Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 ...(COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.
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 membrane attack complex (MAC) is the pore-forming toxin of the complement system, a relatively early evolutionary acquisition that confers upon complement the capacity to directly kill pathogens. ...The MAC is more than just a bactericidal missile, having the capacity when formed on self-cells to initiate a host of cell activation events that can have profound consequences for tissue homeostasis in the face of infection or injury. Although the capacity of complement to directly kill pathogens has been recognised for over a century, and the pore-forming killing mechanism for at least 50 years, there remains considerable uncertainty regarding precisely how MAC mediates its killing and cell activation activities. A recent burst of new information on MAC structure provides context and opportunity to re-assess the ways in which MAC kills bacteria and modulates cell functions. In this brief review we will describe key aspects of MAC evolution, function and structure and seek to use the new structural information to better explain how the MAC works.
Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated ...C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.
Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal ...structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.
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► Crystal structure of C5b6 defines a platform for MAC assembly ► Cryo-EM map of sC5b9 reveals immune protection mechanism by host proteins ► MAC immune pores resemble those of bacterial toxins ► This assembly model provides a structural basis for fighting microbial infections
Activation of the mammalian complement system initiates the formation of membrane attack complexes that lyse microbes. Hadders, Bubeck, et al. reveal the stepwise buildup of the pore-forming complex and how host cells are protected from bystander damage. X-ray and cryo-EM data show how C6 captures C5b and how C7, C8αβγ, and C9 dock sideways onto C6, aligning their β-barrel forming domains in an arc. Vitronectin and clusterin enwrap the membrane perforating segments and block polymerization of C9.
Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding ...and membrane insertion of C5b6, C7, C8, and C9. Using cryo-electron tomography with a Volta phase plate and subtomogram averaging, we imaged C5b-7, C5b-8, and C5b-9 complexes and determined the C5b-9 pore structure in lipid bilayers. The in situ C5b-9 pore structure at 2.3-nm resolution reveals a 10- to 11.5-nm cone-shaped pore starting with C5b678 and multiple copies of C9 that is poorly closed, yielding a seam between C9 and C6 substituting for the shorter β strands in C6 and C7. However, large variations of composite pore complexes are apparent in subtomograms. Oligomerized initiator complexes C5b-7 and C5b-8 show stages of membrane binding, deformation, and perforation that yield ∼3.5-nm-wide pores. These data indicate a dynamic process of pore formation that likely adapts to biological membranes under attack.
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•MAC pores are cone shaped, twisted, and poorly sealed between C9 end and C5b-8 start•Complement activation yields single and joined MAC pores in lipid membranes•Oligomerization of three to four C5b-8 complexes causes ∼3.5-nm pores in membranes•Phase plates markedly increase interpretability of cryo-electron tomograms
Immune defense by complement forms membrane attack complex pores in membranes. Using phase-plate cryo-electron tomography, Sharp et al. find heterogeneous oligomeric MAC structures in membranes. Subtomogram averaging of single pores at 2.3-nm resolution reveals a cone-shaped, twisted barrel that is incompletely closed.
Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is ...down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.
Studies in mouse models implicate complement activation as a causative factor in adverse pregnancy outcomes (APOs). We investigated whether activation of complement early in pregnancy predicts APOs ...in women with systemic lupus erythematosus (SLE) and/or antiphospholipid (aPL) antibodies.
The PROMISSE Study enrolled pregnant women with SLE and/or aPL antibodies (n=487) and pregnant healthy controls (n=204) at <12 weeks gestation and evaluated them monthly. APOs were: fetal/neonatal death, preterm delivery <36 weeks because of placental insufficiency or preeclampsia and/or growth restriction <5th percentile. Complement activation products were measured on serial blood samples obtained at each monthly visit.
APO occurred in 20.5% of SLE and/or aPL pregnancies. As early as 12-15 weeks, levels of Bb and sC5b-9 were significantly higher in patients with APOs and remained elevated through 31 weeks compared with those with normal outcomes. Moreover, Bb and sC5b-9 were significantly higher in patients with SLE and/or aPL without APOs compared with healthy controls. In logistic regression analyses, Bb and sC5b-9 at 12-15 weeks remained significantly associated with APO (OR
=1.41 per SD increase; 95% CI 1.06 to 1.89; P=0.019 and OR
=1.37 per SD increase; 95% CI 1.05 to 1.80; P=0.022, respectively) after controlling for demographic and clinical risk factors for APOs in PROMISSE. When analyses were restricted to patients with aPL (n=161), associations between Bb at 12-15 weeks and APOs became stronger (OR
=2.01 per SD increase; 95% CI 1.16 to 3.49; P=0.013).
In pregnant patients with SLE and/or aPL, increased Bb and sC5b-9 detectable early in pregnancy are strongly predictive of APOs and support activation of complement, particularly the alternative pathway, as a contributor to APOs.
Background. Patients with terminal complement pathway deficiency (TPD) are susceptible to recurrent invasive meningococcal disease (IMD). Neisseria meningitidis (Nm) strains infecting these patients ...are poorly documented in the literature. Methods. We identified patients with TPD and available Nm strains isolated during IMD. We investigated the genetic basis of the different TPDs and the characteristics of the Nm strains. Results. We included 56 patients with C5 (n = 8), C6 (n = 20), C7 (n = 18), C8 (n = 9), or C9 (n = 1) deficiency. Genetic study was performed in 47 patients and 30 pathogenic variants were identified in the genes coding for C5 (n = 4), C6 (n = 5), C7 (n = 12), C8 (n = 7), and C9 (n = 2). We characterized 61 Nm strains responsible for IMD in the 56 patients with TPD. The most frequent strains belonged to groups Y (n = 27 44%), B (n = 18 30%), and W (n = 8 13%). Hyperinvasive clonal complexes (CC11, CC32, CC41/44, and CC269) were responsible for 21% of IMD cases. The CC23 predominates and represented 26% of all invasive isolates. Eleven of the 15 clonal complexes identified fit to 12 different clonal complexes belonging to carriage strains. Conclusions. Unusual meningococcal strains with low level of virulence similar to carriage strains are most frequently responsible for IMD in patients with TPD.
Age-related macular degeneration (AMD) is a common disease that can result in severe visual impairment. Abnormal regulation of the complement system has been implicated in its pathogenesis, and CFH ...polymorphisms contribute substantially to risk. How these polymorphisms exert their effects is poorly understood. We performed enzyme-linked immunosorbent assay (ELISA) analysis on young, aged, and AMD choroids to determine the abundance of the membrane attack complex (MAC) and performed immunofluorescence studies on eyes from 117 donors to evaluate the MAC in aging, early AMD, and advanced AMD. Morphometric studies were performed on eyes with high- or low-risk CFH genotypes. ELISA confirmed that MAC increases significantly with aging and with AMD. MAC was localized to Bruch's membrane and the choriocapillaris and was detectable at low levels as early as 5 years of age. Hard drusen were labeled with anti-MAC antibody, but large or confluent drusen and basal deposits were generally unlabeled. Labeling of retinal pigment epithelium was observed in some cases of advanced AMD, but not in early disease. Eyes homozygous for the high-risk CFH genotype had thinner choroids than low-risk homozygotes (P < 0.05). These findings suggest that increased complement activation in AMD and in high-risk genotypes can lead to loss of endothelial cells in early AMD. Treatments to protect the choriocapillaris in early AMD are needed.
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