Leptospiral Immunoglobulin-like Proteins Interact With Human Complement Regulators Factor H, FHL-1, FHR-1, and C4BP

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC

Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities

The complement system of the goat: haemolytic assays and isolation of major proteins

[EN] Background: The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus) and to characterize the major goat complement system proteins. Results: The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions: Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS.

IVIG blocks complement deposition mediated by anti-GM1 antibodies in multifocal motor neuropathy

The pathogenesis of multifocal motor neuropathy (MMN) has yet to be established. MMN patients often carry anti-GM1 IgM antibodies, suggesting an autoimmune process involving complement. Intravenous immunoglobulin (IVIG) is the first line treatment, but its action mechanism is unknown.

A hypoxia complement differentiates the muscle response to endurance exercise

Metabolic stress is believed to constitute an important signal for training-induced adjustments of gene expression and oxidative capacity in skeletal muscle. We hypothesized that the effects of endurance training on expression of muscle-relevant transcripts and ultrastructure would be specifically modified by a hypoxia complement during exercise due to enhanced glycolytic strain. Endurance training of untrained male subjects in conditions of hypoxia increased subsarcolemmal mitochondrial density in the recruited vastus lateralis muscle and power output in hypoxia more than training in normoxia, i.e. 169 versus 91% and 10 versus 6%, respectively, and tended to differentially elevate sarcoplasmic volume density (42 versus 20%, P = 0.07). The hypoxia-specific ultrastructural adjustments with training corresponded to differential regulation of the muscle transcriptome by single and repeated exercise between both oxygenation conditions. Fine-tuning by exercise in hypoxia comprised gene ontologies connected to energy provision by glycolysis and fat metabolism in mitochondria, remodelling of capillaries and the extracellular matrix, and cell cycle regulation, but not fibre structure. In the untrained state, the transcriptome response during the first 24 h of recovery from a single exercise bout correlated positively with changes in arterial oxygen saturation during exercise and negatively with blood lactate. This correspondence was inverted in the trained state. The observations highlight that the expression response of myocellular energy pathways to endurance work is graded with regard to metabolic stress and the training state. The exposed mechanistic relationship implies that the altitude specificity of improvements in aerobic performance with a 'living low-training high' regime has a myocellular basis.

Role of complement and perspectives for intervention in ischemia-reperfusion damage

Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation.

Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule playing a nonredundant role in resistance against Aspergillus fumigatus. The present study was designed to investigate the molecular pathways involved in the opsonic activity of PTX3. The PTX3 N-terminal domain was responsible for conidia recognition, but the full-length molecule was necessary for opsonic activity. The PTX3-dependent pathway of enhanced neutrophil phagocytic activity involved complement activation via the alternative pathway; Fc receptor (Fc R) IIA/CD32 recognition of PTX3-sensitized conidia and complement receptor 3 (CR3) activation; and CR3 and CD32 localization to the phagocytic cup. Gene targeted mice (ptx3, FcR common chain, C3, C1q) validated the in vivo relevance of the pathway. In particular, the protective activity of exogenous PTX3 against A fumigatus was abolished in FcR common chain-deficient mice. Thus, the opsonic and antifungal activity of PTX3 is at the crossroad between complement, complement receptor 3-, and Fc R-mediated recognition. Because short pentraxins (eg, C-reactive protein) interact with complement and Fc R, the present results may have general significance for the mode of action of these components of the humoral arm of innate immunity.

Progressive dopamine and hypocretin deficiencies in Parkinson's disease: is there an impact on sleep and wakefulness?

Sleep-wake disturbances are frequent in patients with Parkinson's disease, but prospective controlled electrophysiological studies of sleep in those patients are surprisingly sparse, and the pathophysiology of sleep-wake disturbances in Parkinson's disease remains largely elusive. In particular, the impact of impaired dopaminergic and hypocretin (orexin) signalling on sleep and wakefulness in Parkinson's disease is still unknown. We performed a prospective, controlled electrophysiological study in patients with early and advanced Parkinson's disease, e.g. in subjects with presumably different levels of dopamine and hypocretin cell loss. We compared sleep laboratory tests and cerebrospinal fluid levels with hypocretin-deficient patients with narcolepsy with cataplexy, and with matched controls. Nocturnal sleep efficiency was most decreased in advanced Parkinson patients, and still lower in early Parkinson patients than in narcolepsy subjects. Excessive daytime sleepiness was most severe in narcolepsy patients. In Parkinson patients, objective sleepiness correlated with decrease of cerebrospinal fluid hypocretin levels, and repeated hypocretin measurements in two Parkinson patients revealed a decrease of levels over years. This suggests that dopamine and hypocretin deficiency differentially affect sleep and wakefulness in Parkinson's disease. Poorer sleep quality is linked to dopamine deficiency and other disease-related factors. Despite hypocretin cell loss in Parkinson's disease being only partial, disturbed hypocretin signalling is likely to contribute to excessive daytime sleepiness in Parkinson patients.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system

Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion.