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BACKGROUND: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. METHODS: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. RESULTS: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. CONCLUSIONS: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.

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The skin is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during feeding. Tick salivary glands have evolved a complex and sophisticated pharmacological arsenal, consisting of bioactive molecules, to assist blood feeding and pathogen transmission. In this work, persulcatin, a multifunctional molecule that targets keratinocyte function and hemostasis, was identified from Ixodes persulcatus female ticks. The recombinant persulcatin was expressed and purified and is a 25-kDa acidic protein with 2 Kunitz-type domains. Persulcatin is a classical tight-binding competitive inhibitor of proteases, targeting plasmin (Ki: 28 nM) and thrombin (Ki: 115 nM). It blocks plasmin generation on keratinocytes and inhibits their migration and matrix protein degradation; downregulates matrix metalloproteinase 2 and matrix metalloproteinase 9; and causes a delay in blood coagulation, endothelial cell activation, and thrombin-induced fibrinocoagulation. It interacts with exosite I of thrombin and reduces thrombin-induced endothelial cell permeability by inhibiting vascular endothelial-cadherin disruption. The multifaceted roles of persulcatin as an inhibitor and modulator within the plasminogen-plasmin system and thrombin not only unveil further insights into the intricate mechanisms governing wound healing but also provide a fresh perspective on the intricate interactions between ticks and their host organisms.

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Acaricide resistance is a global problem that has impacts worldwide. Tick populations with broad resistance to all commercially available acaricides have been reported. Since resistance selection in ticks and their role in pathogen transmission to animals and humans result in important economic and public health burden, it is essential to develop new strategies for their control (i.e., novel chemical compounds, vaccines, biological control). The synganglion is the tick central nervous system and it is responsible for synthesizing and releasing signaling molecules with different physiological functions. Synganglion proteins are the targets of the majority of available acaricides. In this review we provide an overview of the mode-of-action and resistance mechanisms against neurotoxic acaricides in ticks, as well as putative target sites in synganglion, as a supporting tool to identify new target proteins and to develop new strategies for tick control.

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The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.

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BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.

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To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.

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Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.

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The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

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Here we present the proteomic profile datasets of two Fasciola hepatica NEJ isolates derived from different snail hosts: Lymnaea viatrix and Pseudosuccinea columella. The data used in the analysis are related to the article 'A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella' (Di Maggio et al., 2019).

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In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ±â€¯11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.

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