquired in the