Alternative Pathway Convertase Research Articles

#1940 Plasma complement activation profile in kidney transplant recipients with IgAN supports the importance of the complement alternative pathway in IgAN

Abstract Background and Aims The alternative complement and lectin pathways are key players in IgA nephropathy (IgAN). However, the precise dynamics of complement activation in IgAN remain poorly understood. Non-invasive complement biomarkers are urgently needed in view of the evolving landscape of complement-targeting therapies under evaluation in IgAN. Method We analyzed plasma complement activation biomarkers in a cohort of 87 kidney transplant recipients (KTR) with IgAN and 30 controls consisting of autosomal dominant polycystic kidney disease (ADPKD) KTR (NCT05234463, DC-2013-1990). Patients were excluded if 1) they had a biological inflammatory syndrome at the time of sampling or 2) they had experienced a cellular/humoral rejection in the year prior to or after sampling. We simultaneously measured a large panel of complement activation and regulation fragments (C3a, C5a, C4a, sC5b-9, Ba, Bb) and intact proteins (C3, C4, FI, FH, properdin) in plasma using multiplex ELISA (Quidel Ortho®, A900), nephelometry and in-house ELISA. Results There was no consumption of C3 and C4 proteins in IgAN patients. However, C3a and Bb concentrations were elevated in IgAN patients compared to ADPKD patients (161 [98-227] vs. 102 [66-172] ng/mL, p = 0.007; 3.0 [2.6-4.0] vs. 2.5 [2.0-2.8] µg/mL, p < 0.0001). Overall, 70/87 IgAN patients (80.5%) had elevated Bb levels. The plasma concentration of C5a was elevated in IgAN patients compared to ADPKD patients (6.3 [3.8-9.2] vs. 4.9 [2.8-6.3] ng/mL, p = 0.0274), with values within the reference range in 92.0% of IgAN patients. There was no difference in plasma levels of sC5b-9 between the two groups (p = 0.186). The combined analysis of C3a, C5a and sC5b-9 allowed us to distinguish 4 clusters of patients (Fig. 1). A total of 48 patients (55.2%) show evidence of C3 convertase activity in plasma (clusters 3 and 4), including 16 patients (18.4%) with the highest plasma concentrations of sC5b-9, C3a and Bb. In contrast, 15 patients (17.2%, cluster 1) have no (or very low) complement activation. The last group (n = 32, 36.8%, cluster 2) shows an isolated increase in sC5b-9 without evidence of plasma C3 convertase activity. Proteinuria was significantly increased in groups 3 and 4, which had the highest C3a concentrations, compared to groups 1 and 2 (p = 0.0051). Conclusion This study provides insights into the mechanisms of complement activation in IgAN, with a pivotal role for the activity and regulation of the alternative C3 pathway convertase. In addition, the combined analysis of complement biomarkers identified, for the first time, clusters of patients who may benefit from complement targeting therapies.

A Cell-Based Assay to Measure the Activity of the Complement Convertases

IntroductionThe complement system serves as a crucial defense mechanism against invading pathogens, but dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical/lectin and the alternative pathway convertases. Aberrant regulation of the convertases is often implicated in the development of rare complement-related diseases. However, analyzing convertase activity poses a significant challenge due to their labile nature and intricate interactions with serum proteins. MethodsIn this study, we propose a novel assay for the functional evaluation of these complexes. Our approach leverages a widely available human lymphoma cell line, which, when sensitized with antibodies, triggers activation of the classical pathway with a substantial amplification by the alternative pathway. The combined action of two C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors are washed away and guinea pig serum in EDTA buffer supports the development of lytic sites on the platform of pre-existing convertases. ResultsThe assay detects recombinant gain-of-function components of both convertase types within human serum or plasma. Furthermore, we demonstrate the assay's practical utility in analyzing nephrological patients harboring C3 genetic variants and illustrate its capacity to distinguish between patients and asymptomatic relatives carrying the same pathogenic C3 variant. ConclusionsWe provided a proof of concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or these of unknown significance in ubiquitous complement proteins like C3.

Heat-inactivated Factor B inhibits alternative pathway fluid-phase activation and convertase formation on endothelial cell-secreted ultra-large von Willebrand factor strings

Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.

Complement factors B, D, C3bBbP and risk of future venous thromboembolism.

