Novel Amphibian Bowman-Birk-Like Inhibitor with Antioxidant and Anticoagulant Effects Ameliorates Pancreatitis Symptoms in Mice.

Acute pancreatitis (AP) is a serious inflammatory disorder and still lacks effective therapy globally. In this study, a novel Ranacyclin peptide, Ranacin, was identified from the skin of Pelophylax nigromaculatus frog. Ranacin adopted a compact ß-hairpin conformation with a disulfide bond (Cys5-Cys15). Ranacin was also demonstrated effectively to inhibit trypsin and have anticoagulant and antioxidant activities in vitro. Furthermore, the severity of pancreatitis was significantly alleviated in l-Arg-induced AP mice after treatment with Ranacin. In addition, structure-activity studies of Ranacin analogues confirmed that the sequences outside the trypsin inhibitory loop (TIL), especially at the C-terminal side, might be closely associated with the efficacy of its trypsin inhibitory activity. In conclusion, our data suggest that Ranacin can improve pancreatic injury in mice with severe AP through its multi-activity. Therefore, Ranacin is considered a potential drug candidate in AP therapy.

Transcriptomic analysis of the tick midgut and salivary gland responses upon repeated blood-feeding on a vertebrate host.

Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host's hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.

Identification and transcriptional profile of Culex quinquefasciatus Say (Diptera: Culicidae) lipophorin in different stages.

Lipophorin is a major hemolymph lipoprotein found in insects with a molecular native mass of 700 kDa. In mosquitoes, two different types of apolipoproteins are characterized, apolipophorin-I (ApoLp-I, ~250 kDa) and apolipophorin-II (ApoLp-II, ~80 kDa). This concentration depends on the stage of development and the age of the insects. Lipophorins are best studied in mosquitoes of the genus Aedes and Anopheles. In this study, we analyze the lipophorin sequence and show the lipophorin purification of the Culex quinquefasciatus and the transcriptional profile of the lipophorin gene in different life cycle stages. Similar amino acid composition and molecular weights are founded in three mosquitoes species lipophorins amino acid sequence. The two subunits of purified lipophorin (Apo I and Apo II) showed molecular masses of approximately 248 and 93 kDa, like that found in other mosquitoes. A gradual increase in the lipophorin expression gene was obtained during the previtellogenic period and after feeding we obtained peak expression at 24 h after feeding. With our results, we conclude that C. quinquefasciatus protein sequence has the same characteristics as those observed in other mosquitoes and that the expression of its apolipophorins is induced by blood feeding.

Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus.

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Characterization and functional analysis of cathelicidin-MH, a novel frog-derived peptide with anti-septicemic properties.

Antimicrobial peptides form part of the innate immune response and play a vital role in host defense against pathogens. Here we report a new antimicrobial peptide belonging to the cathelicidin family, cathelicidin-MH (cath-MH), from the skin of Microhyla heymonsivogt frog. Cath-MH has a single α-helical structure in membrane-mimetic environments and is antimicrobial against fungi and bacteria, especially Gram-negative bacteria. In contrast to other cathelicidins, cath-MH suppresses coagulation by affecting the enzymatic activities of tissue plasminogen activator, plasmin, ß-tryptase, elastase, thrombin, and chymase. Cath-MH protects against lipopolysaccharide (LPS)- and cecal ligation and puncture-induced sepsis, effectively ameliorating multiorgan pathology and inflammatory cytokine through its antimicrobial, LPS-neutralizing, coagulation suppressing effects as well as suppression of MAPK signaling. Taken together, these data suggest that cath-MH is an attractive candidate therapeutic agent for the treatment of septic shock.

Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro.

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

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.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

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.

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.

Small protease inhibitors in tick saliva and salivary glands and their role in tick-host-pathogen interactions.

Ticks must durably suppress vertebrate host responses (hemostasis, inflammation, immunity) to avoid rejection and act as vectors of many pathogenic microorganisms that cause disease in humans and animals. Transcriptomics and proteomics studies have been used to study tick-host-pathogen interactions and have facilitated the systematic characterization of salivary composition and molecular dynamics throughout tick feeding. Tick saliva contains a complement of protease inhibitors that are differentially produced during feeding, many of which inhibit blood coagulation, platelet aggregation, vasodilation, and immunity. Here we focus on two major groups of protease inhibitors, the small molecular weight Kunitz inhibitors and cystatins. We discuss their role in tick-host-pathogen interactions, how they mediate the interaction between ticks and their hosts, and how they might be exploited both by pathogens to invade hosts and as candidates for the treatment of various human pathologies.

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.