Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Editorial: Special Issue on the "Molecular Biology of Disease Vectors".

Arthropod disease vectors not only transmit malaria but many other serious diseases, many of which are, to a greater or lesser degree, neglected [...].

Recent Advances in Tick Antigen Discovery and Anti-Tick Vaccine Development.

Ticks can seriously affect human and animal health around the globe, causing significant economic losses each year. Chemical acaricides are widely used to control ticks, which negatively impact the environment and result in the emergence of acaricide-resistant tick populations. A vaccine is considered as one of the best alternative approaches to control ticks and tick-borne diseases, as it is less expensive and more effective than chemical controls. Many antigen-based vaccines have been developed as a result of current advances in transcriptomics, genomics, and proteomic techniques. A few of these (e.g., Gavac® and TickGARD®) are commercially available and are commonly used in different countries. Furthermore, a significant number of novel antigens are being investigated with the perspective of developing new anti-tick vaccines. However, more research is required to develop new and more efficient antigen-based vaccines, including on assessing the efficiency of various epitopes against different tick species to confirm their cross-reactivity and their high immunogenicity. In this review, we discuss the recent advancements in the development of antigen-based vaccines (traditional and RNA-based) and provide a brief overview of recent discoveries of novel antigens, along with their sources, characteristics, and the methods used to test their efficiency.

Tick Salivary Kunitz-Type Inhibitors: Targeting Host Hemostasis and Immunity to Mediate Successful Blood Feeding.

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 ß-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.

Emerging roles of non-coding RNAs in vector-borne infections.

Non-coding RNAs (ncRNAs) are nucleotide sequences that are known to assume regulatory roles previously thought to be reserved for proteins. Their functions include the regulation of protein activity and localization and the organization of subcellular structures. Sequencing studies have now identified thousands of ncRNAs encoded within the prokaryotic and eukaryotic genomes, leading to advances in several fields including parasitology. ncRNAs play major roles in several aspects of vector-host-pathogen interactions. Arthropod vector ncRNAs are secreted through extracellular vesicles into vertebrate hosts to counteract host defense systems and ensure arthropod survival. Conversely, hosts can use specific ncRNAs as one of several strategies to overcome arthropod vector invasion. In addition, pathogens transmitted through vector saliva into vertebrate hosts also possess ncRNAs thought to contribute to their pathogenicity. Recent studies have addressed ncRNAs in vectors or vertebrate hosts, with relatively few studies investigating the role of ncRNAs derived from pathogens and their involvement in establishing infections, especially in the context of vector-borne diseases. This Review summarizes recent data focusing on pathogen-derived ncRNAs and their role in modulating the cellular responses that favor pathogen survival in the vertebrate host and the arthropod vector, as well as host ncRNAs that interact with vector-borne pathogens.

Bioinformatic Analysis of Ixodes ricinus Long Non-Coding RNAs Predicts Their Binding Ability of Host miRNAs.

Ixodes ricinus ticks are distributed across Europe and are a vector of tick-borne diseases. Although I. ricinus transcriptome studies have focused exclusively on protein coding genes, the last decade witnessed a strong increase in long non-coding RNA (lncRNA) research and characterization. Here, we report for the first time an exhaustive analysis of these non-coding molecules in I. ricinus based on 131 RNA-seq datasets from three different BioProjects. Using this data, we obtained a consensus set of lncRNAs and showed that lncRNA expression is stable among different studies. While the length distribution of lncRNAs from the individual data sets is biased toward short length values, implying the existence of technical artefacts, the consensus lncRNAs show a more homogeneous distribution emphasizing the importance to incorporate data from different sources to generate a solid reference set of lncRNAs. KEGG enrichment analysis of host miRNAs putatively targeting lncRNAs upregulated upon feeding showed that these miRNAs are involved in several relevant functions for the tick-host interaction. The possibility that at least some tick lncRNAs act as host miRNA sponges was further explored by identifying lncRNAs with many target regions for a given host miRNA or sets of host miRNAs that consistently target lncRNAs together. Overall, our findings suggest that lncRNAs that may act as sponges have diverse biological roles related to the tick-host interaction in different tissues.

