Entamoeba histolytica develops resistance to complement deposition and lysis after acquisition of human complement regulatory proteins through trogocytosis

Entamoeba histolytica is the cause of amoebiasis. The trophozoite (amoeba) form of this parasite is capable of invading the intestine, and can disseminate through the bloodstream to other organs. The mechanisms that allow amoebae to evade complement deposition during dissemination have not been well characterized. We previously discovered a novel complement-evasion mechanism employed by E. histolytica. E. histolytica ingests small bites of living human cells in a process termed trogocytosis. We demonstrated that amoebae were protected from lysis by human serum following trogocytosis of human cells, and that amoebae acquired and displayed human membrane proteins from the cells they ingested. Here, we aimed to define how amoebae are protected from complement lysis after performing trogocytosis. We found that amoebae were protected from complement lysis after ingestion of both human Jurkat T cells and red blood cells, and that the level of protection correlated with the amount of material ingested. Trogocytosis of human cells led to a reduction in deposition of C3b on the surface of amoebae. We asked whether display of human complement regulators is involved in amoebic protection, and found that CD59 was displayed by amoebae after trogocytosis. Deletion of a single complement regulatory protein, CD59 or CD46, from Jurkat cells was not sufficient to alter amoebic protection. Removal of all GPI-anchored proteins, including CD59 and CD55, from the surface of amoebae that had undergone trogocytosis suggested that multiple, redundant complement regulators mediate amoebic protection. These studies shed light on a novel strategy for immune evasion by a pathogen.

ErpY-like lipoprotein of Leptospira outsmart host complement regulation by acquiring complement regulators, activating alternate pathway, and intervening membrane attack complex

The survival of pathogenic Leptospira in the host pivots on its proficiency to circumvent the immune response. These pathogens evade the complement system in serum by enticing and amassing the serum complement regulators onto their surface. ErpY-like lipoprotein, a surface-exposed protein of Leptospira spp., is conserved and exclusively present in the pathogenic spirochete. The recombinant form of this protein is comprehended to interact with multiple extracellular matrix (ECM) components and serum proteins like soluble complement regulators factor H (FH) and factor I (FI). Here, we document that the supplementation of recombinant ErpY-like protein (40 µg/mL) in the host (humans) serum augments the viability of E. coli and saprophytic L. biflexa by more than 2-fold. Pure complement regulators FH and FI, when bound to rErpY-like protein, preserve their respective cofactor and protease activity mandated to cleave the complement component C3b. The supplementation of rErpY-like protein (40 µg/mL) in serum ensued in ~90 % reduction of membrane attack complex (C5b-9/MAC) deposition through alternate complement pathway (AP) activation. However, rErpY-like protein could moderately reduce (~16%) MAC deposition in serum through the classical pathway (CP). In addition, the rErpY-like protein solely activated the AP, suggesting its role in the rapid consumption and depletion of the complement components. Blocking the pathogenic L. interrogans surface with anti-rErpY resulted in an increase in MAC formation on the bacterial surface, indicating a specific role of the ErpY-like lipoprotein in complement-mediated immune evasion. This study underscores the role of the ErpY-like lipoprotein of Leptospira in complement evasion.

Hijacking Factor H for Complement Immune Evasion

The complement system is an essential player in innate and adaptive immunity. It consists of three pathways (alternative, classical, and lectin) that initiate either spontaneously (alternative) or in response to danger (all pathways). Complement leads to numerous outcomes detrimental to invaders, including direct killing by formation of the pore-forming membrane attack complex, recruitment of immune cells to sites of invasion, facilitation of phagocytosis, and enhancement of cellular immune responses. Pathogens must overcome the complement system to survive in the host. A common strategy used by pathogens to evade complement is hijacking host complement regulators. Complement regulators prevent attack of host cells and include a collection of membrane-bound and fluid phase proteins. Factor H (FH), a fluid phase complement regulatory protein, controls the alternative pathway (AP) both in the fluid phase of the human body and on cell surfaces. In order to prevent complement activation and amplification on host cells and tissues, FH recognizes host cell-specific polyanionic markers in combination with complement C3 fragments. FH suppresses AP complement-mediated attack by accelerating decay of convertases and by helping to inactivate C3 fragments on host cells. Pathogens, most of which do not have polyanionic markers, are not recognized by FH. Numerous pathogens, including certain bacteria, viruses, protozoa, helminths, and fungi, can recruit FH to protect themselves against host-mediated complement attack, using either specific receptors and/or molecular mimicry to appear more like a host cell. This review will explore pathogen complement evasion mechanisms involving FH recruitment with an emphasis on: (a) characterizing the structural properties and expression patterns of pathogen FH binding proteins, as well as other strategies used by pathogens to capture FH; (b) classifying domains of FH important in pathogen interaction; and (c) discussing existing and potential treatment strategies that target FH interactions with pathogens. Overall, many pathogens use FH to avoid complement attack and appreciating the commonalities across these diverse microorganisms deepens the understanding of complement in microbiology.

Therapeutic Lessons to be Learned From the Role of Complement Regulators as Double-Edged Sword in Health and Disease

The complement system is an important part of the innate immune system, providing a strong defense against pathogens and removing apoptotic cells and immune complexes. Due to its strength, it is important that healthy human cells are protected against damage induced by the complement system. To be protected from complement, each cell type relies on a specific combination of both soluble and membrane-bound regulators. Their importance is indicated by the amount of pathologies associated with abnormalities in these complement regulators. Here, we will discuss the current knowledge on complement regulatory protein polymorphisms and expression levels together with their link to disease. These diseases often result in red blood cell destruction or occur in the eye, kidney or brain, which are tissues known for aberrant complement activity or regulation. In addition, complement regulators have also been associated with different types of cancer, although their mechanisms here have not been elucidated yet. In most of these pathologies, treatments are limited and do not prevent the complement system from attacking host cells, but rather fight the consequences of the complement-mediated damage, using for example blood transfusions in anemic patients. Currently only few drugs targeting the complement system are used in the clinic. With further demand for therapeutics rising linked to the wide range of complement-mediated disease we should broaden our horizon towards treatments that can actually protect the host cells against complement. Here, we will discuss the latest insights on how complement regulators can benefit therapeutics. Such therapeutics are currently being developed extensively, and can be categorized into full-length complement regulators, engineered complement system regulators and antibodies targeting complement regulators. In conclusion, this review provides an overview of the complement regulatory proteins and their links to disease, together with their potential in the development of novel therapeutics.

Leptospira surface protein LigA plays a multifaceted role in modulating the host innate immune response

Leptospira, a zoonotic pathogen is known to infect a variety of hosts and capable of establishing persistent infection. This remarkable ability of bacteria is attributed to its potential to evade or modulate the host immune response by exploiting its surface proteins. We have identified and characterized the domain of Leptospira immunoglobulin-like protein A (LigA) that is involved in modulating the host innate immune response. We identified that the 11th domain (A11) of the variable region of LigA (LAV) induces strong TLR4 dependent innate response in mouse macrophages via signalling through MAP kinase pathway leading to the production of pro-inflammatory cytokines (IL-6 and TNF-α) and expression of costimulatory molecules (CD80, CD86, CD40) and maturation marker (MHC-II). A11 is also involved in acquiring complement regulators like FH, C4b binding protein and Plasminogen and mediating functional activity to escape from both classical and alternate pathways of complement-mediated killing. The deletion of A11 significantly impaired TLR4 signalling and subsequent activation of innate immune cells and also inhibited the binding of complement regulators leading to the killing of bacteria. Our study discovered an unprecedented role of LAV as nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was mediated by A11 and was inhibited with anti-LAV antibodies. These results highlight the moonlighting function of LigA and demonstrates that a single domain of a surface protein is involved in evading a myriad of host innate immune defences, which might allow the persistence of Leptospira in different hosts for a long term without clearance.

THE INTERACTION OF ARENICIN-1 WITH C3B COMPLEMENT PROTEIN

The complement system and antimicrobial peptides (AMPs) are known to be vital humoral factors of innate immunity. Earlier we showed the double-sided influence of arenicin-1 (Ar-1), the AMP from a sea polychaeta Arenicola marina, on the complement activation. In this work we studied the binding of Ar-1 to C3b protein, the fragment of the central complement component C3, using surface plasmon resonance. We also performed molecular docking and molecular dynamics of interaction between C3b fragment - C3c - and Ar-1. All these data showed that the influence of Ar-1 on complement activation might be realized through the interaction with C3b, the most important component for complement activation. Ar-1 may be used for the design of new complement regulators for treating complement-related diseases.