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Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease. The gene function studies in I. scapularis and other ticks are hampered by the lack of genetic tools, including an inducible promoter for temporal control over transgene-encoding protein or double-stranded RNA. We characterized an intergenic sequence upstream of a heat shock protein 70 (HSP70) gene that can drive Renilla luciferase and mCherry expression in the I. scapularis cell line ISE6 (IsHSP70). In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter of the 3xP3 promoter with a minimal portion of IsHSP70 promoter and generated an I. scapularis -specific 3xP3 (Is3xP3) promoter. Both IsHSP70 and Is3xP3 have a heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent cells upon 2 h heat shock. These promoters described will be valuable tools for gene function studies., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Importance: Ticks are second only to mosquitoes in their importance as vectors of disease agents; however, tick-borne diseases (TBDs) account for the majority of all vector-borne disease cases in the United States (approximately 76.5%), according to Centers for Disease Control and Prevention reports. Newly discovered tick species and their associated disease-causing pathogens, and anthropogenic and demographic factors also contribute to the emergence and re-emergence of TBDs. Thus, incorporating different tick control approaches based on a thorough knowledge of tick biology has great potential to prevent and eliminate TBDs in the future. Here we demonstrate that replication of a transovarially transmitted rickettsial endosymbiont depends on the tick's autophagy machinery but not on apoptosis. Our findings improve our understanding of the role of symbionts in tick biology and the potential to discover tick control approaches to prevent or manage TBDs., Competing Interests: The authors declare no conflict of interest.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Laukaitis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here, we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis IrR/Munich mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with various portions of the region surrounding these genes and a selection reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate's Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA . PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-carried gene (RM_p5) in comparison to chromosomally carried gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis strains, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. IMPORTANCE Rickettsia spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii, can cause severe illness or death. Plasmids are found in a large percentage of nonpathogenic rickettsiae, but not in species that cause severe disease. Studying these plasmids can reveal their role in the biology of these bacteria, as well as the molecular mechanism whereby they are maintained and replicate in rickettsiae. Here, we describe a new series of shuttle plasmids for the transformation of rickettsiae based on parA and dnaA sequences of plasmid pRM from Rickettsia monacensis. These shuttle vectors support transformation of diverse rickettsiae, including the native host of pRM, and are useful for investigating genetic determinants that govern rickettsial virulence or their ability to function as symbionts.

Ixodes scapularis is the primary vector of tick-borne pathogens in North America but notably does not transmit pathogenic Rickettsia species. This tick harbors the transovarially transmitted endosymbiont Rickettsia buchneri , which is widespread in I. scapularis populations, suggesting that it confers a selective advantage for tick survival such as providing essential nutrients. The R. buchneri genome includes genes with similarity to those involved in antibiotic synthesis. There are two gene clusters not found in other Rickettsiaceae, raising the possibility that these may be involved in excluding pathogenic bacteria from the tick. This study explored whether the R. buchneri antibiotic genes might exert antibiotic effects on pathogens associated with I. scapularis . Markedly reduced infectivity and replication of the tick-borne pathogens Anaplasma phagocytophilum, R. monacensis , and R. parkeri were observed in IRE11 tick cells hosting R. buchneri . Using a fluorescent plate reader assay to follow infection dynamics revealed that the presence of R. buchneri in tick cells, even at low infection rates, inhibited the growth of R. parkeri by 86-100% relative to R. buchneri -free cells. In contrast, presence of the low-pathogenic species R. amblyommatis or the endosymbiont R. peacockii only partially reduced the infection and replication of R. parkeri . Addition of host-cell free R. buchneri , cell lysate of R. buchneri- infected IRE11, or supernatant from R. buchneri- infected IRE11 cultures had no effect on R. parkeri infection and replication in IRE11, nor did these treatments show any antibiotic effect against non-obligate intracellular bacteria E. coli and S. aureus . However, lysate from R. buchneri- infected IRE11 challenged with R. parkeri showed some inhibitory effect on R. parkeri infection of treated IRE11, suggesting that challenge by pathogenic rickettsiae may induce the antibiotic effect of R. buchneri . This research suggests a potential role of the endosymbiont in preventing other rickettsiae from colonizing I. scapularis and/or being transmitted transovarially. The confirmation that the observed inhibition is linked to R. buchneri 's antibiotic clusters requires further investigation but could have important implications for our understanding of rickettsial competition and vector competence of I. scapularis for rickettsiae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cull, Burkhardt, Wang, Thorpe, Oliver, Kurtti and Munderloh.)

