Mammalian Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs). Although TLRs are clearly involved in the detection of bacteria and viruses, relatively little is known about their function in the innate response to eukaryotic microorganisms. Here we identify a profilin-like molecule from the protozoan parasite Toxoplasma gondii that generates a potent interleukin-12 (IL-12) response in murine DCs that is dependent on myeloid differentiation factor 88. T. gondii profilin activates DCs through TLR11 and is the first chemically defined ligand for this TLR. Moreover, TLR11 is required in vivo for parasite-induced IL-12 production and optimal resistance to infection, thereby establishing a role for the receptor in host recognition of protozoan pathogens.

Saliva of blood-sucking arthropods contains a complex and diverse mixture of antihemostatic, antiinflammatory, and immunomodulatory compounds. The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein superfamily. In mosquitoes, two subfamilies exist, the long and short D7 proteins. Ticks and kissing bugs evolved salivary lipocalins that act as efficient scavengers of biogenic amines, and a similar function was postulated for the D7 proteins. Accordingly, we expressed the five members of the small D7 family of the African malaria vector Anopheles gambiae and a D7 long form from Aedes aegypti and showed by isothermal microcalorimetry, a modified and very sensitive non-equilibrium chromatography/spectrum distortion method, and by smooth muscle bioassay that four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine. The nonbinding D7 protein is poorly expressed in the salivary glands and appears to be on the path to becoming a pseudogene. Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties. It appears that counteracting biogenic amines is of strong adaptive value in the convergent evolution of arthropods to hematophagy. This adaptation has been solved independently in ticks, bugs, and mosquitoes by co-option of either member of the lipocalin or, as shown here, by the odorant-binding protein families.At least 14 orders or families of arthropods (containing over 400 different genera and more than 15,000 species) independently evolved to feed on vertebrate blood (1). To accomplish this task, these animals evolved sophisticated cocktails of salivary pharmacologic reagents that affect blood clotting, platelet aggregation, vascular contraction, host immunity, inflammation, and angiogenesis. With the development of transcriptome analysis, the salivary compositional diversity of several hematophagous arthropods is being revealed at a fast pace; however, the majority of these proteins have no known function (2).Among many different families of proteins unique to hematophagous arthropods, the D7 family has been recognized to be specifically expressed in the salivary glands of adult Diptera. These proteins are distant relatives of the odorant-binding protein superfamily, of which they are a distinct branch. In mosquitoes, two D7 subfamilies exist, the short family having molecular mass of 15-20 kDa and the long with 27-30 kDa, whereas sand flies appear to have only the long forms (3-5). According to a recent sialotranscriptome analysis, Anopheles gambiae, the main African malaria vector, has three long and five short D7 proteins in chromosome arm 3R (6), arranged in an inverted tandem repeat. In the closely related mosquito Anopheles stephensi, one short D7 protein, named hamadarin, has been expressed and shown to have anticlotting activity (7). No other function has been described for the other protein memb...

The nitrophorins are heme-based proteins from the salivary glands of the blood-sucking insect Rhodnius prolixus that deliver nitric oxide gas (NO) to the victim while feeding, resulting in vasodilation and inhibition of platelet aggregation. The nitrophorins also bind tightly to histamine, which is released by the host to induce wound healing. Here we present three crystal structures of nitrophorin 1 (NP1): bound to cyanide, which binds in a manner similar to NO (2.3 A resolution); bound to histamine (2.0 A resolution); and bound to what appears to be NH3 from the crystallization solution (2.0 A resolution). The NP1 structures reveal heme to be sandwiched between strands of a lipocalin-like beta-barrel, and in an arrangement unlike any other gas-transport protein discovered to date. The heme is six-coordinate with a histidine (His 59) on the proximal side, and ligand in a spacious pocket on the distal side. The structures confirm that NO and histamine compete for the same binding pocket and become buried on binding. The dissociation constant for histamine binding was found to be 19 nM, approximately 100-fold lower than that for NO.

