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.

The complement system (C) present in circulating blood is an effective mechanism of host defense, responsible for the killing of pathogens and the production of potent anaphylatoxins. Inhibitors of the C have been described in the saliva of hematophagous arthropods that are involved in the protection of digestive tissues against C-mediated damage. Here we describe albicin, a novel inhibitor of the alternative pathway of complement from the salivary glands of the malaria vector, Anopheles albimanus. The inhibitor was purified from salivary gland homogenates by reverse phase HPLC, and identified by mass spectrometry as a small (13.4 kDa) protein related to the gSG7 protein of An. gambiae and An. stephensi. Recombinant albicin was produced in Escherichia coli and found to potently inhibit lysis of rabbit erythrocytes in assays of the alternative pathway while having no inhibitory effect on the classical or lectin pathways. Albicin also inhibited the deposition of complement components on agarose-coated plates, although it could not remove previously bound components. Antisera produced against recombinant albicin recognized both the native and recombinant inhibitors and also blocked their activities in in vitro assays. Using surface plasmon resonance and enzymatic assays, we found that albicin binds and stabilizes the C3-convertase complex (C3bBb) formed on a properdin surface, and inhibits the convertase activity of a reconstituted C3bBb complex in solution. The data indicate that albicin specifically recognizes the activated form of the complex allowing more efficient inhibition by an inhibitor whose quantity is limited.

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Background Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host’s complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva.ResultsThe saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays.ConclusionSalivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.

Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni2+ increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system.

BackgroundRhipicephalus (Boophilus) microplus is the main ectoparasite affecting livestock worldwide. For a successful parasitism, ticks need to evade several immune responses of their hosts, including the activation of the complement system. In spite of the importance of R. microplus, previous work only identified one salivary molecule that blocks the complement system. The current study describes complement inhibitory activities induced by R. microplus salivary components and mechanisms elicited by putative salivary proteins on both classical and alternative complement pathways.ResultsWe found that R. microplus saliva from fully- and partially engorged females was able to inhibit both pathways. Saliva acts strongly at the initial steps of both complement activation pathways. In the classical pathway, the saliva blocked C4 cleavage, and hence, deposition of C4b on the activation surface, suggesting that the inhibition occurs at some point between C1q and C4. In the alternative pathway, saliva acts by binding to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and reducing C3b production and deposition as well as cleavage of factor B. Saliva has no effect on formation or decay of the C6 to C8 components of the membrane attack complex.ConclusionThe saliva of R. microplus is able to inhibit the early steps of classical and alternative pathways of the complement system. Saliva acts by blocking C4 cleavage and deposition of C4b on the classical pathway activation surface and, in the alternative pathway, saliva bind to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and the production and deposition of additional C3b.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1726-8) contains supplementary material, which is available to authorized users.

Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.