Inhibition of the complement system by saliva of Anopheles (Nyssorhynchus) aquasalis

https://doi.org/10.1016/j.ibmb.2017.11.004Get rights and content

Highlights

  • Anopheles aquasalis' saliva inhibits the complement system.

  • The inhibitor acts in the initial steps of the alternative pathway cascade.

  • The salivary inhibitor is probably a protein from the SG7 family.

Abstract

Anopheline mosquitoes are vectors of malaria parasites. Their saliva contains anti-hemostatic and immune-modulator molecules that favor blood feeding and parasite transmission. In this study, we describe the inhibition of the alternative pathway of the complement system (AP) by Anopheles aquasalis salivary gland extracts (SGE). According to our results, the inhibitor present in SGE acts on the initial step of the AP blocking deposition of C3b on the activation surfaces. Properdin, which is a positive regulatory molecule of the AP, binds to SGE. When SGE was treated with an excess of properdin, it was unable to inhibit the AP. Through SDS-PAGE analysis, A. aquasalis presented a salivary protein with the same molecular weight as recombinant complement inhibitors belonging to the SG7 family described in the saliva of other anopheline species. At least some SG7 proteins bind to properdin and are AP inhibitors. Searching for SG7 proteins in the A. aquasalis genome, we retrieved a salivary protein that shared an 85% identity with albicin, which is the salivary alternative pathway inhibitor from A. albimanus. This A. aquasalis sequence was also very similar (81% ID) to the SG7 protein from A. darlingi, which is also an AP inhibitor. Our results suggest that the salivary complement inhibitor from A. aquasalis is an SG7 protein that can inhibit the AP by binding to properdin and abrogating its stabilizing activity. Albicin, which is the SG7 from A. albimanus, can directly inhibit AP convertase. Given the high similarity of SG7 proteins, the SG7 from A. aquasalis may also directly inhibit AP convertase in the absence of properdin.

Introduction

Anopheline mosquitoes are the natural vectors of Plasmodium parasites, which are the causative agents of human malaria. Transmission occurs when infected female mosquitoes inject the infective forms of the parasite into the vertebrate host along with saliva, which is stored in a pair of trilobed salivary glands located in the interior of the thorax (Amino et al., 2006, Jariyapan and Choochote, 2007, World Health Organization, 2014). During their solenophagic bite, the damage caused by the penetration of the mosquitoes’ mouthparts elicits host homeostatic responses, such as platelet activation, blood clotting, vascular contraction, local inflammation and immune responses. To overcome these responses and complete the blood meal, hematophagous arthropods have a repertoire of pharmacologically active molecules in their saliva, including vasodilator, anti-coagulant, anti-platelet and immune-mediator molecules (Schneider and Higgs, 2009, Fontaine et al., 2011). Complement inhibitors are some of the immune-mediator molecules present in the saliva of several blood-sucking arthropods (Schroeder et al., 2009, Ferreira et al., 2016, Franco et al., 2016, Mendes-Sousa et al., 2016, Silva et al., 2016).

The complement system is an important component of the innate immune defense that is promptly triggered by microorganisms and non-self molecules or particles. In addition to its direct action against pathogens, the complement system is involved with other functions, such as the clearance of immune complexes, removal of necrotic or apoptotic cells, release of inflammatory anaphylatoxins and optimization of antigen presentation for a humoral response (Carroll, 1998). There are three major routes to complement activation, including the classical, the alternative and the lectin pathways. These pathways converge into a single sequence of events that promote opsonization of the targets by specialized proteins (C4b or especially C3b) to facilitate phagocytosis as well as leading to the formation of the membrane attack complex (MAC) on the pathogen surface (Ricklin et al., 2010).

