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

BACKGROUND: Ixodes scapularis 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 in vitro, no global understanding of the host response to ticks has emerged. METHODS: 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 I. scapularis nymphs. Selected results were then validated at the mRNA and protein levels by additional real-time PCR and bioplex assay. RESULTS: 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. CONCLUSIONS: 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 I. scapularis bite site and suggests both a potential model of the host cutaneous response and candidate genes for further description and investigation.

Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells.

BACKGROUND: Tick modulation of host defenses facilitates both blood feeding and pathogen transmission. Several tick species deviate host T cell responses toward a Th2 cytokine profile. The majority of studies of modulation of T cell cytokine expression by ticks were performed with lymphocytes from infested mice stimulated in vitro with polyclonal T cell activators. Those reports did not examine tick modulation of antigen specific responses. We report use of a transgenic T cell receptor (TCR) adoptive transfer model reactive with influenza hemagglutinin peptide (110-120) to examine CD4+ T cell intracellular cytokine responses during infestation with the metastriate tick, Dermacentor andersoni, or exposure to salivary gland extracts. RESULTS: Infestation with pathogen-free D. andersoni nymphs or administration of an intradermal injection of female or male tick salivary gland extract induced significant increases of IL-4 transcripts in skin and draining lymph nodes of BALB/c mice as measured by quantitative real-time RT-PCR. Furthermore, IL-10 transcripts were significantly increased in skin while IL-2 and IFN-gamma transcripts were not significantly changed by tick feeding or intradermal injection of salivary gland proteins, suggesting a superimposed Th2 response. Infestation induced TCR transgenic CD4+ T cells to divide more frequently as measured by CFSE dilution, but more notably these CD4+ T cells also gained the capacity to express IL-4. Intracellular levels of IL-4 were significantly increased. A second infestation administered 14 days after a primary exposure to ticks resulted in partially reduced CFSE dilution with no change in IL-4 expression when compared to one exposure to ticks. Intradermal inoculation of salivary gland extracts from both male and female ticks also induced IL-4 expression. CONCLUSION: This is the first report of the influence of a metastriate tick on the cytokine profile of antigen specific CD4+ T cells. Blood feeding by D. andersoni pathogen-free nymphs or intradermal injection of salivary gland extracts programs influenza hemagglutinin influenza peptide specific TCR transgenic CD4+ T cells to express IL-4.

Dermatologic changes induced by repeated Ixodes scapularis bites and implications for prevention of tick-borne infection.

Previous studies in rodents and people have demonstrated that repeated tick exposure is associated with reduced Borrelia burgdorferi transmission but the mechanism of prevention remains unclear. We examined the acute histopathologic reactions to initial and repeated Ixodes scapularis bites in BALB/c mice and in people. Skin biopsies of BALB/c mice infested for the first time by I. scapularis nymphs revealed vascular dilatation and an accumulation of inflammatory cells adjacent to the bite site but absent at the site of tick attachment. Such changes would enhance tick-borne pathogen transmission. Mice reexposed to I. scapularis nymphs experienced a decrease in vascular dilatation and a marked increase in inflammatory cells at the site of tick attachment. Skin biopsies of people with attached I. scapularis nymphs revealed similar histologic patterns. These results indicate that cellular changes at the tick-dermal interface following I. scapularis attachment are likely to allow for successful transmission of tick-borne pathogens in non-tick-immune hosts and to inhibit tick-borne pathogen transmission in hosts that have developed tick immunity.

Transcriptome analysis of the salivary glands of Dermacentor andersoni Stiles (Acari: Ixodidae).

Amongst blood-feeding arthropods, ticks of the family Ixodidae (hard ticks) are vectors and reservoirs of a greater variety of infectious agents than any other ectoparasite. Salivary glands of ixodid ticks secrete a large number of pharmacologically active molecules that not only facilitate feeding but also promote establishment of infectious agents. Genomic, proteomic and immunologic characterization of bioactive salivary gland molecules are, therefore, important as they offer new insights into molecular events occurring at the tick-host interface and they have implications for development of novel control strategies. The present work uses complementary DNA (cDNA) sequence analysis to identify salivary gland transcripts expressed by the Rocky Mountain wood tick, Dermacentor andersoni, a vector of the human pathogens causing Rocky Mountain spotted fever, Colorado tick fever, tularemia, and Powassan encephalitis as well as the veterinary pathogen Anaplasma marginale. Dermacentor andersoni is also capable of inducing tick paralysis. Automated single-pass DNA sequencing was conducted on 1440 randomly selected cDNA clones from the salivary glands of adult female D. andersoni collected during the early stages of feeding (18-24h). Analysis of the expressed sequence tags (ESTs) resulted in 544 singletons and 218 clusters with more than one quality read and attempts were made to assign putative functions to tick genes based on amino acid identity to published protein databases. Approximately 25.6% (195) of the sequences showed limited or no homology to previously identified gene products. A number of novel sequences were identified which presented significant sequence similarity to mammalian genes normally associated with extracellular matrix (ECM), regulation of immune responses, tumor suppression, and wound healing. Several coding sequences possessed various degrees of homology to previously described proteins from other tick species. Preliminary nucleotide variation analysis of these and other tick sequences suggests extensive nucleotide diversity, which has implications for evolution of tick feeding. Intra-species diversity studies can be a promising tool for identifying sequence variations potentially associated with phenotypic traits affecting vector-host-pathogen interactions.