The complement system appears to be involved in the pathogenesis of venous thromboembolism (VTE). We investigated the association of complement factors (CF) B, D, and the alternative pathway convertase, C3bBbP, measured at inclusion, with the risk of future VTE in a nested case-control study; 380 VTE patients and 804 age- and sex-matched controls derived from the Tromsø study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE across tertiles of CF concentrations were estimated using logistic regression. There was no association between CFB or CFD and risk of future VTE. Higher levels of C3bBbP gave an increased risk of provoked VTE; subjects in Q4 had a 1.68-fold higher OR compared with Q1 in the age-, sex- and BMI-adjusted model (OR 1.68; 95% CI 1.08-2.64). There was no increased risk of future VTE in individuals with higher levels of complement factors B or D of the alternative pathway. Increased levels of the alternative pathway activation product, C3bBbP, showed an association with future risk of provoked VTE.

A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors.

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

The susceptibility of the kidney to alternative pathway activation-A hypothesis.

The glomerulus is often the prime target of dysregulated alternative pathway (AP) activation. In particular, AP activation is the key driver of two severe kidney diseases: atypical hemolytic uremic syndrome and C3 glomerulopathy. Both conditions are associated with a variety of predisposing molecular defects in AP regulation, such as genetic variants in complement regulators, autoantibodies targeting AP proteins, or autoantibodies that stabilize the AP convertases (C3- and C5-activating enzymes). It is noteworthy that these are systemic AP defects, yet in both diseases pathologic complement activation primarily affects the kidneys. In particular, AP activation is often limited to the glomerular capillaries. This tropism of AP-mediated inflammation for the glomerulus points to a unique interaction between AP proteins in plasma and this particular anatomic structure. In this review, we discuss the pre-clinical and clinical data linking the molecular causes of aberrant control of the AP with activation in the glomerulus, and the possible causes of this tropism. Based on these data, we propose a model for why the kidney is so uniquely and frequently targeted in patients with AP defects. Finally, we discuss possible strategies for preventing pathologic AP activation in the kidney.

Experimentally Induced Anti-Myeloperoxidase Vasculitis Is Not Attenuated in Factor B or VISTA Deficient Mice

Background: Anti-neutrophil cytoplasmic antibody vasculitis is characterized by antibodies to myeloperoxidase or proteinase 3. Previous work in murine anti-myeloperoxidase vasculitis has shown a role for the alternative pathway complement component factor B and the anaphylatoxin C5a. However, mice deficient in properdin, which stabilizes the alternative pathway convertase, were not protected. V-Type immunoglobulin domain-containing suppressor of T-cell activation (VISTA)-deficient mice were protected in the nephrotoxic nephritis model but the role of VISTA in anti-myeloperoxidase vasculitis is unknown. Objectives: This study had 2 aims. First, we attempted to reproduce previous findings on the role of factor B in anti-myeloperoxidase vasculitis. Second, we examined the role of VISTA in this model, in order to see if the protection in the nephrotoxic nephritis model extended to anti-myeloperoxidase vasculitis. Methods: Anti-myeloperoxidase vasculitis was induced in wild type, factor B, or VISTA deficient mice. Disease was assessed by quantifying glomerular crescents and macrophages, in addition to albuminuria and serum creatinine. Results: When wild type and factor B deficient mice were compared, there were no differences in any of the histological or biochemical parameters of disease assessed. Similarly, when wild type or VISTA deficient mice were compared, there were no differences. Conclusions: Factor B deficient mice were not protected which is in contrast to previous studies. Therefore alternative pathway activation is not essential in this model, under the conditions used in this study. VISTA deficient mice were not protected, suggesting that therapies targeting VISTA may not be effective in vasculitis.

The Role of Properdin in C5 Convertase Activity and C5b-9 Formation in the Complement Alternative Pathway

Abstract The complement system is an important part of innate immunity. Complement activation leads to formation of convertase enzymes, switch of their specificity from C3 to C5 cleavage, and generation of lytic membrane attack complexes (C5b-9) on surfaces of pathogens. Most C5 cleavage occurs via the complement alternative pathway (AP). The regulator properdin promotes generation and stabilization of AP convertases. However, its role in C5 activation is not yet understood. In this work, we showed that serum properdin is essential for LPS- and zymosan-induced C5b-9 generation and C5b-9–mediated lysis of rabbit erythrocytes. Furthermore, we demonstrated its essential role in C5 cleavage by AP convertases. To this end, we developed a hemolytic assay in which AP convertases were generated on rabbit erythrocytes by using properdin-depleted serum in the presence of C5 inhibitor (step 1), followed by washing and addition of purified C5–C9 components to allow C5b-9 formation (step 2). In this assay, addition of purified properdin to properdin-depleted serum during convertase formation (step 1) was required to restore C5 cleavage and C5b-9–mediated hemolysis. Importantly, C5 convertase activity was also fully restored when properdin was added together with C5b-9 components (step 2), thus after convertase formation. Moreover, with C3-depleted serum, not capable of forming new convertases but containing properdin, in step 2 of the assay, again full C5b-9 formation was observed and blocked by addition of properdin inhibitor Salp20. Thus, properdin is essential for the convertase specificity switch toward C5, and this function is independent of properdin’s role in new convertase formation.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

Functional Hemolytic Test for Complement Alternative Pathway Convertase Activity.

