Differential expression of Aedes aegypti salivary transcriptome upon blood feeding

Saliva of Aedes aegypti contains a complex array of proteins essential for both blood feeding and pathogen transmission. A large numbers of those proteins are classified as unknown in regard to their function(s). Understanding the dynamic interactions at the mosquito-host interface can be achieved in part by characterizing mosquito salivary gland gene expression relative to blood feeding. Towards this end, we developed an oligonucleotide microarray representing 463 transcripts to determine differential regulation of salivary gland genes. This microarray was used to investigate the temporal gene expression pattern of Ae. aegypti salivary gland transcriptome at different times post-blood feeding. Expression of the majority of salivary gland genes (77–87%) did not change significantly as a result of blood feeding, while 8 to 20% of genes were down-regulated and 2.8 to 11.6% genes were up-regulated. Up-regulated genes included defensins, mucins and other immune related proteins. Odorant-binding protein was significantly down-regulated. Among unknown function proteins, several were up-regulated during the first three hours post-blood feeding and one was significantly down-regulated. Quantitative real-time RT-PCR was used to substantiate differential expression patterns of five randomly selected genes. Linear regression analysis revealed a high degree of correlation (R2 > 0.89) between oligonucleotide microarray and quantitative RT-PCR data. To our knowledge, this is the first study to investigate differential expression of the Ae. aegypti salivary gland transcriptome upon blood feeding. A microarray provides a robust, sensitive way to investigate differential regulation of mosquito salivary gland genes

Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis nymphs

<p>Abstract</p> <p>Background</p> <p><it>Ixodes scapularis </it>ticks are hematophagous arthropods capable of transmitting many infectious agents to humans. The process of blood feeding is an extended and continuous interplay between tick and host responses. While this process has been studied extensively <it>in vitro</it>, no global understanding of the host response to ticks has emerged.</p> <p>Methods</p> <p>To address this issue, we used PCR-arrays to measure skin-specific expression of 233 discrete genes at 8 time points during primary and secondary infestations of mice with pathogen-free <it>I. scapularis </it>nymphs. Selected results were then validated at the mRNA and protein levels by additional real-time PCR and bioplex assay.</p> <p>Results</p> <p>Primary infestation was characterized by the late induction of an innate immune response. Lectin pattern recognition receptors, cytokines, and chemokines were upregulated consistent with increased neutrophil and macrophage migration. Gene ontology and pathway analyses of downregulated genes suggested inhibition of gene transcription and Th17 immunity. During the secondary infestation, additional genes were modulated suggesting a broader involvement of immune cells including CD8 and CD4 positive T lymphocytes. The cytokine response showed a mixed Th1/Th2 profile with a potential for T regulatory cell activity. Key gene ontology clusters observed during the secondary infestation were cell migration and activation. Matrix metalloproteinases were upregulated, apoptosis-related genes were differentially modulated, and immunoreceptor signaling molecules were upregulated. In contrast, transcripts related to mitogenic, WNT, Hedgehog, and stress pathways were downregulated.</p> <p>Conclusions</p> <p>Our results support a model of tick feeding where lectin pattern recognition receptors orchestrate an innate inflammatory response during primary infestation that primes a mixed Th1/Th2 response upon secondary exposure. Tick feeding inhibits gene transcription and Th17 immunity. Salivary molecules may also inhibit upregulation of mitogenic, WNT, Hedgehog, and stress pathways and enhance the activity of T regulatory cells, production of IL-10, and suppressors of cytokine signaling molecules (SOCS). This study provides the first comprehensive transcriptional analysis of the murine host response at the <it>I. scapularis </it>bite site and suggests both a potential model of the host cutaneous response and candidate genes for further description and investigation.</p

Rhipicephalus microplus salivary gland molecules induce differential CD86 expression in murine macrophages

