Kunitz domain-containing proteins are ubiquitous serine protease inhibitors with promising therapeutic potential. They target key proteases involved in major cellular processes such as inflammation ...or hemostasis through competitive inhibition in a substrate-like manner. Protease inhibitors from the Kunitz superfamily have a low molecular weight (18-24 kDa) and are characterized by the presence of one or more Kunitz motifs consisting of α-helices and antiparallel β-sheets stabilized by three disulfide bonds. Kunitz-type inhibitors are an important fraction of the protease inhibitors found in tick saliva. Their roles in inhibiting and/or suppressing host homeostatic responses continue to be shown to be additive or synergistic with other protease inhibitors such as cystatins or serpins, ultimately mediating successful blood feeding for the tick. In this review, we discuss the biochemical features of tick salivary Kunitz-type protease inhibitors. We focus on their various effects on host hemostasis and immunity at the molecular and cellular level and their potential therapeutic applications. In doing so, we highlight that their pharmacological properties can be exploited for the development of novel therapies and vaccines.
•Amblyomma sculptum is one the most important tick species in south America.•Few works have focused on the search for anti-A. sculptum vaccines.•Concealed gut proteins may be promissing vaccine ...antigens against A. sculptum.•A chimeric protein was constructed with promising efficacy against A. sculptum.•Vaccine induced a reduction in female fertility and nymph mortality.
Amblyomma sculptum is widely distributed in Brazil and is the main vector of Rickettsia rickettsii, the causative agent of the Brazilian spotted fever (BSF). Tick gut proteins play an essential role in blood feeding, digestion, and protection of gut epithelium. Therefore, many of these were investigated as potential vaccine targets for tick-control strategies. The present study aimed to select transcripts corresponding to putative immunogenic proteins in the A. sculptum gut epithelial membrane, produce recombinant proteins and evaluate them as antigens against A. sculptum infestations. Three gut proteins − AsMucin, AsAPP, and AsLAMP − and a chimeric protein (rAsChimera) based on 22 peptides containing putative B cell epitopes from seven different gut proteins were evaluated as anti-A. sculptum antigens. Mice immunizations revealed that all recombinant targets elicited humoral response with significantly increased IgG levels compared to controls. For rAsChimera, IgG levels remained significantly higher than controls up to 75 days after the end of the immunization. Challenge trials revealed that vaccination with the chimeric protein was the most effective against A. sculptum, inducing 100 % nymph mortality and reaching 80.8 % efficacy against females. The other three proteins did not induce relevant protection, as AsAPP had only 26.6 % efficacy, whereas AsMucin and AsLAMP induced no protection. These data indicate that targeting gut protein immunogenic regions may be an effective strategy for a vaccine formulation againstA. sculptum.
Clogmia albipunctata (Williston, 1893) is a non-hematophagous insect belonging to the order Diptera, suborder Nematocera (Lower Diptera) and family Psychodidae. In the present work, we investigated ...how C. albipunctata control their midgut pH under different physiological conditions, comparing their midgut physiology with some nematoceran hematophagous species. The C. albipunctata midgut pH was measured after ingestion of sugar, protein and under the effect of the alkalinizing hormone released in the hemolymph of the hematophagous sand fly Lutzomyia longipalpis obtained just after a blood meal. The midgut pH of unfed or sugar-fed C. albipunctata is 5.5–6, and its midgut underwent alkalinization after protein ingestion or under treatment with hemolymph collected from blood fed L. longipalpis. These results suggested that in nematocerans, mechanisms for pH control seem shared between hematophagous and non-hematophagous species. This kind of pH control is convenient for successful blood digestion. The independent evolution of many hematophagous groups from the Lower Diptera suggests that characteristics involved in midgut pH control were already present in non-hematophagous species and represent a readiness for adaptation to this feeding mode.
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•C. albipunctata share midgut pH control mechanisms with hematophagous nematocerans.•It presents an acidic midgut, undergoing alkalinization when feeding on proteins.•Alkalinizing hormones from L. longipalpis hemolymph function in female C. albipunctata.•Exaptation events may be involved in adaptation to hematophagy in nematoceran.
Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules ...in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.
Anti-αGal IgE antibodies mediate a spreading allergic condition known as αGal-syndrome (AGS). People exposed to hard tick bites are sensitized to αGal, producing elevated levels of anti-αGal IgE, ...which are responsible for AGS. This work presents an immunotherapy based on polymeric αGal-glycoconjugates for potentially treating allergic disorders by selectively inhibiting anti-αGal IgE antibodies. We synthesized a set of αGal-glycoconjugates, based on poly-L-lysine of different degrees of polymerization (DP1000, DP600, and DP100), to specifically inhibit
the anti-αGal IgE antibodies in the serum of αGal-sensitized patients (n=13). Moreover, an animal model for αGal sensitization in GalT-KO mice was developed by intradermal administration of hard tick' salivary gland extract, mimicking the sensitization mechanism postulated in humans. The
exposure to all polymeric glycoconjugates (5-10-20-50-100 µg/mL) mainly inhibited anti-αGal IgE and IgM isotypes, with a lower inhibition effect on the IgA and IgG, respectively. We demonstrated a differential anti-αGal isotype inhibition as a function of the length of the poly-L-lysine and the number of αGal residues exposed in the glycoconjugates. These results defined a minimum of 27 αGal residues to inhibit most of the induced anti-αGal IgE
. Furthermore, the αGal-glycoconjugate DP1000-RA0118 (10 mg/kg sc.) showed a high capacity to remove the anti-αGal IgE antibodies (≥75% on average) induced in GalT-KO mice, together with similar inhibition for circulating anti-αGal IgG and IgM. Our study suggests the potential clinical use of poly-L-lysine-based αGal-glycoconjugates for treating allergic disorders mediated by anti-αGal IgE antibodies.
is the main tick associated with human bites in Brazil and the main vector of
, the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are ...directly related to feeding success and vector competence. In the present study, we identified sequences of
salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A.
females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of
.