Message in a vesicle - trans-kingdom intercommunication at the vector-host interface.

Vector-borne diseases cause over 700,000 deaths annually and represent 17% of all infectious illnesses worldwide. This public health menace highlights the importance of understanding how arthropod vectors, microbes and their mammalian hosts interact. Currently, an emphasis of the scientific enterprise is at the vector-host interface where human pathogens are acquired and transmitted. At this spatial junction, arthropod effector molecules are secreted, enabling microbial pathogenesis and disease. Extracellular vesicles manipulate signaling networks by carrying proteins, lipids, carbohydrates and regulatory nucleic acids. Therefore, they are well positioned to aid in cell-to-cell communication and mediate molecular interactions. This Review briefly discusses exosome and microvesicle biogenesis, their cargo, and the role that nanovesicles play during pathogen spread, host colonization and disease pathogenesis. We then focus on the role of extracellular vesicles in dictating microbial pathogenesis and host immunity during transmission of vector-borne pathogens.

Tick salivary gland transcriptomics and proteomics.

'Omics' technologies have facilitated the identification of hundreds to thousands of tick molecules that mediate tick feeding and play a role in the transmission of tick-borne diseases. Deep sequencing methodologies have played a key role in this knowledge accumulation, profoundly facilitating the study of the biology of disease vectors lacking reference genomes. For example, the nucleotide sequences of the entire set of tick salivary effectors, the so-called tick 'sialome', now contain at least one order of magnitude more transcript sequences compared to similar projects based on Sanger sequencing. Tick feeding is a complex and dynamic process, and while the dynamic 'sialome' is thought to mediate tick feeding success, exactly how transcriptome dynamics relate to tick-host-pathogen interactions is still largely unknown. The identification and, importantly, the functional analysis of the tick 'sialome' is expected to shed light on this 'black box'. This information will be crucial for developing strategies to block pathogen transmission, not only for anti-tick vaccine development but also the discovery and development of new, pharmacologically active compounds for human diseases.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion.

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement.

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin.

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

In silico target network analysis of de novo-discovered, tick saliva-specific microRNAs reveals important combinatorial effects in their interference with vertebrate host physiology.

The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood-feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo-discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva, obtaining furthermore molecular indicators that those might be of exosomal origin. Ten out of these microRNAs are at least 100 times more represented in saliva. For the four most expressed microRNAs from this subset, we analyzed their combinatorial effects upon their host transcriptome using a novel in silico target network approach. We show that only the inclusion of combinatorial effects reveals the functions in important pathways related to inflammation and pain sensing. A control set of highly abundant microRNAs in both saliva and salivary glands indicates no significant pathways and a far lower number of shared target genes. Therefore, the analysis of miRNAs from pure tick saliva strongly supports the hypothesis that tick saliva miRNAs can modulate vertebrate host homeostasis and represents the first direct evidence of tick miRNA-mediated regulation of vertebrate host gene expression at the tick-host interface. As such, the herein described miRNAs may support future drug discovery and development projects that will also experimentally question their predicted molecular targets in the vertebrate host.

MicroRNAs as biomarkers of harmful environmental and occupational exposures: a systematic review.

Environmental exposure is a growing public health burden associated with several negative health effects. An estimated 4.2 million deaths occur each year from ambient air pollution alone. Biomarkers that reflect specific exposures have the potential to measure the real integrated internal dose from all routes of complex environmental exposure. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have been studied as biomarkers in various diseases and have also shown potential as environmental exposure biomarkers. Here, we review the available human epidemiological and experimental evidence of miRNA expression changes in response to specific environmental exposures including airborne particulate matter. In doing so, we establish that miRNA exposure biomarker development remains in its infancy and future studies will need to carefully consider biological and analytical 'design rules' in order to facilitate clinical translation.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding.

