Regulation Of Alternative Pathway Research Articles

Disease-associated N-terminal Complement Factor H Mutations Perturb Cofactor and Decay-accelerating Activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19–20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1–4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1–4V62 and FH1–4I62 bind immobilized C3b with similar affinities (KD = 10–14 μm), whereas FH1–4I62 is slightly more effective than FH1–4V62 as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1–4V62 binds to C3b with comparable affinity (KD ∼12 μm) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1–4V62, binds poorly to immobilized C3b (KD >35 μm) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.

Meningococcal Group W-135 and Y Capsular Polysaccharides Paradoxically Enhance Activation of the Alternative Pathway of Complement

Although capsular polysaccharide (CPS) is critical for meningococcal virulence, the molecular basis of alternative complement pathway (AP) regulation by meningococcal CPSs remains unclear. Using serum with only the AP active, the ability of strains to generate C3a (a measure of C3 activation) and subsequently deposit C3 fragments on bacteria was studied in encapsulated group A, B, C, W-135, and Y strains and their isogenic unencapsulated mutants. To eliminate confounding AP regulation by membrane-bound factor H (fH; AP inhibitor) and lipooligosaccharide sialic acid, the meningococcal fH ligands (fHbp and NspA) and lipooligosaccharide sialylation were deleted in all strains. Group A CPS expression did not affect C3a generation or C3 deposition. C3a generated by encapsulated and unencapsulated group B and C strains was similar, but CPS expression was associated with reduced C3 deposition, suggesting that these CPSs blocked C3 deposition on membrane targets. Paradoxically, encapsulated W-135 and Y strains (including the wild-type parent strains) enhanced C3 activation and showed marked C3 deposition as early as 10 min; at this time point C3 was barely activated by the unencapsulated mutants. W-135 and Y CPSs themselves served as a site for C3 deposition; this observation was confirmed using immobilized purified CPSs. Purified CPSs bound to unencapsulated meningococci, simulated findings with naturally encapsulated strains. These data highlight the heterogeneity of AP activation on the various meningococcal serogroups that may contribute to differences in their pathogenic mechanisms.

Atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is defined by the triad of mechanical hemolytic anemia, thrombocytopenia and renal impairment. Atypical HUS (aHUS) defines non Shiga-toxin-HUS and even if some authors include secondary aHUS due to Streptococcus pneumoniae or other causes, aHUS designates a primary disease due to a disorder in complement alternative pathway regulation. Atypical HUS represents 5 -10% of HUS in children, but the majority of HUS in adults. The incidence of complement-aHUS is not known precisely. However, more than 1000 aHUS patients investigated for complement abnormalities have been reported. Onset is from the neonatal period to the adult age. Most patients present with hemolytic anemia, thrombocytopenia and renal failure and 20% have extra renal manifestations. Two to 10% die and one third progress to end-stage renal failure at first episode. Half of patients have relapses. Mutations in the genes encoding complement regulatory proteins factor H, membrane cofactor protein (MCP), factor I or thrombomodulin have been demonstrated in 20-30%, 5-15%, 4-10% and 3-5% of patients respectively, and mutations in the genes of C3 convertase proteins, C3 and factor B, in 2-10% and 1-4%. In addition, 6-10% of patients have anti-factor H antibodies. Diagnosis of aHUS relies on 1) No associated disease 2) No criteria for Shigatoxin-HUS (stool culture and PCR for Shiga-toxins; serology for anti-lipopolysaccharides antibodies) 3) No criteria for thrombotic thrombocytopenic purpura (serum ADAMTS 13 activity > 10%). Investigation of the complement system is required (C3, C4, factor H and factor I plasma concentration, MCP expression on leukocytes and anti-factor H antibodies; genetic screening to identify risk factors). The disease is familial in approximately 20% of pedigrees, with an autosomal recessive or dominant mode of transmission. As penetrance of the disease is 50%, genetic counseling is difficult. Plasmatherapy has been first line treatment until presently, without unquestionable demonstration of efficiency. There is a high risk of post-transplant recurrence, except in MCP-HUS. Case reports and two phase II trials show an impressive efficacy of the complement C5 blocker eculizumab, suggesting it will be the next standard of care. Except for patients treated by intensive plasmatherapy or eculizumab, the worst prognosis is in factor H-HUS, as mortality can reach 20% and 50% of survivors do not recover renal function. Half of factor I-HUS progress to end-stage renal failure. Conversely, most patients with MCP-HUS have preserved renal function. Anti-factor H antibodies-HUS has favourable outcome if treated early.