Blood-sucking vectors must overcome thermal stress caused by intake of proportionally large amounts of warm blood from their hosts. In response to this, Heat Shock Proteins (HSPs) such as the widely ...studied HSP70 family (the inducible HSP70 and the cognate form HSC70, known for their role in preserving essential cellular functions) are rapidly up-regulated in their tissues. The triatomine Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative pathogen of Chagas’ disease, and is also a model organism for studying insect biology and physiology. In this work, we observed that the expression of Rhodnius prolixus HSP70 was rapidly up-regulated in response to thermal shocks (0 °C and 40 °C) and also during the first hours after feeding on blood. HSP70/HSC70 RNAi knockdown elicited important alterations in R. prolixus physiological responses triggered by blood meal and starvation. HSP70/HSC70 knockdown insects showed lower resistance to prolonged starvation in comparison to appropriate controls, dying between 32 and 40 days after dsRNA injection. After blood feeding, the physiological effects of HSP70/HSC70 knockdown were more prominent and the insects died even earlier, within 14–20 days after feeding (21–27 days after dsRNA injection). These bugs showed impaired blood processing and digestion, reduced energetic metabolism and the midgut immune responses were compromised. Our findings suggest that HSP70/HSC70 depletion affected R. prolixus in starvation or fed conditions. After feeding, the arrival of blood in the digestive tract of knockdown insects fails to activate essential signaling pathways involved in blood processing, producing several alterations in their physiological processes enough to generate a premature death.
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•Expression of HSP70 in Rhodnius prolixus was up-regulated in response to thermal stress.•HSP70 depletion elicited important systemic alterations in R. prolixus after blood digestion.•Blood processing and digestion were impaired, energetic metabolism was reduced and immune response genes were not activated.•HSP70 knockdown led these insects to a premature death.
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•Soft ticks eliminate abundant fluid through their coxal glands during feeding.•The fluid spread by capillarity over the cuticular surface and evaporates.•The evaporation of the fluid ...dissipates the excess heat gathered with warm blood.•Ornithodoros reduce thermal stress during feeding y evaporative cooling.•This is the first demonstration of physiological thermoregulation in ticks.
Feeding on the blood of warm-blooded vertebrates is associated to thermal stress in haematophagous arthropods. It has been demonstrated that blood-sucking insects protect their physiological integrity either by synthesising heat-shock proteins or by means of thermoregulatory mechanisms. In this work, we describe the first thermoregulatory mechanism in a tick species, Ornithodoros rostratus. By performing real-time infrared thermography during feeding on mice we found that this acarian eliminates big amounts of fluid (urine) through their coxal glands; this fluid quickly spreads over the cuticular surface and its evaporation cools-down the body of the tick. The spread of the fluid is possible thanks to capillary diffusion through the sculptured exoskeleton of Ornithodoros. We discuss our findings in the frame of the adaptive strategies to cope with the thermal stress experienced by blood-sucking arthropods at each feeding event on warm-blooded hosts.
The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize ...the anterior midgut of T. infestans. To obtain insight into T. cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T. infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T. cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T. cruzi in T. infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T. infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T. cruzi trypomastigotes. Moreover, in antimicrobial experiments using E. coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T. cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T. infestans anterior midgut.
•A new protein (TiAP) from anterior midgut of Triatoma infestans is modulated by Trypanosoma cruzi.•TiAP doesn't interfere in the viability of T. cruzi trypomastigotes.•TiAP is able to control bacterial load inside anterior midgut of T. infestans.•TiAP is important in maintenance of T. cruzi – microbiota balance in the vector.
The haematophagy process by arthropods has been one of the main targets of studies in the parasite-host interaction, and the kissing-bug Rhodnius prolixus, vector of the protozoan Trypanosoma cruzi, ...has been one of the main models for such studies. Still in the 1980s, it was identified that among the salivary proteins for disrupting vertebrate host homeostasis, lipocalins were among the most relevant proteins for this process. Since then, 30 lipocalins have been identified in the salivary glands of R. prolixus, that promotes vasodilatation, prevents inflammation, act as anticoagulants and inhibits platelet aggregation. The present work aims to identify new lipocalins from R. prolixus, combining transcriptome and genome data. Identified new genes were mapped and had their structure annotated. To infer an evolutionary relationship between lipocalins, and to support the predicted functions for each lipocalin, all amino acid sequences were used to construct phylogenetic trees. We identified a total of 29 new lipocalins, 3 new bioaminogenic-biding proteins (which act to inhibit platelet aggregation and vasodilation), 9 new inhibitors of platelet aggregation, 7 new apolipoproteins and 10 lipocalins with no putative function. In addition, we observed that several of the lipocalins are also expressed in different R. prolxius tissues, including gut, central nervous system, antennae, and reproductive organs. In addition to newly identified lipocalins and a mapping the new and old lipocalins in the genome of R. prolixus, our study also carried out a review on functional status and nomenclature of some of the already identified lipocalins. Our study reinforces that we are far from understanding the role of lipocalins in the physiology of R. prolixus, and that studies of this family are still of great relevance.
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•29 new lipocalins identified in Rhodnius prolixus genome and transcriptome.•Many salivary lipocalins are also expressed in other tissues, such as central nervous system, gut, testis, etc.•A new family of apolipoproteins is described.•Evolutionary analysis suggests the change in name for some R. prolixus salivary lipocalins.
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