Confirmation of tick bite by detection of antibody to Ixodes calreticulin salivary protein.

Ticks introduce a variety of pharmacologically active molecules into their host during attachment and feeding in order to obtain a blood meal. People who are repeatedly exposed to ticks may develop an immune response to tick salivary proteins. Despite this response, people usually are unaware of having been bitten, especially if they are not repeatedly exposed to ticks. In order to develop a laboratory marker of tick exposure that would be useful in understanding the epidemiology of tick-borne infection and the immune response to tick bite, we developed an enzyme-linked immunosorbent assay (ELISA) to detect antibody to a recombinant form of calreticulin protein found in the salivary glands of Ixodes scapularis, a member of a complex of Ixodes ticks that serve as the vectors for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Using this assay, we tested sera obtained from C3H/HeN and BALB/c mice before and after experimental deer tick infestation. These mice developed antibody to Ixodes calreticulin antigen after infestation. We then used the same assay to test sera obtained from people before and after they experienced deer tick bite(s). People experiencing deer tick bite(s) developed Ixodes calreticulin-specific antibody responses that persisted for up to 17 months. This Ixodes recombinant calreticulin ELISA provides objective evidence of deer tick exposure in people.

An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks.

Over 8000 expressed sequence tags from six different salivary gland cDNA libraries from the tick Ixodes scapularis were analyzed. These libraries derive from feeding nymphs infected or not with the Lyme disease agent, Borrelia burgdorferi, from unfed adults, and from adults feeding on a rabbit for 6-12 h, 18-24 h, and 3-4 days. Comparisons of the several libraries led to identification of several significantly differentially expressed transcripts. Additionally, over 500 new predicted protein sequences are described, including several novel gene families unique to ticks; no function can be presently ascribed to most of these novel families. Among the housekeeping-associated transcripts, we highlight those enzymes associated with post translation modification of amino acids, particularly those forming sulfotyrosine, hydroxyproline, and carboxyl-glutamic acid. Results support the hypothesis that gene duplication, most possibly including genome duplications, is a major player in tick evolution.

Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

Characterization of a recombinant immunomodulatory protein from the salivary glands of Dermacentor andersoni.

The gene encoding a 36-kDa (p36) immunomodulatory protein present in saliva of Dermacentor andersoni was cloned in prokaryotic and eukaryotic expression vectors. A polymerase chain reaction (PCR)-generated cDNA lacking signal peptide was cloned into the Escherichia coli expression vector pET28 and a similar sequence was cloned into pIB/V5-His-TOPO expression vector for stable transfection of insect cells, High 5 trade mark. The 26-kDa molecular mass of p36 expressed by bacteria is in agreement with that predicted from the translated full-length cDNA sequence. Eukaryotic-cell-expressed p36 consisted of multiple forms with molecular masses between 34 and 36 kDa. These multiple forms were attributed to differences in post-translational modifications. N-linked mannose was detected on insect-cell-expressed and tick-derived p36. Multiple bands remained after endoglycosidase removal of N-linked sugars, indicating the presence of other modifications. Both bacterial- and insect-cell-expressed p36 reacted on immunoblots with polyclonal antibodies raised against tick-derived p36. Insect-cell-expressed p36 suppressed T-lymphocyte-mitogen-driven in vitro proliferation of splenocytes from tick-naïve mice in a dose-dependent manner. Bacterial-cell-expressed p36 lacked immunomodulatory activity.

RpoN (sigma(54)) is required for plasmid-encoded coronatine biosynthesis in Pseudomonas syringae.

The plant pathogen Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a phytotoxin which functions as a virulence factor in bacterial blight of soybeans. The COR biosynthetic gene cluster in PG4180 is borne on a 90-kb plasmid named p4180A. Although pathway-specific regulatory genes for COR have been identified, global regulatory genes for COR production in PG4180 remain undefined. In the present study, we evaluated the role of rpoN, which encodes sigma(54), in the virulence of strain PG4180. A rpoN mutant of PG4180, designated PG4180.K2, was unable to grow in M9 minimal medium; however, the addition of exogenous glutamate, glutamine or aspartate to M9 medium enabled PG4180.K2 to grow in vitro. PG4180.K2 could not induce disease symptoms or multiply in soybean plants and was defective in COR production and cor gene expression. Furthermore, PG4180.K2 was impaired in transcription of hrpL, an alternate sigma factor that mediates expression of genes in the type III secretion system of P. syringae. PG4180.K2 transconjugants with a wild-type copy of rpoN were complemented for hrpL and cor gene expression, COR biosynthesis, and growth in vitro. Our results indicate that rpoN is required for growth and the expression of both chromosomal and plasmid-encoded virulence factors in P. syringae pv. glycinea PG4180.