<p>Abstract</p> <p>Background</p> <p>Tick parasitism is a major impediment for cattle production in many parts of the world. The southern cattle tick, <it>Rhipicephalus </it>(<it>Boophilus</it>) <it>microplus</it>, is an obligate hematophagous parasite of domestic and wild animals that serves as vector of infectious agents lethal to cattle. Tick saliva contains molecules evolved to modulate host innate and adaptive immune responses which facilitates blood feeding and pathogen transmission. Tick feeding promotes CD4 T cell polarization to a Th2 profile usually accompanied by down-regulation of Th1 cytokines through as yet undefined mechanisms. Co-stimulatory molecules on antigen presenting cells are central to development of T cell responses including Th1 and Th2 responses. Tick induced changes to antigen presenting cell signal transduction pathways are largely unknown. Here we document the ability of <it>R</it>. <it>microplus </it>salivary gland extracts (SGE) to effect differential CD86 expression.</p> <p>Results</p> <p>We examined changes in co-stimulatory molecule expression in murine RAW 264.7 cells in response to <it>R</it>. <it>microplus </it>SGE exposure in the presence of the toll-like receptor 4 (TLR4) ligand, LPS. After 24 hrs, CD86, but not CD80, was preferentially up-regulated on mouse macrophage RAW 264.7 cells when treated with SGE and then LPS, but not SGE alone. CD80 and CD40 expression was increased with LPS, but the addition of SGE did not alter expression. Higher concentrations of SGE were less effective at increasing CD86 RNA expression. The addition of mitogen or extracellular kinase (MEK) inhibitor, PD98059, significantly reduced the ability for SGE to induce CD86 expression, indicating activation of MEK is necessary for SGE induced up-regulation.</p> <p>Conclusions</p> <p>Molecules in SGE of <it>R. microplus </it>have a concentration-dependent effect on differential up-regulation of CD86 in a macrophage cell line activated by the TLR4 ligand, LPS. This CD86 up-regulation is at least partially dependent on the ERK1/2 pathway and may serve to promote Th2 polarization of the immune response.</p

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti*

BACKGROUND: Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes. RESULTS: A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes. CONCLUSION: Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the Æ. ægypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect

Changes in Temporal and Spatial Patterns of Outer Surface Lipoprotein Expression Generate Population Heterogeneity and Antigenic Diversity in the Lyme Disease Spirochete, Borrelia burgdorferi

This is the published version. Copyright 2015 by the American Society for Microbiology.Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi

Hypersensitivity to Ticks and Lyme Disease Risk

Persons who report frequent tick-associated itch are less likely to contract Lyme disease than those who do not

Regulation of OspE-Related, OspF-Related, and Elp Lipoproteins of Borrelia burgdorferi Strain 297 by Mammalian Host-Specific Signals

This is the published version. Copyright 2001 by the American Society for Microbiology.In previous studies we have characterized the cp32/18 loci inBorrelia burgdorferi 297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37°C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that B. burgdorferi-infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the −10 and −35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as B. burgdorferi is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease

Analyzing ligation mixtures using a PCR based method

We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been demonstrated using ligation mixtures of two cDNA’s derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods

Amblyomma imitator Ticks as Vectors of Rickettsia rickettsii, Mexico

Real-time PCR of Amblyomma imitator tick egg masses obtained in Nuevo Leon State, Mexico, identified a Rickettsia species. Sequence analyses of 17-kD common antigen and outer membrane protein A and B gene fragments showed to it to be R. rickettsii, which suggested a potential new vector for this bacterium

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission

BACKGROUND: Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model. METHODS: Cattle were inoculated with a multivalent antigen cocktail containing recombinant tick protective antigen subolesin as well as two additional R. appendiculatus saliva antigens: the cement protein TRP64, and three different histamine binding proteins. The cocktail also contained the T. parva sporozoite antigen p67C. The effect of vaccination on the feeding success of nymphal and adult R. appendiculatus ticks was evaluated together with the effect on transmission of T. parva using a tick challenge model. RESULTS: To our knowledge, this is the first evaluation of the anti-tick effects of these antigens in the natural host-tick-parasite combination. In spite of evidence of strong immune responses to all of the antigens in the cocktail, vaccination with this combination of tick and parasite antigens did not appear to effect tick feeding success or reduce transmission of T. parva. CONCLUSION: The results of this study highlight the importance of early evaluation of anti-tick vaccine candidates in biologically relevant challenge systems using the natural tick-host-parasite combination