SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Viviana P Ferreira,Michalis Kotsyfakis,Nelder F Gontijo,Vladimir Fazito Vale,Iliano V Coutinho-Abreu,Antonio Ferreira Mendes-Sousa,Jose M C Ribeiro,Manoochehr Rasouli,Marcos Horácio Pereira,Jesus G Valenzuela,Nicolas Collin,Michael K Pangburn,Fabiano Oliveira,Maha Abdeladhim,Kolyvan Ferreira Lima,Elizabeth A Brandt,Claudio Meneses,Shaden Kamhawi,Ricardo Nascimento Araújo

doi:10.1038/srep19300

Abstract

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Highlights

We first reproduced previous findings and show that the equivalent of one pair of Lu. longipalpis salivary gland homogenate (SGH) is sufficient to inhibit the hemolytic activity of the human classical pathway of complement (Fig. 1A)
SALO is a protein of a predicted molecular weight of 10.8 KDa, a theoretical pI of 4.15 with no sequence similarities to other salivary proteins apart from New World sand flies
Antibodies against rSALO under reducing conditions recognize, mostly, a single form of approximately 15 kDa (Fig. 1E, right panel), these antibodies recognize different forms of rSALO when this protein is under non-reducing conditions Fig. 1E, right panel); there is a strong recognition of a protein of 10kDa and other at much higher molecular weight can be observed (Fig. 1E, right panel), suggesting rSALO under non-reducing conditions forms multimers

Summary

Introduction

Sub-products generated during complement activation (the anaphylatoxins C3a and C5a) are important for phagocyte recruitment to infection sites[11,14] and produce microcirculatory alterations. These alterations include vasoconstriction, platelet aggregation, and increased vascular permeability[15]. Activation of the classical complement pathway begins when the C1 complex (composed of C1q, C1r, and C1s proteins) recognizes properly oriented Fc fragments of immunoglobulins bound to antigens as well as other surface patterns including PAMPS or altered self surfaces[16]. The objective of this work is to identify the salivary protein responsible for the inhibition of the classical pathway of complement in this sand fly species and partially characterize its mechanism of action

Objectives

Methods

Results

Conclusion