Treatment of Typical (Enteropathic) Hemolytic Uremic Syndrome

Typical enteropathic HUS (eHUS) is triggered by Shiga toxin (Stx)-producing bacteria (STPB), predominantly Stx-producing ESCHERICHIA COLI O157. The cell biological aspects of Stx have been well defined, but host factors potentially predisposing to the development or severity of HUS remain elusive. Treatment of eHUS includes supportive measures and invasive extracorporeal therapies. Thirty to 60% of children with eHUS require dialysis. Peritoneal and hemodialysis appear equally effective. Patient age, center experience, and equipment availability determine the choice of the modality; circulatory instability may require continuous renal replacement therapies. At present, no evidence indicates that plasma infusion or exchange therapies improve outcome of Stx-induced HUS. However, the traditional separation between diarrhea-positive (D (+)) and negative (D (-)) HUS, implying two entirely different pathological pathways, requires a fresh look: Atypical HUS may follow nonspecific diarrhea, and, conversely, STPB and fecal Stx may not be detected anymore at the time of the diagnosis of HUS. Recently, Stx has been found to directly interfere with the alternative complement pathway regulator factor H in vitro, whereas some patients with Stx-HUS demonstrate evidence of complement activation. Among newer treatments for eHUS, development of Stx-neutralizing monoclonal antibodies is the most advanced. This review concludes with a discussion of the rationale, mode of action, and status of presently available therapeutic antibodies against Stx2 and Stx1.

Regulation of Energy Partitioning and Alternative Electron Transport Pathways During Cold Acclimation of Lodgepole Pine is Oxygen Dependent

Second year needles of Lodgepole pine (Pinus contorta L.) were exposed for 6 weeks to either simulated control summer ['summer'; 25 °C/250 photon flux denisty (PFD)], autumn ('autumn'; 15°C/250 PFD) or winter conditions ('winter'; 5 °C/250 PFD). We report that the proportion of linear electron transport utilized in carbon assimilation (ETR(CO2)) was 40% lower in both 'autumn' and 'winter' pine when compared with the 'summer' pine. In contrast, the proportion of excess photosynthetic linear electron transport (ETR(excess)) not used for carbon assimilation within the total ETR(Jf) increased by 30% in both 'autumn' and 'winter' pine. In 'autumn' pine acclimated to 15°C, the increased amounts of 'excess' electrons were directed equally to 21 kPa O2-dependent and 2 kPa O2-dependent alternative electron transport pathways and the fractions of excitation light energy utilized by PSII photochemistry (Φ(PSII)), thermally dissipated through Φ(NPQ) and dissipated by additional quenching mechanism(s) (Φ(f,D)) were similar to those in 'summer' pine. In contrast, in 'winter' needles acclimated to 5 °C, 60% of photosynthetically generated 'excess' electrons were utilized through the 2 kPa O2-dependent electron sink and only 15% by the photorespiratory (21 kPa O2) electron pathway. Needles exposed to 'winter' conditions led to a 3-fold lower Φ(PSII), only a marginal increase in Φ(NPQ) and a 2-fold higher Φ(f,D), which was O2 dependent compared with the 'summer' and 'autumn' pine. Our results demonstrate that the employment of a variety of alternative pathways for utilization of photosynthetically generated electrons by Lodgepole pine depends on the acclimation temperature. Furthermore, dissipation of excess light energy through constitutive non-photochemical quenching mechanisms is O2 dependent.

Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome

AbstractComplement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D254G and K325N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.

