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The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions. The complement membrane attack complex (MAC) is a lytic immune pore that kills pathogens. Here the authors use cryoEM to provide a structural and biophysical mechanism for how β-pore forming proteins breach the lipid bilayer, providing pathways to explore pore-formation in molecular detail.

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells. CD59 protects human cells from damage by the MAC immune pore. The authors show how CD59 inhibits MAC, by deflecting pore-forming β-hairpins of complement proteins. As well as how the membrane environment influences the role of CD59 in complement regulation and in host-pathogen interactions.

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion. The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. Here, the authors use atomic force microscopy to show that MAC proteins oligomerize within the membrane, allowing them to identify the kinetic bottleneck of MAC formation.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/“tick-over” mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

Paramyosin is a thick myofibrillar protein found exclusively in invertebrates. Evidence suggested that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent. We previously reported that recombinant Trichinella spiralis paramyosin (Ts-Pmy) elicited a partial protective immunity in mice. In this study, the ability of Ts-Pmy to bind host complement components and protect against host complement attack was investigated. In this study, the transcriptional and protein expression levels of Ts-Pmy were determined in T. spiralis newborn larva (NBL), muscle larva (ML) and adult worm developmental stages by RT-PCR and western blot analysis. Expression of Ts-Pmy at the outer membrane was observed in NBL and adult worms using immunogold electron microscopy and immunofluorescence staining. Functional analysis revealed that recombinant Ts-Pmy(rTs-Pmy) strongly bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). rTs-Pmy also inhibited the lysis of rabbit erythrocytes (E(R)) elicited by an alternative pathway-activated complement from guinea pig serum. Inhibition of native Ts-Pmy on the surface of NBL with a specific antiserum reduced larvae viability when under the attack of complement in vitro. In vivo passive transfer of anti-Ts-Pmy antiserum and complement-treated larvae into mice also significantly reduced the number of larvae that developed to ML. These studies suggest that the outer membrane form of T. spiralis paramyosin plays an important role in the evasion of the host complement attack.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]- N -5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Introduction Complement component C8 is one of the five terminal complement components required for the formation of the membrane attack complex. Complete absence of C8 results in increased susceptibility to gram-negative bacteria such as Neisseria species. Materials and Methods Two functionally distinct C8 deficiency states have been described: C8 α–γ deficiency has been predominantly reported amongst Afro-Caribbeans, Hispanics, and Japanese and C8β mainly in Caucasians. Results We report a case of functional and immunochemical deficiency of the complement component C8, diagnosed in a Caucasian adult following three episodes of meningitis. Western blotting and hemolytic assay demonstrated absence of C8β. In genetic studies, the common exon 9 C > T transition responsible for 85% of C8β deficiencies was not found. Two mutations were identified: a novel duplication mutation, c.1047_1053 dupGGCTGTG in exon 7 that introduces a frame shift, resulting in the addition of seven novel amino acid residues and a premature stop codon, and a previously reported mutation, c.271C > T in exon 3. The parents each expressed one of these mutations, confirming compound heterozygosity. Discussion This is the first report of a duplication mutation in C8β deficiency and extends the molecular heterogeneity of the disorder.

CD59 protects rat kidney from complement mediated injury in collaboration with Crry. As previously reported, the membrane-bound complement regulator at the C3 level (Crry/p65) is important in maintaining normal integrity of the kidney in rats. However, the role of a complement regulator at the C8/9 level (CD59) is not clear, especially when activation of complement occurs at the C3 level. The aim of this work was to elucidate the in vivo role of CD59 under C3 activating conditions. Two monoclonal antibodies, 5I2 and 6D1, were used to suppress the function of Crry and CD59, respectively. In order to activate alternative the pathway of complement, the left kidney was perfused with 5I2 and/or 6D1 and was recirculated. In the kidneys perfused with 5I2 alone, deposition of C3 and membrane attack complex (MAC) was observed in the peritubular capillaries, vasa recta, and tubular basement membranes. Cast formation, tubular dilation and degeneration, and cellular infiltration were observed at days 1 and 4, and they recovered by day 7. Further suppression of CD59 by 6D1 significantly enhanced the deposition of MAC and worsened the already exacerbated tubulointerstitial injury. These effects of 6D1 were dose dependent. Perfusion with 6D1 alone did not induce histologic damage or MAC deposition in the tubulointerstitium. In rats, CD59 maintains normal integrity of the kidney in collaboration with Crry in rats against complement-mediated damage in vivo.

Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8alpha at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane beta hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8alpha is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.