The blacklegged tick, Ixodes scapularis , a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus . The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri . qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis . We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Apoptosis is an innate immune response induced by infection in eukaryotes that contributes significantly to protection from pathogens. However, little is known about the role of apoptosis in the interactions of arthropod vectors with the rickettsiae that they transmit. Rickettsia spp. are vector-borne obligately intracellular bacteria and display different degrees of virulence in their eukaryotic hosts. In this study, we found that infection with Rickettsia parkeri ( Rp ) activated the apoptosis pathway in an Amblyomma americanum tick cell line (AAE2), as evidenced by the loss of phospholipid membrane asymmetry and DNA fragmentations. Additionally, infection with Rp also led to apoptosis activation in cell lines of different tick species. Interestingly, suppressing apoptosis decreased Rp infection and replication, while the activation of apoptosis increased Rp accumulation at the early stage of infection. Moreover, mitochondrion-dependent apoptosis was essential for Rp infection and replication in vector cells, and apoptosis induction required intracellular rickettsia replication. We further showed that Rp utilizes two different survival strategies to modulate apoptosis in the arthropod vectors and mammalian host cells. There was no direct correlation between apoptosis activation in vector cells and rickettsial pathogenicity. These novel findings indicate a possible mechanism whereby apoptosis facilitates infection and replication of a Rickettsia sp. in an arthropod vector. These results contribute to our understanding of how the vector's responses to pathogen infection affect pathogen replication and therefore transmission. IMPORTANCE Rickettsioses, infections caused by the genus Rickettsia , are among the oldest known infectious diseases. Ticks are essential arthropod vectors for rickettsiae, and knowledge about the interactions between ticks, their hosts, and pathogens is fundamental for identifying drivers of tick-borne rickettsioses. Despite the rapid development in apoptosis research with rickettsiae, little is known regarding the role of apoptosis in the interactions between Rickettsia spp., vertebrate hosts, and arthropod vectors. Here, we demonstrated that mitochondrion-dependent apoptosis is essential for rickettsial infection and replication in vector cells and that apoptosis induction requires intracellular rickettsial replication. However, rickettsial pathogenicity is not linked with apoptosis activation in tick cells. Our findings improve understanding of the apoptosis mechanism in arthropods exploited by rickettsiae and also the potential to discover specific targets for new vaccines and drugs to prevent or treat rickettsial infections., (Copyright © 2021 Wang et al.)

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri , it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny. IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks., (Copyright © 2021 American Society for Microbiology.)