Although pathogens are usually transmitted within the first 24-48 h of attachment of the castor bean tick Ixodes ricinus, little is known about the tick's biological responses at these earliest phases of attachment. Tick midgut and salivary glands are the main tissues involved in tick blood feeding and pathogen transmission but the limited genomic information for I. ricinus delays the application of high-throughput methods to study their physiology. We took advantage of the latest advances in the fields of Next Generation RNA-Sequencing and Label-free Quantitative Proteomics to deliver an unprecedented, quantitative description of the gene expression dynamics in the midgut and salivary glands of this disease vector upon attachment to the vertebrate host. A total of 373 of 1510 identified proteins had higher expression in the salivary glands, but only 110 had correspondingly high transcript levels in the same tissue. Furthermore, there was midgut-specific expression of 217 genes at both the transcriptome and proteome level. Tissue-dependent transcript, but not protein, accumulation was revealed for 552 of 885 genes. Moreover, we discovered the enrichment of tick salivary glands in proteins involved in gene transcription and translation, which agrees with the secretory role of this tissue; this finding also agrees with our finding of lower tick t-RNA representation in the salivary glands when compared with the midgut. The midgut, in turn, is enriched in metabolic components and proteins that support its mechanical integrity in order to accommodate and metabolize the ingested blood. Beyond understanding the physiological events that support hematophagy by arthropod ectoparasites, we discovered more than 1500 proteins located at the interface between ticks, the vertebrate host, and the tick-borne pathogens. Thus, our work significantly improves the knowledge of the genetics underlying the transmission lifecycle of this tick species, which is an essential step for developing alternative methods to better control tick-borne diseases.

Desmolaris, a novel factor XIa anticoagulant from the salivary gland of the vampire bat (Desmodus rotundus) inhibits inflammation and thrombosis in vivo.

The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.

The tick salivary protein sialostatin L2 inhibits caspase-1-mediated inflammation during Anaplasma phagocytophilum infection.

Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1ß (IL-1ß) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.

Tick cysteine protease inhibitors suppress immune responses in mannan-induced psoriasis-like inflammation.

Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.

Cathelicidin-HG Alleviates Sepsis-Induced Platelet Dysfunction by Inhibiting GPVI-Mediated Platelet Activation.

Platelet activation contributes to sepsis development, leading to microthrombosis and increased inflammation, which results in disseminated intravascular coagulation and multiple organ dysfunction. Although Cathelicidin can alleviate sepsis, its role in sepsis regulation remains largely unexplored. In this study, we identified Cath-HG, a novel Cathelicidin from Hylarana guentheri skin, and analyzed its structure using nuclear magnetic resonance spectroscopy. The modulatory effect of Cath-HG on the symptoms of mice with sepsis induced by cecal ligation and puncture was evaluated in vivo, and the platelet count, degree of organ damage, and microthrombosis were measured. The antiplatelet aggregation activity of Cath-HG was studied in vitro, and its target was verified. Finally, we further investigated whether Cath-HG could regulate thrombosis in vivo in a FeCl3 injury-induced carotid artery model. The results showed that Cath-HG exhibited an α-helical structure in sodium dodecyl sulfate solution and effectively reduced organ inflammation and damage, improving survival in septic mice. It alleviated sepsis-induced thrombocytopenia and microthrombosis. In vitro, Cath-HG specifically inhibited collagen-induced platelet aggregation and modulated glycoprotein VI (GPVI) signaling pathways. Dot blotting, enzyme-linked immunosorbent assay, and pull-down experiments confirmed GPVI as the target of Cath-HG. Molecular docking and amino acid residue truncations/mutations identified crucial sites of Cath-HG. These findings suggest that GPVI represents a promising therapeutic target for sepsis, and Cath-HG may serve as a potential treatment for sepsis-related thrombocytopenia and thrombotic events. Additionally, identifying Cath-HG as a GPVI inhibitor provides insights for developing novel antithrombotic therapies targeting platelet activation mediated by GPVI.