The recombinant NO-binding heme protein, nitrophorin 1 (NP1) from the saliva of the blood-sucking insect, Rhodnius prolixus, has been studied by spectroelectrochemistry, EPR, NMR, and FTIR spectroscopies and X-ray crystallography. It is found that NP1 readily binds NO in solution and in the crystalline state, but the protein is not readily autoreduced by excess NO. Likewise, dithionite is not a very effective reductant of NP1. However, the protein can be photoreduced by illumination with visible light in the presence of excess NO, deazaflavin, and EDTA. Optical spectra of the FeIIINO and FeIINO complexes of NP1 are extremely similar, which makes it difficult to characterize the oxidation state of the NO complex by UV−visible spectroscopy. The reduction potential of NP1 in the absence of NO is ∼300 mV more negative than that of metmyoglobin (metMb). In the presence of NO, the reduction potential shifts ∼+430 mV for NP1−NO, but the reduction potential of metMb−NO cannot be measured for comparison. Based on estimated values of K d for NP1III−NO, the K d values for the FeII−NO complex are 20.8 and 80.6 fM at pH 5.5 and 7.5, respectively. The lower driving force for NP1 reduction is qualitatively consistent with the slower rate of autoreduction of NP1−NO; the negative charges surrounding the heme probably also play a role in determining the much slower rate of autoreduction. The N−O stretching frequencies of NP1III−NO and NP1II−NO were measured by FTIR spectroscopy. The values obtained are very typical of other heme−NO stretching frequencies in the two oxidation states:  νNO = 1917 and 1904 cm-1 for two species of FeIIINO and 1611 cm-1 for FeIINO; the values of νNO are consistent with 6-coordinate “base-on” heme−NO centers for both oxidation states. The breadths of the IR bands are consistent with the large solvent accessibility of the bound NO of NP1 and also with the possibility of minor dissociation of the protein-provided histidine ligand on the IR time scale. The ratio of the two FeIII−NO species changes with pH and the nature of the buffer. The CO complex of the Fe(II) form of NP1 has νCO = 1960 and 1936 cm-1, again showing the presence of two species. Both NMR and X-ray crystallography show that the protohemin center of NP1 imidazole has a very high preference for a single orientation of the unsymmetrical protoheme moiety. The structure shows the Fe−N−O unit to be quite bent, which is consistent with its being the FeII−NO form of the protein, presumably formed by photoreduction in the X-ray beam. The proximal base, His-59, is clearly coordinated to the iron in the crystalline state and in solution at ambient temperatures, based on FTIR data, but EPR studies of dithionite-reduced samples show that a percentage of the protein has lost the histidine ligand from the FeIINO center in frozen solution.

Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition mouse dendritic cells (DCs) to promote Th2 lymphocyte differentiation. Using an in vitro bystander polarization assay as the readout, we purified and identified the major Th2-inducing component from soluble egg extract (SEA) as the secreted T2 ribonuclease, omega-1. The Th2-promoting activity of omega-1 was found to be sensitive to ribonuclease inhibition and did not require MyD88/TRIF signaling in DCs. In common with unfractioned SEA, the purified native protein suppresses lipopolysaccharide-induced DC activation, but unlike SEA, it fails to trigger interleukin 4 production from basophils. Importantly, omega-1–exposed DCs displayed pronounced cytoskeletal changes and exhibited decreased antigen-dependent conjugate formation with CD4+ T cells. Based on this evidence, we hypothesize that S. mansoni omega-1 acts by limiting the interaction of DCs with CD4+ T lymphocytes, thereby lowering the strength of the activation signal delivered.

Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.

Toxoplasma gondii releases factors that potently stimulate production of interleukin-12 (IL-12) from dendritic cells (DCs). Purification of this activity showed that cyclophilin-18 (C-18) was its principal component, and antibodies generated against recombinant C-18 inhibited tachyzoite extract-induced synthesis of IL-12. Recombinant C-18 showed high affinity for and triggered cell signaling through CCR5, a chemokine receptor important in parasite-induced IL-12 production by DCs. These findings suggest that the unusual potency of T. gondii in inducing IL-12 from DCs results from its synthesis of a unique chemokine mimic that signals through CCR5. The ability to generate this strong protective response may benefit parasite transmission by preventing the protozoan from overwhelming its intermediate hosts.

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target.