Plasma Complement Components and Activation Fragments: Associations with Age-Related Macular Degeneration Genotypes and Phenotypes

Several genes encoding complement system components and fragments are associated with age-related macular degeneration (AMD). This study was conducted to determine whether alterations in circulating levels of these markers of complement activation and regulation are also independently associated with advanced AMD and whether they are related to AMD genotypes. Plasma and DNA samples were selected from individuals in our AMD registry who had progressed to or developed the advanced stages of AMD, including 58 with geographic atrophy and 62 with neovascular disease. Subjects of similar age and sex, but without AMD, and who did not progress were included as controls (n = 60). Plasma complement components (C3, CFB, CFI, CFH, and factor D) and activation fragments (Bb, C3a, C5a, iC3b, and SC5b-9) were analyzed. DNA samples were genotyped for seven single-nucleotide polymorphisms in six genes previously shown to be associated with AMD: CFB, CFH, C2, C3, and CFI and the LOC387715/ARMS2 gene region. The association between AMD and each complement biomarker was assessed by using logistic regression, controlling for age, sex, and proinflammatory risk factors: smoking and body mass index (BMI). Functional genomic analyses were performed to assess the relationship between the complement markers and genotypes. Concordance, or C, statistics were calculated to assess the effect of complement components and activation fragments in an AMD gene-environment prediction model. The highest quartiles of Bb and C5a were significantly associated with advanced AMD, when compared with the lowest quartiles. In multivariate models without genetic variants, the odds ratio (OR) for Bb was 3.3 (95% confidence interval [CI] = 1.3-8.6), and the OR for C5a was 3.6 (95% CI = 1.2-10.3). With adjustment for genetic variants, these ORs were substantially higher. The alternative pathway regulator CFH was inversely associated with AMD in the model without genotypes (OR = 0.3; P = 0.01). Positive associations were found between BMI and plasma C3, CFB, CFH, iC3b, and C3a. There were also significant associations between C5a fragment and LOC387715/ARMS2 and C3 genotypes (P for trend = 0.02, 0.04), respectively. C statistics for models with behavioral and genetic factors increased to 0.94 +/- 0.20 with the addition of C3a, Bb, and C5a. Increased levels of activation fragments Bb and C5a are independently associated with AMD. Higher BMI is related to increased levels of complement components. C5a is associated with AMD genotypes. C statistics are stronger with the addition of C3a, Bb, and C5a in predictive models. Results implicate ongoing activation of the alternative complement pathway in AMD pathogenesis.

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19-20 (FH19-20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19-20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19-20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19-20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19-20 to C3b/C3d is essential for target discrimination by the alternative pathway.

Complement factor H related proteins in immune diseases

The complement system is a powerful part of the host innate immune defense and is aimed to damage and eliminate microbes and modified self-cells. To protect host cells and biological surfaces from damage mediated by complement activation products a tight control of the complement system is necessary. Imbalances in complement regulation contribute to tissue injury and can result in autoimmune diseases such as hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (MPGN) or age related macular degeneration (AMD). Disease associated mutations have been identified in several complement regulators or components, such as members of the factor H protein family. This group includes the major alternative pathway regulator, complement factor H (CFH) and five complement factor H related proteins (CFHR). Homozygous chromosomal deletion of a genomic 84kb, chromosomal fragment which includes the genes CFHR1/CFHR3 is a risk factor for hemolytic uremic syndrome (HUS) at young age and is predominantly associated with the generation of autoantibodies to CFH, leading to a specific type of HUS, called DEAP (deficiency of CFHR and autoantibody positive)—HUS. The same deletion however is protective to the development of age related macular degeneration (AMD) in elderly people. Thus CFHR1 and CFHR3 proteins, and likely also the other members of this gene family are linked to human diseases. We here summarize the current knowledge about the role or association of CFHR1 and CFHR3 in the human diseases HUS and AMD.

Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice

Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin-producing enteropathic bacteria (Stx-associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx-associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx-2. Stx-2 was administered together with lipopolysaccharide to wild-type and Cfh(+/-) C57BL/6 animals. Forty-eight hours after administration of the first Stx-2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild-type or Cfh(+/-) Stx-2-treated mice. Uraemia and renal tubular apoptosis did not develop in wild-type or Cfh(+/-) animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx-2 treated animals. In summary, Stx-2 administration did not result in TMA in either Cfh(+/-) or wild-type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx-2-induced renal tubular injury.

Functional Mapping of YadA- and Ail-Mediated Binding of Human Factor H toYersinia enterocoliticaSerotype O:3

Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37 degrees C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding.

Complement Factor H Deficiency and Posttransplantation Glomerulonephritis With Isolated C3 Deposits

We report the first cases of atypical hemolytic and uremic syndrome associated with complement factor H (CFH) deficiency in native kidneys and glomerulonephritis with isolated C3 deposits after kidney transplantation. Two boys developed atypical hemolytic and uremic syndrome at 16 and 11 months of age, associated with low C3 and CFH levels. Both rapidly progressed to end-stage renal failure and received a kidney transplant. Patient 1 had combined CFH and complement factor I (CFI) heterozygous mutations and a membrane cofactor protein (gene symbol, CD46) gene polymorphism. Five years posttransplantation, an allograft biopsy specimen showed numerous mesangial and extramembranous C3 deposits, although the patient had no biological sign of glomerulopathy. Nine years after transplantation, he was well with stable kidney function. Patient 2, who had a homozygous CFH mutation, developed glomerulonephritis with isolated C3 deposits 5 months after kidney transplantation while he was treated for early recurrence of hemolytic anemia. Four years later, the second kidney transplant biopsy specimen showed recurrence of thrombotic microangiopathy. Six years posttransplantation, kidney function was stable and complete blood cell count was normal with regular plasma therapy. These observations suggest that constitutional dysregulation of the alternative pathway is associated with a wide spectrum of kidney diseases, and glomerulonephritis with isolated C3 deposits and thrombotic microangiopathy may be different expressions of the same condition. Several factors could influence the disease, such as degree of CFH haploinsufficiency and other complement alternative pathway regulator abnormalities, such as a membrane cofactor protein polymorphism.

Gpm1p Is a Factor H-, FHL-1-, and Plasminogen-binding Surface Protein of Candida albicans

The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.

Human PathogenicBorrelia spielmaniisp. nov. Resists Complement-Mediated Killing by Direct Binding of Immune Regulators Factor H and Factor H-Like Protein 1

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.

Influence of irradiation on cyanide-resistant respiration and AOX1 multi-gene family expression during greening of etiolated rice seedlings

We investigated the differential expression of AOX1 multi-gene family and the regulation of alternative respiratory pathway during initial greening development in leaves of rice (Oryza sativa L.) seedlings. After exposing the dark-grown rice seedlings to continuous irradiation, total respiration (Vt), capacity of alternative pathway (Valt), and their ratio (Valt/Vt) increased with the greening of leaves. In this process, AOX1c transcript increased under constant irradiation, while AOX1a and AOX1b transcripts were hardly detected. Thus AOX1c in rice presents a similar expression pattern as AOX2 does in many dicotyledonous species during greening development. Compared with the rapid increase of cyanideresistant respiration in the presence of photon energy, CO2 fixation was not observed until 8 h after the onset of irradiation. The AOX inhibitor salicylhydroxamic acid (SHAM; 1 mM) inhibited 67.3 % of cyanide-insensitive oxygen uptake in dark-grown leaves and 69.4 % of it in leaves grown under irradiation. Dark-grown plants pre-treated with SHAM were then irradiated for 12 h. SHAM did not obviously modify photosynthetic CO2 fixation rate on a chlorophyll (Chl) content basis in both leaves and simultaneously isolated chloroplasts. Hence during initial greening steps of the plants, the induction of alternative pathway and AOX1 expression by irradiation is not directly linked with carbon assimilation of photosynthesis. The application of SHAM partially limited Chl production in rapidly greening leaves, indicating that Chl synthesis in the process of greening might be medicated to some extent by alternative respiratory pathway.

