Antonio Frederico Michel Pinto

Proteomic analysis of cattle tick Rhipicephalus (Boophilus) microplus saliva: a comparison between partially and fully engorged females.

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.

Interaction of the cysteine‐rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site‐specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor‐mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factoru2003A (VWA) domains, including the extracellular matrix proteins collagenu2003XII, collagenu2003XIV, matrilinsu20031, 3 and 4, and von Willebrand factor (VWF) via their cysteine‐rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin‐like and cysteine‐rich domains, catrocollastatinu2003C, a disintegrin‐like/cysteine‐rich protein, and the recombinant cysteine‐rich domain of atrolysinu2003A (A/C) all interacted with immobilized VWF in a dose‐dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF‐induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysinu2003C, a SVMP lacking the cysteine‐rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatinu2003C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine‐rich domain‐containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2‐DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N‐glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P‐III‐rich to a P‐I‐rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in‐depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.

Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom.

Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombins activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.

Wound exudate as a proteomic window to reveal different mechanisms of tissue damage by snake venom toxins.

In light of the complexity of wound tissue, proteomic analysis may not clearly reveal the nature of the wound or the processes involved in healing. However, exudate associated with wounds may provide a window on cellular events leading to the development of the wound and/or its healing. In this investigation we performed proteomic analysis on wound exudates from muscular wounds in mice caused by two very different types of snake venom toxins: BaP1, a snake venom metalloproteinase and Mtx-I, a snake venom phospholipase A2. Proteomic analysis of the exudates associated with these wounds clearly differentiated them and offered new perspectives on functional mechanisms by which these toxins cause tissue damage. In the case of wounds caused by the metalloproteinase, there was evidence of degradation of nonfibrillar collagens whereas the phospholipase wound exudate was noted by the presence of fibrillar collagen type I, apolipoproteins A-I, A-IV, and E, and fibronectin. These results suggest that the hemorrhage caused by snake venom metalloproteinases may be associated with the degradation of specific extracellular matrix proteins which play a role in matrix/capillary stabilization and that release of apolipoproteins from their complexes may be involved with the dysfunctional hemostasis observed following snake envenoming.

Metarhizium anisopliae host–pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.

Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding

Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the ticks antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the ticks strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick feeding phases. These data set the foundation for in depth I. scapularis tick feeding physiology and TBD transmission studies.

A thrombin inhibitor from the gut of Boophilus microplus ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin–Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26xa0kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B.xa0microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B.xa0microplus molecules.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BackgroundHaemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods.MethodsH. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences.ResultsA total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products.ConclusionsThis study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

Proteomic profiling of snake venom metalloproteinases (SVMPs): Insights into venom induced pathology

Bothrops sp. snakebites account for the majority of envenomations in South and Central America. Bothrops jararaca accidents are characterized by edema, hemorrhage and necrosis, mainly attributed to the action of hemorrhagic snake venom metalloproteinases (SVMPs). Interestingly, accidents involving Bothrops lanceolatus (Fer-de-Lance) have a prothrombotic profile with necrosis and hemorrhage rarely reported. Here we describe biochemical and proteomic approaches to compare the venom composition of these snakes, focusing on the presence and activity of SVMPs. The total relative amount of SVMPs was found to be approximately the same in the venom of both species, the difference being in the distribution of SVMPs subgroups. Fer-de-Lance venom has relatively more PI SVMPs peptides identified (23-16%) while Jararaca venom has a higher amount of PIII SVMPs (54-43%). Gelatinolytic activity in the PIII mass range is also higher in Jararaca venom. Interestingly, the homologous band region in the Fer-de-Lance zymogram was only very weakly gelatinolytic. According to these findings it is feasible that the different distribution of SVMPs subgroups and their particular biochemical and pharmacological characteristics are two of the main factors contributing to these two radically different venom induced pathologies.