The complement system is a key part of innate immunity. However, if the system becomes dysregulated, damage to healthy host cells can occur, especially to the glomerular cells of the kidney. The convertases of the alternative pathway of the complement system play a crucial role in complement activation. In healthy conditions, their activity is strictly regulated. In patients with diseases caused by complement alternative pathway dysregulation, such as C3 glomerulopathy and atypical hemolytic uremic syndrome, factors can be present in the blood that disturb this delicate balance, leading to convertase overactivity. Such factors include C3 nephritic factors, which are autoantibodies against the C3 convertase that prolong its activity, or genetic variants resulting in a stabilized convertase complex. This chapter describes a method in which the activity and stability of the alternative pathway convertases can be measured to detect aberrant serum factors causing convertase overactivity.

Anti-Factor B Antibodies and Acute Postinfectious GN in Children.

The pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that, unlike acute postinfectious GN, has a poor prognosis. This retrospective study investigated mechanisms of complement activation in 34 children with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement protein autoantibodies, carried out related functional characterization, and compared results with those of 60 children from the National French Registry who had C3 glomerulopathy and persistent hypocomplementemia. All children with acute postinfectious GN had activation of the alternative pathway of the complement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3 convertase) were found in a significantly higher proportion of children with the disorder versus children with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase activity in vitro, confirming their pathogenic effect. We also identified crucial antibody binding sites on factor B, including one correlated to disease severity. These findings elucidate the pathophysiologic mechanisms underlying acute postinfectious GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as C3 glomerulopathy, and to help determine therapy.

Properdin: A Novel Target for Neuroprotection in Neonatal Hypoxic-Ischemic Brain Injury.

Background: Hypoxic-ischemic (HI) encephalopathy is a major cause of neonatal mortality and morbidity, with a global incidence of 3 per 1,000 live births. Intrauterine or perinatal complications, including maternal infection, constitute a major risk for the development of neonatal HI brain damage. During HI, inflammatory response and oxidative stress occur, causing subsequent cell death. The presence of an infection sensitizes the neonatal brain, making it more vulnerable to the HI damage. Currently, therapeutic hypothermia is the only clinically approved treatment available for HI encephalopathy, however it is only partially effective in HI alone and its application in infection-sensitized HI is debatable. Therefore, there is an unmet clinical need for the development of novel therapeutic interventions for the treatment of HI. Such an alternative is targeting the complement system. Properdin, which is involved in stabilization of the alternative pathway convertases, is the only known positive regulator of alternative complement activation. Absence of the classical pathway in the neonatal HI brain is neuroprotective. However, there is a paucity of data on the participation of the alternative pathway and in particular the role of properdin in HI brain damage.Objectives: Our study aimed to validate the effect of global properdin deletion in two mouse models: HI alone and LPS-sensitized HI, thus addressing two different clinical scenarios.Results: Our results indicate that global properdin deletion in a Rice-Vannucci model of neonatal HI and LPS-sensitized HI brain damage, in the short term, clearly reduced forebrain cell death and microglial activation, as well as tissue loss. In HI alone, deletion of properdin reduced TUNEL+ cell death and microglial post-HI response at 48 h post insult. Under the conditions of LPS-sensitized HI, properdin deletion diminished TUNEL+ cell death, tissue loss and microglial activation at 48 h post-HI.Conclusion: Overall, our data suggests a critical role for properdin, and possibly also a contribution in neonatal HI alone and in infection-sensitized HI brain damage. Thus, properdin can be considered a novel target for treatment of neonatal HI brain damage.

Crystallization and X-ray analysis of monodisperse human properdin.

The 54 kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be crystallized, and complete data sets extending to 2.8 Å resolution for the intact FP monomer and to 3.5 Å resolution for the truncated variant were collected. The principle of specific monomer excision and domain removal by the insertion of a protease cleavage site may be broadly applicable to structural studies of oligomeric, flexible and modular proteins.

