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Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

ABSTRACT: Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens' ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In the present study, the expression of genes involved in immune signaling pathways of R. microplus adults' guts when challenged with a high or low parasitic load of T. equi was evaluated. This research demonstrates divergences in the immune gene expression pattern linked to T. equi infection in R. microplus since the Toll, IMD, and JNK signaling pathways were transcriptionally repressed in the guts of adult ticks infected with T. equi. Moreover, the results showed that different infectious doses of T. equi induce differential gene expression of key components of immune signaling cascades in R. microplus gut, suggesting a link between the intensity of infection and the activation of tick immunity response. The present study adds knowledge to elucidate the gut immune signaling response of R. microplus to T. equi infection. In addition, the generated data can serve as a basis for further investigations to develop strategies for controlling and preventing equine piroplasmosis.

SUBMITTER: Paulino PG

PROVIDER: S-EPMC9782190 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

Paulino Patrícia Gonzaga PG Peckle Maristela M Mendonça Leo Paulis LP Massard Carlos Luiz CL Antunes Sandra S Couto Joana J Domingos Ana A Guedes Junior Daniel da Silva DDS Cabezas-Cruz Alejandro A Santos Huarrisson Azevedo HA

Pathogens (Basel, Switzerland) 20221206 12

Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens' ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In ...[more]

PMID: 36558812

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Project description:BACKGROUND: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. RESULTS: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. CONCLUSIONS: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Project description:Arthropods transmit important infectious diseases of humans and animals. Importantly, replication and the development of pathogen infectivity are tightly linked to vector feeding on the mammalian host; thus analysis of the transcriptomes of both vector and pathogen during feeding is fundamental to understanding transmission. Using Anaplasma marginale infection of Rhipicephalus microplus as the experimental model, we tested three hypotheses exploring the temporal and organ-specific nature of the tick midgut and salivary gland transcriptomes during feeding and in response to infection. Numerous R. microplus genes were regulated in response to feeding and were differentially regulated between the midgut and salivary gland; additionally, there was a progression in regulated gene expression in the salivary gland over time. In contrast, relatively few tick genes were specifically regulated in response to A. marginale infection and these genes were predominantly annotated as hypothetical or were of unknown function. Notable among the genes with informative annotation was that several ribosomal proteins were down-regulated, suggesting that there may be a corresponding decrease in translation. The hypotheses that R. microplus midgut and salivary gland genes are differentially regulated and that the salivary gland transcriptome is dynamic over time were accepted. This is consistent with, and important for understanding the roles of, the two organs, the midgut serving as an initial site of uptake and replication while the salivary gland serves as the final site of replication and secretion. The nominal effect of A. marginale on the tick transcriptome in terms of numbers of regulated genes and fold of regulation supports the view that the vector-pathogen relationship is well established with minimal deleterious effect on the tick. The small set of predominantly hypothetical genes regulated by infection suggests that A. marginale is affecting a novel set of tick genes and may provide new opportunities for blocking transmission from the tick.

Project description:BackgroundIt is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the control of pathogens and microbiota in insects. However, the knowledge of the role of ROS and RNS in tick-pathogen and tick-microbiota interactions is limited. Here, we evaluated the immune-related redox metabolism of the embryonic cell line BME26 from the cattle tick Rhipicephalus microplus in response to Anaplasma marginale infection.MethodsA high-throughput qPCR approach was used to determine the expression profile of 16 genes encoding proteins involved in either production or detoxification of ROS and RNS in response to different microbial challenges. In addition, the effect of RNAi-mediated gene silencing of catalase, glutathione peroxidase, thioredoxin and protein oxidation resistance 1 in the control of infection with A. marginale was evaluated.ResultsInfection with A. marginale resulted in downregulation of the genes encoding ROS-generating enzymes dual oxidase and endoplasmic reticulum oxidase. In contrast, the genes encoding the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, thioredoxin reductase and peroxiredoxin were upregulated. The gene expression pattern in response to infection with Rickettsia rickettsii and exposure to heat-killed microorganisms, Micrococcus luteus, Enterobacter cloacae or S. cerevisiae was the opposite of that triggered by A. marginale challenge. The simultaneous silencing of three genes, catalase, glutathione peroxidase, and thioredoxin as well as the oxidation resistance 1 gene by RNAi apparently favoured the colonization of BME26 cells by A. marginale, suggesting that the antioxidant response might play a role in the control of infection.ConclusionsTaken together, our results suggest that a general response of tick cells upon microbial stimuli is to increase ROS/RNS production. In contrast, A. marginale infection triggers an opposite profile, suggesting that this pathogen might manipulate the tick redox metabolism to evade the deleterious effect of the oxidant-based innate immune response.

Project description:BackgroundCystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively.MethodsHere we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins.ResultsEnzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins.ConclusionThe presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.

Project description:BackgroundTicks are obligate bloodsucking parasites responsible for significant economic losses and concerns with human and animal health, mainly due to the transmission of pathogens. Entomopathogenic fungi have been intensively studied as an alternative strategy for tick control that can be used in combination with synthetic acaricides in the integrated management of ticks. Here, we investigated how the gut bacterial community of Rhipicephalus microplus is shaped after Metarhizium anisopliae treatment and how the tick susceptibility to the fungus is affected after disrupting gut bacterial microbiota.MethodsPartially engorged tick females were artificially fed with pure bovine blood or blood plus tetracycline. Two other groups received the same diet and were topically treated with M. anisopliae. The guts were dissected, and the genomic DNA was extracted 3 days after the treatment; the V3-V4 variable region of the bacterial 16S rRNA gene was amplified.ResultsThe gut of ticks that received no antibiotic but were treated with M. anisopliae exhibited lower bacterial diversity and a higher occurrence of Coxiella species. The Simpson diversity index and Pielou equability coefficient were higher in the gut bacterial community when R. microplus were fed with tetracycline and fungus-treated. Ticks from fungus-treated groups (with or without tetracycline) exhibited lower survival than untreated females. Previous feeding of ticks with the antibiotic did not change their susceptibility to the fungus. Ehrlichia spp. were not detected in the gueated groups.ConclusionsThese findings suggest that myco-acaricidal action would not be impacted if the calf hosting these ticks is under antibiotic therapy. Moreover, the hypothesis that entomopathogenic fungi can affect the bacterial community in the gut of R. microplus engorged females is endorsed by the fact that ticks exposed to M. anisopliae exhibited a dramatic reduction in bacterial diversity. This is the first report of an entomopathogenic fungus affecting the tick gut microbiota.

Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission.

Dataset Information

Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

Publications

Differential Expression of Immune Genes in the <i>Rhipicephalus microplus</i> Gut in Response to <i>Theileria equi</i> Infection.

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