Factor H-related protein 1 promotes complement-mediated opsonization of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for a wide range of infections. The complement system is the main early host defense mechanism to control these infections. P. aeruginosa counteracts complement attack by binding Factor H (FH), a complement regulator that inactivates C3b, preventing the formation of the C3-convertase and complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere in this bacterial mechanism of resisting complement. Here, we show that FHR-1 binds to P. aeruginosa via the outer membrane protein OprG in a lipopolysaccharide (LPS) O antigen-dependent manner. Binding assays with purified components or with FHR-1-deficient serum supplemented with FHR-1 show that FHR-1 competes with FH for binding to P. aeruginosa. Blockage of FH binding to C3b deposited on the bacteria reduces FH-mediated cofactor activity of C3b degradation, increasing the opsonization of the bacteria and the formation of the potent chemoattractant C5a. Overall, our findings indicate that FHR-1 is a host factor that promotes complement activation, facilitating clearance of P. aeruginosa by opsonophagocytosis.

The crystal structure of iC3b-CR3 αI reveals a modular recognition of the main opsonin iC3b by the CR3 integrin receptor.

Complement activation on cell surfaces leads to the massive deposition of C3b, iC3b, and C3dg, the main complement opsonins. Recognition of iC3b by complement receptor type 3 (CR3) fosters pathogen opsonophagocytosis by macrophages and the stimulation of adaptive immunity by complement-opsonized antigens. Here, we present the crystallographic structure of the complex between human iC3b and the von Willebrand A inserted domain of the α chain of CR3 (αI). The crystal contains two composite interfaces for CR3 αI, encompassing distinct sets of contiguous macroglobulin (MG) domains on the C3c moiety, MG1-MG2 and MG6-MG7 domains. These composite binding sites define two iC3b-CR3 αI complexes characterized by specific rearrangements of the two semi-independent modules, C3c moiety and TED domain. Furthermore, we show the structure of iC3b in a physiologically-relevant extended conformation. Based on previously available data and novel insights reported herein, we propose an integrative model that reconciles conflicting facts about iC3b structure and function and explains the molecular basis for iC3b selective recognition by CR3 on opsonized surfaces.

Functional characterization of 105 factor H variants associated with aHUS: lessons for variant classification.

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.

Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases.

Factor H (FH)-related proteins are a group of partly characterized complement proteins thought to promote complement activation by competing with FH in binding to surface-bound C3b. Among them, FH-related protein 1 (FHR-1) is remarkable because of its association with atypical hemolytic uremic syndrome (aHUS) and other important diseases. Using a combination of biochemical, immunological, nuclear magnetic resonance, and computational approaches, we characterized a series of FHR-1 mutants (including 2 associated with aHUS) and unraveled the molecular bases of the so-called deregulation activity of FHR-1. In contrast with FH, FHR-1 lacks the capacity to bind sialic acids, which prevents C3b-binding competition between FH and FHR-1 in host-cell surfaces. aHUS-associated FHR-1 mutants are pathogenic because they have acquired the capacity to bind sialic acids, which increases FHR-1 avidity for surface-bound C3-activated fragments and results in C3b-binding competition with FH. FHR-1 binds to native C3, in addition to C3b, iC3b, and C3dg. This unexpected finding suggests that the mechanism by which surface-bound FHR-1 promotes complement activation is the attraction of native C3 to the cell surface. Although C3b-binding competition with FH is limited to aHUS-associated mutants, all surface-bound FHR-1 promotes complement activation, which is delimited by the FHR-1/FH activity ratio. Our data indicate that FHR-1 deregulation activity is important to sustain complement activation and C3 deposition at complement-activating surfaces. They also support that abnormally elevated FHR-1/FH activity ratios would perpetuate pathological complement dysregulation at complement-activating surfaces, which may explain the association of FHR-1 quantitative variations with diseases.

Extravascular hemolysis and complement consumption in Paroxysmal Nocturnal Hemoglobinuria patients undergoing eculizumab treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.