Properdin binds independent of complement activation in an in vivo model of anti–glomerular basement membrane disease

Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent invitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin invivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues invivo independent of its main ligand C3.

Generation of an alternative pathway convertase by contact-activated C3 is dependent on the conformation of C3

Generation of an alternative pathway convertase by contact-activated C3 is dependent on the conformation of C3

The role of properdin in complement-mediated renal diseases: a new player in complement-inhibiting therapy?

Properdin is known as the only positive regulator of the complement system. Properdin promotes the activity of this defense system by stabilizing its key enzymatic complexes: the complement alternative pathway (AP) convertases. Besides, some studies have indicated a role for properdin as an initiator of complement activity. Though the AP is a powerful activation route of the complement system, it is also involved in a wide variety of autoimmune and inflammatory diseases, many of which affect the kidneys. The role of properdin in regulating complement in health and disease has not received as much appraisal as the many negative AP regulators, such as factor H. Historically, properdin deficiency has been strongly associated with an increased risk for meningococcal disease. Yet only recently had studies begun to link properdin to other complement-related diseases, including renal diseases. In the light of the upcoming complement-inhibiting therapies, it is interesting whether properdin can be a therapeutic target to attenuate AP-mediated injury. A full understanding of the basic concepts of properdin biology is therefore needed. Here, we first provide an overview of the function of properdin in health and disease. Then, we explore its potential as a therapeutic target for the AP-associated renal diseases C3 glomerulopathy, atypical hemolytic uremic syndrome, and proteinuria-induced tubulointerstitial injury. Considering current knowledge, properdin-inhibiting therapy seems promising in certain cases. However, knowing the complexity of properdin’s role in renal pathologies in vivo, further research is required to clarify the exact potential of properdin-targeted therapy in complement-mediated renal diseases.

Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.

Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.

Overactivity of Alternative Pathway Convertases in Patients With Complement-Mediated Renal Diseases.

Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.

Functional and structural insight into properdin control of complement alternative pathway amplification.

Properdin (FP) is an essential positive regulator of the complement alternative pathway (AP) providing stabilization of the C3 and C5 convertases, but its oligomeric nature challenges structural analysis. We describe here a novel FP deficiency (E244K) caused by a single point mutation which results in a very low level of AP activity. Recombinant FP E244K is monomeric, fails to support bacteriolysis, and binds weakly to C3 products. We compare this to a monomeric unit excised from oligomeric FP, which is also dysfunctional in bacteriolysis but binds the AP proconvertase, C3 convertase, C3 products and partially stabilizes the convertase. The crystal structure of such a FP-convertase complex suggests that the major contact between FP and the AP convertase is mediated by a single FP thrombospondin repeat and a small region in C3b. Small angle X-ray scattering indicates that FP E244K is trapped in a compact conformation preventing its oligomerization. Our studies demonstrate an essential role of FP oligomerization invivo while our monomers enable detailed structural insight paving the way for novel modulators of complement.

FACIN, a Double-Edged Sword of the Emerging Periodontal Pathogen Filifactor alocis: A Metabolic Enzyme Moonlighting as a Complement Inhibitor.

Periodontal disease is one of the most common inflammatory infectious diseases worldwide and it is associated with other syndromes, such as cardiovascular disease or rheumatoid arthritis. Recent advances in sequencing allowed for identification of novel periodontopathogens such as Gram-positive Filifactor alocis, but its virulence mechanisms remain largely unknown. We confirmed that F. alocis is a prevalent species in periodontitis patients, and we also observed strong correlation of this bacterium with clinical parameters, highlighting its role in the pathogenesis of the disease. Further, we found that preincubation of human serum with F. alocis resulted in abolished bactericidal activity and that F. alocis was surviving readily in full blood. We demonstrated that one of the key contributors to F. alocis complement resistance is a unique protein, FACIN (F. alocis complement inhibitor), which binds to C3, resulting in suppression of all complement pathways. Interestingly, FACIN is a nonclassical cell surface protein, a cytosolic enzyme acetylornithine transaminase, for which we now identified a moonlighting function. FACIN binds to C3 alone, but more importantly it also captures activated complement factor 3 within the complex with factor B, thereby locking in the convertase in an inactive state. Because of the indispensable role of alternative pathway convertase in amplifying complement cascades, its inhibition by FACIN results in a very potent downregulation of activated complement factor 3 opsonization on the pathogen surface, accompanied by reduction of downstream C5 cleavage.