Mechanism of complement inhibition by a mosquito protein revealed through cryo-EM.

Salivary complement inhibitors occur in many of the blood feeding arthropod species responsible for transmission of pathogens. During feeding, these inhibitors prevent the production of proinflammatory anaphylatoxins, which may interfere with feeding, and limit formation of the membrane attack complex which could damage arthropod gut tissues. Salivary inhibitors are, in many cases, novel proteins which may be pharmaceutically useful or display unusual mechanisms that could be exploited pharmaceutically. Albicin is a potent inhibitor of the alternative pathway of complement from the saliva of the malaria transmitting mosquito, Anopheles albimanus. Here we describe the cryo-EM structure of albicin bound to C3bBb, the alternative C3 convertase, a proteolytic complex that is responsible for cleavage of C3 and amplification of the complement response. Albicin is shown to induce dimerization of C3bBb, in a manner similar to the bacterial inhibitor SCIN, to form an inactive complex unable to bind the substrate C3. Size exclusion chromatography and structures determined after 30 minutes of incubation of C3b, factor B (FB), factor D (FD) and albicin indicate that FBb dissociates from the inhibited dimeric complex leaving a C3b-albicin dimeric complex which apparently decays more slowly.

Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Acid phosphatase-like proteins, a biogenic amine and leukotriene-binding salivary protein family from the flea Xenopsylla cheopis.

The salivary glands of hematophagous arthropods contain pharmacologically active molecules that interfere with host hemostasis and immune responses, favoring blood acquisition and pathogen transmission. Exploration of the salivary gland composition of the rat flea, Xenopsylla cheopis, revealed several abundant acid phosphatase-like proteins whose sequences lacked one or two of their presumed catalytic residues. In this study, we undertook a comprehensive characterization of the tree most abundant X. cheopis salivary acid phosphatase-like proteins. Our findings indicate that the three recombinant proteins lacked the anticipated catalytic activity and instead, displayed the ability to bind different biogenic amines and leukotrienes with high affinity. Moreover, X-ray crystallography data from the XcAP-1 complexed with serotonin revealed insights into their binding mechanisms.

A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

A Deeper Insight into the Tick Salivary Protein Families under the Light of Alphafold2 and Dali: Introducing the TickSialoFam 2.0 Database.

Hard ticks feed for several days or weeks on their hosts and their saliva contains thousands of polypeptides belonging to dozens of families, as identified by salivary transcriptomes. Comparison of the coding sequences to protein databases helps to identify putative secreted proteins and their potential functions, directing and focusing future studies, usually done with recombinant proteins that are tested in different bioassays. However, many families of putative secreted peptides have a unique character, not providing significant matches to known sequences. The availability of the Alphafold2 program, which provides in silico predictions of the 3D polypeptide structure, coupled with the Dali program which uses the atomic coordinates of a structural model to search the Protein Data Bank (PDB) allows another layer of investigation to annotate and ascribe a functional role to proteins having so far being characterized as "unique". In this study, we analyzed the classification of tick salivary proteins under the light of the Alphafold2/Dali programs, detecting novel protein families and gaining new insights relating the structure and function of tick salivary proteins.

Functional aspects of evolution in a cluster of salivary protein genes from mosquitoes.

The D7 proteins are highly expressed in the saliva of hematophagous Nematocera and bind biogenic amines and eicosanoid compounds produced by the host during blood feeding. These proteins are encoded by gene clusters expressing forms having one or two odorant-binding protein-like domains. Here we examine functional diversity within the D7 group in the genus Anopheles and make structural comparisons with D7 proteins from culicine mosquitoes in order to understand aspects of D7 functional evolution. Two domain long form (D7L) and one domain short form (D7S) proteins from anopheline and culicine mosquitoes were characterized to determine their ligand selectivity and binding pocket structures. We previously showed that a D7L protein from Anopheles stephensi, of the subgenus Cellia, could bind eicosanoids at a site in its N-terminal domain but could not bind biogenic amines in its C-terminal domain as does a D7L1 ortholog from the culicine species Aedes aegypti, raising the question of whether anopheline D7L proteins had lost their ability to bind biogenic amines. Here we find that D7L from anopheline species belonging to two other subgenera, Nyssorhynchus and Anopheles, can bind biogenic amines and have a structure much like the Ae. aegypti ortholog. The unusual D7L, D7L3, can also bind serotonin in the Cellia species An. gambiae. We also show through structural comparisons with culicine forms that the biogenic amine binding function of single domain D7S proteins in the genus Anopheles may have evolved through gene conversion of structurally similar proteins, which did not have biogenic amine binding capability. Collectively, the data indicate that D7L proteins had a biogenic amine and eicosanoid binding function in the common ancestor of anopheline and culicine mosquitoes, and that the D7S proteins may have acquired a biogenic amine binding function in anophelines through a gene conversion process.

