Lourdes Isaac

Clinical Aspects and Molecular Basis of Primary Deficiencies of Complement Component C3 and its Regulatory Proteins Factor I and Factor H

The complement system participates in both innate and acquired immune responses. Deficiencies in any of the protein components of this system are generally uncommon and require specialized services for diagnosis. Consequently, complement deficiencies are clinically underscored and may be more common than is normally estimated. As C3 is the major complement component and participates in all three pathways of activation, it is fundamental to understand all the clinical consequences observed in patients for which this protein is below normal concentration or absent in the serum. C3 deficiencies are generally associated with higher susceptibility to severe infections and in some cases with autoimmune diseases such as systemic lupus erythematosus. Here, we review the main clinical aspects and the molecular basis of primary C3 deficiency as well as the mutations in the regulatory proteins factor I and factor H that result in secondary C3 deficiencies. We also discuss the use of animal models to study these deficiencies.

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.

Immune Evasion of Leptospira Species by Acquisition of Human Complement Regulator C4BP

ABSTRACT Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.

Leptospirosis: aspects of innate immunity, immunopathogenesis and immune evasion from the complement system.

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. It constitutes a major public health problem in developing countries, with outcomes ranging from subclinical infections to fatal pulmonary haemorrhage and Weil′s syndrome. To successfully establish an infection, leptospires bind to extracellular matrix compounds and host cells. The interaction of leptospires with pathogen recognition receptors is a fundamental issue in leptospiral immunity as well as in immunophatology. Pathogenic but not saprophytic leptospires are able to evade the host complement system, circulate in the blood and spread into tissues. The target organs in human leptospirosis include the kidneys and the lungs. The association of an autoimmune process with these pathologies has been explored and diverse mechanisms that permit leptospires to survive in the kidneys of reservoir animals have been proposed. However, despite the intense research aimed at the development of a leptospirosis vaccine supported by the genome sequencing of Leptospira strains, there have been relatively few studies focused on leptospiral immunity. The knowledge of evasion strategies employed by pathogenic leptospires to subvert the immune system is of extreme importance as they may represent targets for the development of new treatments and prophylactic approaches in leptospirosis.

Functional characterization of LcpA, a surface-exposed protein of Leptospira spp. that binds the human complement regulator C4BP.

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Ontogeny of complement regulatory proteins - concentrations of factor h, factor I, c4b-binding protein, properdin and vitronectin in healthy children of different ages and in adults.

Previous studies of human in vivo complement protein levels have only compared data for neonates with that from adult sera. Here, we establish the normal concentration ranges of the following complement regulatory proteins in healthy Brazilian children of different age groups (neonates: 1 month−1 year, 1–6 years and 6–13 years) and in adults: factor H (fH), factor I (fI), C4b‐binding protein (C4 BP), properdin and vitronectin. We found that the concentrations of fH, fI, properdin and vitronectin in neonates are significantly lower than in adults. Remarkably, the concentration of C4 BP is below the method resolution (<50 µg/ml) in 76% of the sera from neonates, while adults presented 199–532 µg/ml of C4 BP in their sera. The concentration of properdin in the sera from neonates and up to 1‐year‐old children was less than that observed in older children. Adults presented vitronectin levels significantly higher than all the other age groups in the study. No significant sex differences in the concentrations of all the studied regulatory proteins were detected. This study reveals the ontogeny of complement system in greater detail than previously available and may point to the reasons why neonates have higher susceptibility to develop life‐threatening pyogenic infections. These reference values will be of use in clinical and laboratory investigations of disorders associated with low levels of these regulatory proteins.

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients.

Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement proteins

Leptospirosis is an infectious disease of public health importance. To successfully colonize the host, pathogens have evolved multiple strategies to escape the complement system. Here we demonstrate that the culture supernatant of pathogenic but not saprophytic Leptospira inhibit the three complement pathways. We showed that the proteolytic activity in the supernatants of pathogenic strains targets the central complement molecule C3 and specific proteins from each pathway, such as factor B, C2, and C4b. The proteases cleaved α and β chains of C3 and work in synergy with host regulators to inactivate C3b. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. A recombinant leptospiral metalloprotease from the thermolysin family cleaved C3 in serum and could be one of the proteases responsible for the supernatant activity. We conclude that pathogenic leptospiral proteases can deactivate immune effector molecules and represent potential targets to the development of new therapies in leptospirosis.

Human monocyte-derived dendritic cells are a source of several complement proteins

Abstract.ObjectiveLittle is known about the role of local production of complement components by dendritic cells (DCs) during the generation of specifi c immune responses. In this study, we demonstrate that human DCs are an extrahepatic source of several soluble complement proteins.MethodsReverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the expression and production of several complement proteins.ResultsWe show that DCs produce C3, C5, C9, Factor (F)I, FH, FB, FD and properdin at levels similar to macrophages. Treatment of DCs with lipopolysaccharide (LPS) promoted an increase in the expression of C3 and FI mRNAs and a decrease in C5 mRNA, while C9, FH, FB, FD and properdin mRNA levels were not affected. Treatment with interleukin (IL) −1 or dexamethasone induced a modest increase in C3 mRNA levels and did not affect the expression of other complement components.ConclusionDCs are a source of complement proteins whose synthesis may be regulated in response to different infl ammatory stimuli.

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.