Unlike other complement pathways that depend on pathogen recognition molecules to become activated, the alternative pathway undergoes constant activation. This activation occurs when a small fraction of the C3 component (which is present in all body fluids) exposes a highly reactive thioester group to the medium that combines with water molecules to form C3-H2O. These hydrated molecules undergo conformational modification binding to factor B, which is activated by a specific protease called factor D that releases the portion Ba of the molecule. The soluble C3-H2O-Bb complex acts as a specific protease that is capable of specifically cleaving other C3 molecules. Each activated C3 generates a C3b with its reactive thioester group and the anaphylatoxin fragment C3a. Most of the C3b that is produced will combine with water or self-molecules that are rapidly inactivated by complement control factors. However, if a C3b randomly binds to the surface of a pathogen, it becomes a binding site for factor B. Immediately, the C3b-B that is formed will be converted to C3b-Bb by factor D (a very specific and highly active serine protease). The C3b-Bb complex, which is now linked to the surface of a pathogen, is a protease capable of efficiently activating many C3 molecules that create various C3b-Bb-C3b on the pathogen surface. These complexes are very efficient C3- and C5-convertases when stabilized by factor P, which is also called properdin, and without factor P, the convertase has a considerably shorter half-life. Activation of C5 will culminate with the assembly of the membrane attack complex (Harboe and Mollnes, 2008, Ricklin et al., 2010).

Complement inhibition seems to be essential for blood-sucking arthropods since the occurrence of salivary and intestinal inhibitors is widespread among unrelated species (Ribeiro, 1987, Valenzuela et al., 2000, Couvreur et al., 2008, Barros et al., 2009, Mendes-Sousa et al., 2013, Mendes-Sousa et al., 2016, Ferreira et al., 2016, Franco et al., 2016, Silva et al., 2016). It seems that the main role of complement inhibitors is to protect the insect intestinal cells against complement attack (Barros et al., 2009, Khattab et al., 2015). Considering the presence of complement inhibitors and other immunomodulatory molecules in arthropod's saliva, multiple pathogens could benefit from their depressant action during transmission by the vectors (Wikel and Allen, 1978, Kamhawi et al., 2000, Rohousová and Volf, 2006, Gomes et al., 2008, Schuijt et al., 2011, Gomes and Oliveira, 2012).

We recently described anti-complement molecules in Anopheles albimanus and A. darlingi saliva, including two proteins belonging to the SG7 family that are capable of inhibiting the alternative pathway (Mendes-Sousa et al., 2016). In the present study, we investigated the inhibition of the complement system by salivary gland extract (SGE) from A. aquasalis, which is a widely distributed malaria vector in the coastal areas of Latin America. The main activity observed in this study is on the alternative pathway. Here, we present some evidence that the alternative pathway inhibitor from A. aquasalis saliva may be a protein from the SG7 family that is similar to the inhibitors described in A. albimanus and A. darlingi salivary glands (Mendes-Sousa et al., 2016). The salivary activity against the alternative pathway was partially characterized.

Section snippets

Ethics statement

This study was conducted according to ethical principles for animal experimentation at the Universidade Federal de Minas Gerais and the procedures were approved by the Ethics Committee in Animal Experimentation at this university (CETEA/UFMG) under protocol number 087/11.

Mosquito rearing and salivary gland extract (SGE) preparation

The insects were housed in Barripel® cages covered with netting and fed a 10% sugar solution. Females were blood-fed on anesthetized mice. Eggs, larvae and pupae were reared in 1:10 diluted seawater. The insectary conditions

Results

A. aquasalis salivary glands were dissected for SGE preparation. The total protein concentration in the SGE was 1.5 ± 0.1 μg/pair of glands corresponding to one insect. SGE inhibited the alternative pathway-mediated hemolysis in a dose-dependent manner. With amounts of saliva equivalent to 10 and 15 glands, the inhibition by the SGE was close to 80% and 90%, respectively (Fig. 1-A). Interestingly, the classical pathway was only slightly inhibited (less than 40% inhibition) by the highest tested

Discussion

Anopheles (Nyssorhynchus) aquasalis is an important malaria vector found in coastal areas of Latin America (Póvoa et al., 2003) and is actually one of the few South American anopheline species maintained in stable laboratory colonies (Silva et al., 2006). These colonies have allowed researchers to use this mosquito species in experiments designed to study insecticide resistance (Molina and Figueroa, 2009), midgut physiology (Dias-lopes et al., 2015) and the immune response against Plasmodium

Acknowledgments

This work was supported by a grant from the Brazilian research agencies FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), INCT Entomologia Molecular - CNPq and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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    Present address: Universidade Federal do Piaui, Picos, PI, Brazil.

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