Biochemical characterization of a novel sphingomyelinase-like protein from the Rhipicephalus microplus tick.

Sphingomyelinase D is a toxin present in venomous spiders and bacteria and is associated with infection symptoms in patients affected by spider bites. It was observed that in Ixodes scapularis ticks, sphingomyelinase-like protein secreted in saliva can modulate the host immune response, affecting the transmission of flavivirus to the host via exosomes. In this work, a sphingomyelinase D-like protein (RmSMase) from R. microplus, a tick responsible for economic losses and a vector of pathogens for cattle, was investigated. The amino acid sequence revealed the lack of important residues for enzymatic activity, but the recombinant protein showed sphingomyelinase D activity. RmSMase shows Ca2+ and Mg2+ dependence in acidic pH, differing from IsSMase, which has Mg2+ dependence in neutral pH. Due to the difference between RmSMase and other SMases described, the data suggest that RmSMase belongs to SMase D class IIc. RmSMase mRNA transcription levels are upregulated during tick feeding, and the recombinant protein was recognized by host antibodies elicited after heavy tick infestation, indicating that RmSMase is present in tick saliva and may play a role in the tick feeding process.

Evaluation of the biological function of ribosomal protein S18 from cattle tick Rhipicephalus microplus.

Rhipicephalus (Boophilus) microplus, also known as the cattle tick, causes severe parasitism and transmits different pathogens to vertebrate hosts, leading to massive economic losses. In the present study, we performed a functional characterization of a ribosomal protein from R. microplus to investigate its importance in blood feeding, egg production and viability. Ribosomal protein S18 (RPS18) is part of the 40S subunit, associated with 18S rRNA, and has been previously pointed to have a secondary role in different organisms. Rhipicephalus microplus RPS18 (RmRPS18) gene expression levels were modulated in female salivary glands during blood feeding. Moreover, mRNA levels in this tissue were 10 times higher than those in the midgut of fully engorged female ticks. Additionally, recombinant RmRPS18 was recognized by IgG antibodies from sera of cattle naturally or experimentally infested with ticks. RNAi-mediated knockdown of the RmRPS18 gene was performed in fully engorged females, leading to a significant (29 %) decrease in egg production. Additionally, egg hatching was completely impaired, suggesting that no viable eggs were produced by the RmRPS18-silenced group. Furthermore, antimicrobial assays revealed inhibitory activities against gram-negative Escherichia coli and gram-positive Staphylococcus aureus bacteria, affecting bacterial growth. Data presented here show the important role of RmRPS18 in tick physiology and suggest that RmRPS18 can be a potential target for the development of novel strategies for tick control.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit.

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus.

Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.

Identification and partial characterization of a gut Rhipicephalus appendiculatus cystatin.

Vaccines are among the alternative tick control methods expected to replace at least in part the volumes of chemical acaricides currently used worldwide. However, a vaccination approach depends on a host immune response against proteins that are essential to tick physiology. The cystatin family is a protein class recently investigated to compose an effective antigen in a tick vaccine. In this study, a cDNA from Rhipicephalus appendiculatus with high sequence similarity to cystatins type 2 was identified by random sequencing analysis and called R. appendiculatus cystatin 1 (Ra-cyst-1). DNA sequence analysis showed that the cloned Ra-cyst-1 has a 423-bp open reading frame and codified to a 140-amino acid polypeptide. The putative mature protein consists of 115 amino acid residues with a deduced molecular weight of 12.8kDa. The highly conserved G (P-I), QxVxG (P-II), and PW (P-III) type 2 cystatins motifs are present in Ra-cyst-1 cDNA. RT-PCR analysis showed that the Ra-cyst-1 gene is expressed in nymph, male, and female midgut following blood feeding, but not in the salivary glands of fed females. In addition, Western blot revealed that recombinant Ra-cyst-1 was not recognized by sera derived from rabbits infested with ticks, suggesting that this cystatin is not secreted into the host during infestation. We hypothesize that Ra-cyst-1 may play a role in the tick feeding process and could be a concealed antigen candidate in further anti-tick vaccination trials.

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.

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.

The quest for a universal vaccine against ticks: cross-immunity insights.

As blood-sucking parasites, ticks inflict great damage to animals and humans in many parts of the world. The continued use of chemical acaricides is not sustainable due to increasing tick resistance, growing public concern over drug residues in food and in the environment, and the high cost of developing new acaricides. Therefore, an alternative control strategy is urgently needed. Vaccines against ticks have been shown to be functionally feasible, as highlighted by the success of Bm86 vaccines against Rhipicephalus (Boophilus) microplus and closely related tick species. However, a limited number of tick antigens with cross-protective epitopes have been characterized so far, limiting widespread deployment of the available vaccines, including those derived from Bm86. Therefore, identifying tick antigens with potential broad-spectrum protection against multiple tick species is subject of vigorous research at present. In this paper, progress towards effective anti-tick vaccines is reviewed in the light of emerging data from studies including heterologous tick challenge. Taken together, these studies indicate that the decades-long search for a universal tick vaccine is making progress, with such a vaccine likely to be based on multiple cross-reactive antigens.

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.