Integrated analysis of the sialotranscriptome and sialoproteome of the rat flea Xenopsylla cheopis.

Over the last 20 years, advances in sequencing technologies paired with biochemical and structural studies have shed light on the unique pharmacological arsenal produced by the salivary glands of hematophagous arthropods that can target host hemostasis and immune response, favoring blood acquisition and, in several cases, enhancing pathogen transmission. Here we provide a deeper insight into Xenopsylla cheopis salivary gland contents pairing transcriptomic and proteomic approaches. Sequencing of 99 pairs of salivary glands from adult female X. cheopis yielded a total of 7432 coding sequences functionally classified into 25 classes, of which the secreted protein class was the largest. The translated transcripts also served as a reference database for the proteomic study, which identified peptides from 610 different proteins. Both approaches revealed that the acid phosphatase family is the most abundant salivary protein group from X. cheopis. Additionally, we report here novel sequences similar to the FS-H family, apyrases, odorant and hormone-binding proteins, antigen 5-like proteins, adenosine deaminases, peptidase inhibitors from different subfamilies, proteins rich in Glu, Gly, and Pro residues, and several potential secreted proteins with unknown function. SIGNIFICANCE: The rat flea X. cheopis is the main vector of Yersinia pestis, the etiological agent of the bubonic plague responsible for three major pandemics that marked human history and remains a burden to human health. In addition to Y. pestis fleas can also transmit other medically relevant pathogens including Rickettsia spp. and Bartonella spp. The studies of salivary proteins from other hematophagous vectors highlighted the importance of such molecules for blood acquisition and pathogen transmission. However, despite the historical and clinical importance of X. cheopis little is known regarding their salivary gland contents and potential activities. Here we provide a comprehensive analysis of X. cheopis salivary composition using next generation sequencing methods paired with LC-MS/MS analysis, revealing its unique composition compared to the sialomes of other blood-feeding arthropods, and highlighting the different pathways taken during the evolution of salivary gland concoctions. In the absence of the X. cheopis genome sequence, this work serves as an extended reference for the identification of potential pharmacological proteins and peptides present in flea saliva.

An Overview of D7 Protein Structure and Physiological Roles in Blood-Feeding Nematocera.

Each time an insect bites a vertebrate host, skin and vascular injury caused by piercing triggers a series of responses including hemostasis, inflammation and immunity. In place, this set of redundant and interconnected responses would ultimately cause blood coagulation, itching and pain leading to host awareness, resulting in feeding interruption in the best-case scenario. Nevertheless, hematophagous arthropod saliva contains a complex cocktail of molecules that are crucial to the success of blood-feeding. Among important protein families described so far in the saliva of blood sucking arthropods, is the D7, abundantly expressed in blood feeding Nematocera. D7 proteins are distantly related to insect Odorant-Binding Proteins (OBP), and despite low sequence identity, observation of structural similarity led to the suggestion that like OBPs, they should bind/sequester small hydrophobic compounds. Members belonging to this family are divided in short forms and long forms, containing one or two OBP-like domains, respectively. Here, we provide a review of D7 proteins structure and function, discussing how gene duplication and some modifications in their OBP-like domains during the course of evolution lead to gain and loss of function among different hematophagous Diptera species.

Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis.

The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.

A sand fly salivary protein acts as a neutrophil chemoattractant.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

Anti-inflammatory effects of FS48, the first potassium channel inhibitor from the salivary glands of the flea Xenopsylla cheopis.

The voltage-gated potassium (Kv) 1.3 channel plays a crucial role in the immune responsiveness of T-lymphocytes and macrophages, presenting a potential target for treatment of immune- and inflammation related-diseases. FS48, a protein from the rodent flea Xenopsylla cheopis, shares the three disulfide bond feature of scorpion toxins. However, its three-dimensional structure and biological function are still unclear. In the present study, the structure of FS48 was evaluated by circular dichroism and homology modeling. We also described its in vitro ion channel activity using patch clamp recording and investigated its anti-inflammatory activity in LPS-induced Raw 264.7 macrophage cells and carrageenan-induced paw edema in mice. FS48 was found to adopt a common αßß structure and contain an atypical dyad motif. It dose-dependently exhibited the Kv1.3 channel in Raw 264.7 and HEK 293T cells, and its ability to block the channel pore was demonstrated by the kinetics of activation and competition binding with tetraethylammonium. FS48 also downregulated the secretion of proinflammatory molecules NO, IL-1ß, TNF-α, and IL-6 by Raw 264.7 cells in a manner dependent on Kv1.3 channel blockage and the subsequent inactivation of the MAPK/NF-κB pathways. Finally, we observed that FS48 inhibited the paw edema formation, tissue myeloperoxidase activity, and inflammatory cell infiltrations in carrageenan-treated mice. We therefore conclude that FS48 identified from the flea saliva is a novel potassium channel inhibitor displaying anti-inflammatory activity. This discovery will promote understanding of the bloodsucking mechanism of the flea and provide a new template molecule for the design of Kv1.3 channel blockers.

Structure-switching aptamer sensors for the specific detection of piperaquine and mefloquine.

Tracking antimalarial drug use and efficacy is essential for monitoring the current spread of antimalarial drug resistance. However, available methods for determining tablet quality and patient drug use are often inaccessible, requiring well-equipped laboratories capable of performing liquid chromatography-mass spectrometry (LC-MS). Here, we report the development of aptamer-based fluorescent sensors for the rapid, specific detection of the antimalarial compounds piperaquine and mefloquine-two slow-clearing partner drugs in current first-line artemisinin-based combination therapies (ACTs). Highly selective DNA aptamers were identified that bind piperaquine and mefloquine with dissociation constants (K d's) measured in the low nanomolar range via two independent methods. The aptamers were isolated from a library of single-stranded DNA molecules using a capture-systematic evolution of ligands by exponential enrichment (SELEX) technique and then adapted into structure-switching aptamer fluorescent sensors. Sensor performance was optimized for the detection of drug from human serum and crushed tablets, resulting in two sensing platforms. The patient sample platform was validated against an LC-MS standard drug detection method in samples from healthy volunteers and patients with malaria. This assay provides a rapid and inexpensive method for tracking antimalarial drug use and quality for the containment and study of parasite resistance, a major priority for malaria elimination campaigns. This sensor platform allows for flexibility of sample matrix and can be easily adapted to detect other small-molecule drugs.

Salivary complement inhibitors from mosquitoes: Structure and mechanism of action.

Inhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family.

A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development.

Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.

Functional and structural similarities of D7 proteins in the independently-evolved salivary secretions of sand flies and mosquitoes.

The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P. duboscqi, respectively, inhibit the activation of platelets by collagen and the thromboxane A2 analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C4, D4 and E4 to the P. papatasi protein. The crystal structure of P. duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.

Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis.

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the "Basic-tail" family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51-104) and (1-50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions.

High-Sensitivity Assays for Plasmodium falciparum Infection by Immuno-Polymerase Chain Reaction Detection of PfIDEh and PfLDH Antigens.

Background: Rapid diagnostic tests based on Plasmodium falciparum histidine-rich protein II (PfHRP-II) and P. falciparum lactate dehydrogenase (PfLDH) antigens are widely deployed for detection of P. falciparum infection; however, these tests often miss cases of low-level parasitemia, and PfHRP-II tests can give false-negative results when P. falciparum strains do not express this antigen. Methods: We screened proteomic data for highly expressed P. falciparum proteins and compared their features to those of PfHRP-II and PfLDH biomarkers. Search criteria included high levels of expression, conservation in all parasite strains, and good correlation of antigen levels with parasitemia and its clearance after drug treatment. Different assay methods were compared for sensitive detection of parasitemia in P. falciparum cultures. Results: Among potential new biomarkers, a P. falciparum homolog of insulin-degrading enzyme (PfIDEh) met our search criteria. Comparative enzyme-linked immunosorbent assays with monoclonal antibodies against PfLDH or PfIDEh showed detection limits of 100-200 parasites/µL and 200-400 parasites/µL, respectively. Detection was dramatically improved by use of real-time immuno-polymerase chain reaction (PCR), to parasitemia limits of 0.02 parasite/µL and 0.78 parasite/µL in PfLDH- and PfIDEh-based assays, respectively. Conclusions: The ability of PfLDH- or PfIDEh-based immuno-PCR assays to detect <1 parasite/µL suggests that improvements of bound antibody sensor technology may greatly increase the sensitivity of malaria rapid diagnostic tests.

The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B.

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.

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.