Background: Haematobia spp., horn flies (HF) and buffalo flies (BF), are economically important ectoparasites of dairy and beef cattle. Control of these flies relies mainly on treating cattle with chemical insecticides. However, the development of resistance to commonly used compounds is compromising the effectiveness of these treatments and alternative methods of control are required. Wolbachia are maternally transmitted endosymbiotic bacteria of arthropods that cause various reproductive distortions and fitness effects, making them a potential candidate for use in the biological control of pests. The first step towards this is the establishment and adaptation of xenobiotic infections of Wolbachia in target host cell lines., Results: Here, we report the successful establishment of a continuous HF cell line (HIE-18) from embryonic cells and its stable transinfection with Wolbachia strains wAlbB native to mosquitoes, and wMel and wMelPop native to Drosophila melanogaster. HIE-18 cells were typically round and diploid with ten chromosomes (2n = 10) or tetraploid with 20 chromosomes (4n = 20), with a doubling time of 67.2 h. Wolbachia density decreased significantly in HIE-18 cells in the first 48 h of infection, possibly due to overexpression of antimicrobial peptides through the Imd immune signalling pathway. However, density recovered after this time and HIE-18 cell lines stably infected with the three strains of Wolbachia have now each been subcultured more than 50 times as persistently infected lines., Conclusion: The amenability of HF cells to infection with different strains of Wolbachia and the establishment of stable sustaining infections suggest the potential for use of Wolbachia in novel approaches for the control of Haematobia spp. Further, the availability of the HIE-18 cell line will provide an important resource for the study of genetics, host-parasite interactions and chemical resistance in Haematobia populations. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis , Ixodes ricinus and Rhipicephalus microplus , were inoculated with Wolbachia strains w Stri and w AlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The w Stri strain infected Ixodes spp. cells and was maintained through 29 passages. The w AlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2-5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated w CfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the "pandemic" A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia .

Ticks are obligate hematophagous arthropods and must tolerate starvation during off-host periods. Macroautophagy (hereafter autophagy) is a well-conserved self-eating mechanism of cell survival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Although the genome sequence of Ixodes scapularis (Say) is available, the characteristics and functions of autophagy-related gene families remain largely unknown. To advance our understanding of autophagy in I. scapularis, we used comprehensive genomic approaches to identify Atg genes. Homologues of 14 Atg genes were identified, and their protein motif compositions were predicted. Phylogenetic analysis indicated that ATGs in I. scapularis were evolutionarily closely related to their homologues in Haemaphysalis longicornis and Rhipicephalus microplus ticks. Expression patterns of Atg genes differed across tick developmental stages. Immunofluorescence results by monodansylcadaverine (MDC) staining indicated that autophagy was activated after amino acid starvation treatments in I. scapularis embryo-derived cell lines ISE6 and IDE8. Subsequently, the expression of key Atg genes involved in autophagy pathway in both cell lines were examined. In ISE6 cells, the expression levels of three Atg genes (Atg4B, Atg6 and Atg8A) increased significantly after amino acid starvation; similarly, four Atg genes (Atg4A, Atg4B, Atg6 and Atg8B) were upregulated in IDE8 cells in response to starvation. In parallel, the MDC and lysotracker staining results indicated that autophagy was triggered after amino acid starvation treatments in R. microplus embryo-derived cell line BME26. Our observations showed that Atg family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tick-borne pathogens., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks. IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis , a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen., (Copyright © 2019 American Society for Microbiology.)

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts- Rickettsia peacockii and R. buchneri -both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris-like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis, and infect nymphs with tick-borne pathogens.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.

Rickettsia bellii is an obligate intracellular bacterium that is one of the few rickettsiae that encode a complete set of conjugative transfer (tra) genes involved in bacterial conjugation and has been shown to exhibit pili-like structures. The reductive genomes of rickettsiae beg the question whether the tra genes are nonfunctional or functioning to enhance the genetic plasticity and biology of rickettsiae. We characterized the transcriptional dynamics of R. bellii tra genes in comparison to genes transcribed stably and above the background level to understand when and at what levels the tra genes are active or whether the tra genes are degenerative. We determined that the best reference genes, out of 10 tested, were methionyl tRNA ligase (metG) or a combination of metG and ribonucleoside diphosphate reductase 2 subunit beta (nrdF), using statistical algorithms from two different programs: Normfinder and BestKeeper. To validate the use of metG with other rickettsial genes exhibiting variable transcriptional patterns we examined its use with sca2 and rickA, genes involved in actin based motility. Both were shown to be up-regulated at different times of replication in Vero cells, showing variable and stable transcription levels of rickA and sca2, respectively. traATi was up-regulated at 72 hours post inoculation in the tick cell line ISE6, but showed no apparent changes in the monkey cell line Vero and mouse cell line L929. The transcription of tra genes was positively correlated with one another and up-regulated from 12 to 72 hours post inoculation (HPI) when compared to RBE_0422 (an inactivated transposase-derivative found within the tra cluster). Thus, the up-regulation of the tra genes indicated that the integrity and activity of each gene were intact and may facilitate the search for the optimal conditions necessary to demonstrate conjugation in rickettsiae.

