Raúl Manzano-Román

Cloning and characterization of a plasminogen-binding surface-associated enolase from Schistosoma bovis.

Schistosoma bovis is a ruminant parasite able to survive prolonged periods in the vasculature of its host without either being cleared by the host defensive systems or inducing thrombotic or coagulation disturbances. This suggests that the parasite modulates both the immune and haemostatic host responses. Previous studies have shown that host plasminogen binds to the surface of S. bovis adult worms, and that a tegument extract from S. bovis fixes and activates host plasminogen, generating plasmin, which in turn could both inhibit blood clotting and dissolve clots. Enolase has been identified among the tegumental proteins that bind plasminogen. The aim of the present study is to determine the physiological role of the enolase found in the tegument of S. bovis adult worms as regards plasminogen-binding and activation, and to confirm its surface exposure on the parasite. The study included the cloning and sequencing of S. bovis enolase cDNA, collection of the corresponding recombinant protein and evaluation of its plasminogen-binding and activation activity, and an exploration of the expression and localization of native enolase in adult worms and lung schistosomulae. Here we show that S. bovis male adult worms express enolase on their tegumental surface and that this protein binds host plasminogen and increases its activation in the presence of host tissue plasminogen activator (t-PA). This suggests that the surface-associated enolase found here is a physiological receptor of plasminogen that plays a role in the activation of the host fibrinolytic system, most probably to avoid blood clot formation on the worms surface.

Conservation and immunogenicity of the mosquito ortholog of the tick-protective antigen, subolesin.

The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. The ortholog of the tick-protective antigen, subolesin, was identified in Aedes albopictus and found to have conserved epitopes in ticks and mosquitoes. RNA interference with the tick and mosquito double-stranded RNA in three tick species resulted in significant gene knockdown and decreased tick weight and/or survival. Feeding Anopheles atroparvus, Aedes caspius, and Culex pipiens female mosquitoes on an A. albopictus subolesin hyperimmune serum resulted in 11 ± 5% to 29 ± 6% survival inhibition when compared to controls fed on preimmune serum. Feeding sand flies, Phlebotomus perniciosus, on antimosquito subolesin ortholog protein antibodies inhibited female survival and the number of larvae and adults obtained after hatching by 28 ± 22% and 16 ± 3%, respectively, when compared to controls. Vaccination with tick and mosquito subolesin ortholog proteins significantly reduced Ixodes scapularis tick infestation and weight in a similar way. However, vaccination with the recombinant mosquito subolesin ortholog antigen did not protect against Amblyomma americanum and Rhipicephalus sanguineus tick infestations. Collectively, these preliminary results provided the first evidence that development of vaccines may be possible for control of multiple arthropod vectors using subolesin orthologs but suggested that multiple antigens may be required to produce an effective vaccine.

Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA

RNA interference (RNAi) has become the most powerful experimental tool for the study of gene function in ticks. Subolesin, initially called 4D8, was found to be protective against tick infestations when used as a vaccine and was shown to be highly conserved among ixodid tick species at the nucleotide and protein levels. RNAi caused systemic silencing of subolesin and demonstrated that this protein is involved in regulation of tick feeding, reproduction, and development. Recently, these results were extended to the one-host tick Rhipicephalus (Boophilus) microplus in which injection of dsRNA into replete females resulted in transovarial silencing of subolesin expression in eggs and larvae. Herein, we report transovarial silencing of subolesin by RNAi in the three-host ticks, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. Silencing of subolesin expression by RNAi in these tick species also affected subolesin expression in eggs and larvae. Transovarial RNAi appears to be a common mechanism in ixodid ticks and provides a simple method for the rapid characterization of tick genes involved in oviposition, embryogenesis, and larval development.

MicroRNAs in parasitic diseases: Potential for diagnosis and targeting

MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs that can down-regulate protein expression by specific mRNA recognition. Evidence is accumulating that the miRNAs are implicated in the course and outcome of infectious and non-infectious diseases. Both parasites specific miRNA sequences and the phenomenon of the alteration of host miRNA levels after parasite infection are known, although detailed information about the direct intervention of parasites in the alteration of host miRNA levels and how this is regulated by parasites at molecular level is still lacking. Circulating miRNAs can be detected in biological fluids as serum, saliva and others, exhibiting a good potential as non-invasive biomarkers. Their ability to function as master regulators of the gene expression and the possibility for a relative easy manipulation of the miRNA machinery and related events, coupled with their apparent lack of adverse events when administered, place miRNAs as promising targets for the treatment of diseases. Moreover, the dependence of parasites over the host cellular machinery to accomplish infection and complete their biological cycles, together with the potential manipulation of hosts responses through parasite miRNAs, point out that the miRNA machinery is particularly interesting to seek for alternative therapeutic approaches against parasites. Additionally, the studies about parasitic manipulation of the host immune responses thought miRNAs could broaden our knowledge about basic aspects of the host-parasite relationships.

Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum

The lone star tick, Amblyomma americanum, vectors pathogens of emerging diseases of humans and animals in the United States. Currently, measures are not available for effective control of A. americanum infestations. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, the limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, we report the application of RNA interference (RNAi) for screening an A. americanum cDNA library for discovery of tick protective antigens that reduce tick survival and weights after feeding. Four cDNA clones, encoding for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7), were selected for vaccine studies in cattle, along with subolesin, a tick protective protein identified previously. In vaccinated cattle, an overall efficacy (E)>30% was obtained when considering the vaccine effect on both nymphs and adults, but only 2D10, 2G7 and subolesin affected both tick stages. The highest efficacy of control for adult ticks (E>55%) was obtained in cattle vaccinated with recombinant 2G7 or subolesin. These collective results demonstrated the feasibility of developing vaccines for the control of lone star tick infestations. The use of RNAi for identification of tick protective antigens proved to be a rapid and cost-effective tool for discovery of candidate vaccine antigens, and this approach could likely be applied to other parasites of veterinary and medical importance.

An insight into the proteome of the saliva of the argasid tick Ornithodoros moubata reveals important differences in saliva protein composition between the sexes

Tick saliva contains pharmacologically active molecules that allow these parasites to obtain a blood meal from the host and facilitate host infection by tick-borne pathogens. Recent transcriptomic and proteomic analyses of the salivary glands of several tick species have provided data sets that are invaluable for a better understanding of tick sialomes and tick-host-pathogen relationships. Here we performed a proteomic study of the saliva from the argasid tick Ornithodoros moubata. Saliva samples from female and male specimens were analyzed separately by LC-MS/MS before and after their equalization to facilitate the identification of the less abundant proteins. We report the array of 193 proteins identified in the saliva of O. moubata showing: (i) the broad and complex composition of the saliva of this tick, in good agreement with the complexity of the argasid and ixodid sialomes described previously; (ii) a notable difference in the saliva proteomes of females and males, since only 10 of the proteins identified appeared to be shared by both sexes; and (iii) the presence in the salivary fluid of a wide range of proteins known to be housekeeping/intracellular, which could be secreted in unconventional ways, including exosome secretion.

Self-assembled protein arrays from an Ornithodoros moubata salivary gland expression library.

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

Subolesin/akirin orthologs from Ornithodoros spp soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.

Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells.

Differential Expression of the Tick Protective Antigen Subolesin in Anaplasma marginale- and A. phagocytophilum-infected Host Cells

Subolesin was recently shown in vaccine and RNA interference (RNAi) studies to protect against tick infestations and to affect tick feeding, reproduction, and development as well as infection of host cells by Anaplasma marginale and A. phagocytophilum. Recent experiments provided evidence that infection of both tick and vertebrate host cells with these two pathogens modified gene expression. We therefore hypothesized that infection of host cells with A. marginale and A. phagocytophilum affects expression of subolesin. Subolesin mRNA levels were determined by real‐time reverse transcriptase (RT)‐PCR in uninfected and A. marginale‐infected Dermacentor variabilis guts and salivary glands and IDE8‐cultured tick cells and in uninfected and A. phagocytophilum‐infected Ixodes scapularis nymphs, ISE6‐cultured tick cells, and the human cell line HL‐60. In addition, the effect of subolesin on Anaplasma spp. infection/multiplication was characterized by RNAi in tick tissues and/or cultured tick and human cells. These experiments presented evidence of differential expression of subolesin in A. marginale‐ and A. phagocytophilum‐infected cells. Subolesin was differentially expressed in A. marginale‐infected ticks in a tissue‐specific manner in which mRNA levels increased in response to A. marginale infection in tick salivary gland cells but not in the gut cells. Subolesin knockdown by RNAi reduced Anaplasma infection/multiplication only in cells in which infection increased subolesin expression, i.e., in A. marginale‐infected D. variabilis salivary glands and IDE8 cells. The results reported herein further support the role of subolesin in Anaplasma–host interactions and suggest a putative role of subolesin in vaccines for the control of pathogen infection/multiplication in ticks.