Role of Complement in Defense of the Middle Ear Revealed by Restoring the Virulence of NontypeableHaemophilus influenzae siaBMutants

Nontypeable (NT) Haemophilus influenzae is an important cause of otitis media in children. We have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding ( approximately 5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an approximately 1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.

Mechanisms and functions of photosystem I-related alternative electron transport pathways in chloroplasts

This review analyzes various alternative pathways of chloroplast electron transport mediated by photoreactions of photosystem I (PSI) and unrelated to activity of photosystem II (PSII). The mechanisms and functional significance of the alternative pathways are considered. These pathways are complexly organized and comprise ferredoxin-dependent electron recycling around PSI, as well as electron donation to noncyclic chain in the region between PSII and PSI from reduced substances localized in the chloroplast stroma. For each of the alternative pathways, the origin of corresponding enzymes and their compartmentalization in the complex membrane system of the chloroplast are discussed. It is shown that operation of alternative electron transport pathways contributes to energy transduction and cell defense function, facilitates the absorption of inorganic carbon, and is significant for chloroplast respiration. Multiple mechanisms for regulation of alternative pathways have been revealed. It is concluded that PSI-related alternative electron transport pathways constitute an integral part of entire system of photosynthetic electron transport, this system being principally responsible for energy supply of phototrophic cells and whole plants.

Relapsing Fever SpirochetesBorrelia recurrentisandB. duttoniiAcquire Complement Regulators C4b-Binding Protein and Factor H

Relapsing fever is a rapidly progressive and severe septic disease caused by certain Borrelia spirochetes. The disease is divided into two forms, i.e., epidemic relapsing fever, caused by Borrelia recurrentis and transmitted by lice, and the endemic form, caused by several Borrelia species, such as B. duttonii, and transmitted by soft-bodied ticks. The spirochetes enter the bloodstream by the vector bite and live persistently in plasma even after the development of specific antibodies. This leads to fever relapses and high mortality and clearly indicates that the Borrelia organisms utilize effective immune evasion strategies. In this study, we show that the epidemic relapsing fever pathogen B. recurrentis and an endemic relapsing fever pathogen, B. duttonii, are serum resistant, i.e., resistant to complement in vitro. They acquire the host alternative complement pathway regulator factor H on their surfaces in a similar way to that of the less serum-resistant Lyme disease pathogen, B. burgdorferi sensu stricto. More importantly, the relapsing fever spirochetes specifically bind host C4b-binding protein, a major regulator of the antibody-mediated classical complement pathway. Both complement regulators retained their functional activities when bound to the surfaces of the spirochetes. In conclusion, this is the first report of complement evasion by Borrelia recurrentis and B. duttonii and the first report showing capture of C4b-binding protein by spirochetes.

The Hyphal and Yeast Forms ofCandida albicansBind the Complement Regulator C4b-Binding Protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

Solution structure of a functionally active fragment of decay-accelerating factor.

The second and third modules of human decay accelerating factor (DAF) are necessary and sufficient to accelerate decay of the classical pathway (CP) convertase of complement. No structure of a mammalian protein with decay-accelerating activity has been available to date. We therefore determined the solution structure of DAF modules 2 and 3 (DAF approximately 2,3). Structure-guided analysis of 24 mutants identified likely contact points between DAF and the CP convertase. Three (R96, R69, and a residue in the vicinity of L171) lie on DAF approximately 2,3's concave face. A fourth, consisting of K127 and nearby R100, is on the opposite face. Regions of module 3 remote from the semiflexible 2-3 interface seem not to be involved in binding to the CP convertase. DAF thus seems to occupy a groove on the CP convertase such that both faces of DAF close to the 2-3 junction (including a positively charged region that encircles the protein at this point) interact simultaneously. Alternative pathway convertase interactions with DAF require additional regions of CCP 3 lying away from the 2-3 interface, consistent with the established additional requirement of module 4 for alternative pathway regulation.