Adriana Seixas

A Boophilus microplus vitellin-degrading cysteine endopeptidase

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4.0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS-PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Vaccine potential of a tick vitellin-degrading enzyme (VTDCE).

VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the ticks eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.

Rhipicephalus (Boophilus) microplus embryo proteins as target for tick vaccine.

Rhipicephalus (Boophilus) microplus is one of the most widely distributed tick in the world. The control of the parasite is based mainly on the use of chemical acaricides, which are produced from a limited set of molecules. These drugs induce selection of acaricide-resistant ticks, and are an important source of environmental pollution. An approach based on anti-tick vaccines may circumvent these obstacles. Characterization of the physiological function of tick molecules may be useful to develop new vaccines. Previously, we reported the ability of some tick proteins as inducers of protective immune response. Vaccination studies using tick proteins like native (nBYC), recombinant (rBYC) egg-yolk aspartic endopeptidase and cysteine endopeptidase (VTDCE) from R. microplus and glutathione S-transferase (Hl-GST) from Haemaphysalis longicornis demonstrated the immunogenicity and antigenicity of these proteins in bovines. Eventually, immunization with these proteins triggered a partial immune response against R. microplus infestation in cattle, manifested mainly as a reduction in egg fertility (7.7% and 13.9% for nBYC, 5.9% for rBYC; 4.7% for VTDCE, 7.9% for Hl-GST), and in the number of fully engorged ticks (18.2% for rBYC, 14.6% for VTDCE, 53% for Hl-GST). The data so far obtained suggest that these proteins have potential to be used as antigens in an anti-tick vaccine. Other proteins involved in tick embryogenesis also have this potential, like THAP and BmCl1, which are enzymes with key roles in vitellin and hemoglobin hydrolysis. Moreover, the identification of analogous proteins present in other tick species may bring information about the way to develop a vaccine against multiple tick species which can help to solve the problem faced by numerous countries where animals are parasitized by more than one tick species. The aim of the present review is to comprehensibly summarize the data obtained in the last few years by our collaborative research, discussing the efforts we have made to find antigens efficient enough for a cattle tick-controlling vaccine. This review discusses tick physiology studies aimed at the selection of possible targets, characterization of the selected proteins with emphasis on their biochemical and immunological aspects and results of vaccine trials on bovines.

Multi-antigenic vaccine against the cattle tick Rhipicephalus (Boophilus) microplus: a field evaluation.

The tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite of cattle that severely impairs livestock production. Studies on tick immunological control address mostly single-antigen vaccines. However, from the commercial standpoint, so far no single-antigen vaccine has afforded appropriate protection against all R. microplus populations. In this context, multi-antigen cocktails have emerged as a way to enhance vaccine efficacy. In this work, a multi-antigenic vaccine against R. microplus was analyzed under field conditions in naturally infested cattle. The vaccine was composed by three tick recombinant proteins from two tick species that in previous single-vaccination reports provided partial protection of confined cattle against R. microplus infestations: vitellin-degrading cysteine endopeptidase (VTDCE) and boophilus yolk pro-cathepsin (BYC) from R. microplus, and glutathione S-transferase from Haemaphysalis longicornis (GST-Hl). Increased antibody levels against three proteins were recorded after immunizations, with a distinct humoral immune response dynamics for each protein. Compared to the control group, a statistically significant lower number of semi-engorged female ticks were observed in vaccinated cattle after two inoculations. This reduction persisted for 3 months, ranging from 35.3 to 61.6%. Furthermore, cattle body weight gain was significantly higher in vaccinated animals when compared to control cattle. Compared to the single-antigen vaccines composed by VTDCE, BYC or GST-Hl, this three-antigen vaccine afforded higher protection levels against R. microplus infestations.

A family of serine protease inhibitors (serpins) in the cattle tick Rhipicephalus (Boophilus) microplus

Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in many organisms. In arthropods these proteins are involved in innate immune system, morphogenesis and development. In mammals serpins regulate pathways that are essential to life such as blood coagulation, fibrinolysis, inflammation and complement activation, some of which are considered the hosts first line of defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use serpins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNA sequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1-18 (R. microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpins are transcribed during feeding, suggesting their participation in tick physiology regulation. We speculate that the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis. Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by putative signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealed that R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptive study enlarges the knowledge on the molecular biology of R. microplus, an important tick species.

Immunoprotective potential of a Rhipicephalus (Boophilus) microplus metalloprotease

Ticks have serious impacts on animal and human health, causing significant economic losses in cattle breeding. Besides damage due to the hematophagous behavior, they transmit several pathogens. Low cost and environmental safety have made vaccines a promising alternative control method against tick infestation. Metalloproteases (MPs) have been shown to be essential for diverse biological functions in hematophagous organisms, inhibiting blood clotting, degrading extracellular matrix proteins, and inhibiting host tissue repair via anti-angiogenic activity. In this study, we analyzed the immunoprotective potential of a recombinant MP against Rhipicephalus (Boophilus) microplus infestation. First, a cDNA encoding R. microplus amino acids sequence with highly conserved regions of the metzincin (reprolysin) group of MP was identified (BrRm-MP4). After expression and purification, recombinant BrRm-MP4 was used as a vaccinal antigen against R. microplus infestation in cattle (Bos taurus taurus). All vaccinated bovines developed immune response to the antigen, resulting in increased antibody level throughout the immunization protocol. Immunization with rBrRm-MP4 reduced tick feeding success, decreasing the number of engorged females and their reproduction potential, representing a 60% overall protection. These results show that rBrRm-MP4 provides protection against tick infestation, placing it is a potential candidate for an anti-tick vaccine.

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.

Proteomic profiling of the influence of iron availability on Cryptococcus gattii

Iron is essential and ubiquitous in living organisms. The competition for this micronutrient between the host and its pathogens has been related to disease establishment. Cryptococcus gattii is an encapsulated yeast that causes cryptococcosis mainly in immunocompetent individuals. In this study, we analyzed the proteomic profile of the C. gattii R265 Vancouver Island isolate under iron-depleted and -repleted conditions by multidimensional protein identification technology (MudPIT) and by 2D-GE. Proteins and key mechanisms affected by alteration of iron levels such as capsule production, cAMP-signaling pathway, response to stress, and metabolic pathways related to mitochondrial function were identified. Our results also show both proteomic methodologies employed to be complementary.

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.

Sequence characterization and immunogenicity of cystatins from the cattle tick Rhipicephalus (Boophilus) microplus

Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies.