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Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

ABSTRACT: 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.

SUBMITTER: Zivkovic Z

PROVIDER: S-EPMC2848250 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

Zivkovic Zorica Z Esteves Eliane E Almazán Consuelo C Daffre Sirlei S Nijhof Ard M AM Kocan Katherine M KM Jongejan Frans F de la Fuente José J

BMC genomics 20100318

<h4>Background</h4>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 cu ...[more]

PMID: 20298599

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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.

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:It 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.A 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.Infection 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.Taken 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:BackgroundAlternative strategies are required to control the southern cattle tick, Rhipicephalus microplus, due to evolving resistance to commercially available acaricides. This invasive ectoparasite is a vector of economically important diseases of cattle such as bovine babesiosis and anaplasmosis. An understanding of the biological intricacies underlying vector-host-pathogen interactions is required to innovate sustainable tick management strategies that can ultimately mitigate the impact of animal and zoonotic tick-borne diseases. Tick saliva contains molecules evolved to impair host innate and adaptive immune responses, which facilitates blood feeding and pathogen transmission. Antigen presenting cells are central to the development of robust T cell responses including Th1 and Th2 determination. In this study we examined changes in co-stimulatory molecule expression and cytokine response of bovine macrophages exposed to salivary gland extracts (SGE) obtained from 2-3 day fed, pathogen-free adult R. microplus.MethodsPeripheral blood-derived macrophages were treated for 1 hr with 1, 5, or 10 μg/mL of SGE followed by 1, 6, 24 hr of 1 μg/mL of lipopolysaccharide (LPS). Real-time PCR and cytokine ELISA were used to measure changes in co-stimulatory molecule expression and cytokine response.ResultsChanges were observed in co-stimulatory molecule expression of bovine macrophages in response to R. microplus SGE exposure. After 6 hrs, CD86, but not CD80, was preferentially up-regulated on bovine macrophages when treated with 1 μg/ml SGE and then LPS, but not SGE alone. At 24 hrs CD80, CD86, and CD69 expression was increased with LPS, but was inhibited by the addition of SGE. SGE also inhibited LPS induced upregulation of TNFα, IFNγ and IL-12 cytokines, but did not alter IL-4 or CD40 mRNA expression.ConclusionsMolecules from the salivary glands of adult R. microplus showed bimodal concentration-, and time-dependent effects on differential up-regulation of CD86 in bovine macrophages activated by the TLR4-ligand, LPS. Up regulation of proinflammatory cytokines and IL-12, a Th1 promoting cytokine, were inhibited in a dose-dependent manner. The co-stimulatory molecules CD80, as well as the cell activation marker, CD69, were also suppressed in macrophages exposed to SGE. Continued investigation of the immunomodulatory factors will provide the knowledge base to research and develop therapeutic or prophylactic interventions targeting R. microplus-cattle interactions at the blood-feeding interface.

Dataset Information

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

Publications

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

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