The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 β-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.

Human pathogens transmitted by ticks undergo complex life cycles alternating between the arthropod vector and a mammalian host. While the latter has been investigated to a greater extent, examination of the biological interactions between microbes and the ticks that carry them presents an equally important opportunity for disruption of the disease cycle. In this study, we used in situ hybridization to demonstrate infection by the Ehrlichia muris-like organism, a newly recognized human pathogen, of Ixodes scapularis ticks, a primary vector for several important human disease agents. This allowed us to assess whole sectioned ticks for the patterns of tissue invasion, and demonstrate generalized dissemination of ehrlichiae in a variety of cell types and organs within ticks infected naturally via blood feeding. Electron microscopy was used to confirm these results. Here we describe a strong ehrlichial affinity for epithelial cells, neuronal cells of the synganglion, salivary glands, and male accessory glands.

We obtained a rickettsial isolate from the ovaries of the blacklegged tick, Ixodes scapularis. The isolate (ISO7(T)) was grown in the Ixodes ricinus embryonic cell line IRE11. We characterized the isolate by transmission electron microscopy and gene sequencing. Phylogenetic analysis of 11 housekeeping genes demonstrated that the isolate fulfils the criteria to be classified as a representative of a novel rickettsial species closely related to 'Rickettsia monacensis'. These rickettsiae form a clade separate from other species of rickettsiae. Gene sequences indicated that several genes important in rickettsial motility, invasiveness and temperature adaptation were mutated (e.g. sca2, rickA, hsp22, pldA and htrA). We propose the name Rickettsia buchneri sp. nov. for this bacterium that infects the ovaries of the tick I. scapularis to acknowledge the pioneering contributions of Professor Paul Buchner (1886-1978) to research on bacterial symbionts. The type strain of R. buchneri sp. nov. is strain ISO-7(T) ( = DSM 29016(T) = ATCC VR-1814(T))., (© 2015 IUMS.)

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The rickA gene sequence and protein vary substantially between Rickettsia species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three Rickettsia bellii variants. These included two shuttle vector-transformed R. bellii strains and the wild-type isolate from which they were derived, R. bellii RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained Rickettsia monacensis-derived rickA, and the other lacked the rickA gene. Rickettsia bellii transformed to express R. monacensis rickA highly overexpressed this transcript in comparison to its native rickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of R. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques.

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P<0.001). Using Western blots probed with two Msp2-monospecific and one Msp2-monoclonal antibodies, we detected expression of variants with differences in molecular weight. Immunofluorescence-assay revealed that specific antibodies bound from 25 to 60% of colonies, depending on the host cell-line (P<0.001). Molecular analysis of cloned variant-encoding genes demonstrated expression of different predominant variants in tick (V1) and mammalian (V2) cell-lines. Analysis of the putative secondary structure of the variants revealed a change in structure when A. marginale was transferred from one cell-type to another, suggesting that the expression of particular Msp2 variants depended on the cell-type (tick or mammalian) in which A. marginale developed. Similarly, analysis of the putative secondary structure of over 200 Msp2 variants from ticks, blood samples, and other mammalian cells available in GenBank showed the predominance of a specific structure during infection of a host type (tick versus blood sample), demonstrating that selection of a possible structure also occurred in vivo. The selection of a specific structure in surface proteins may indicate that Msp2 fulfils an important role in infection and adaptation to diverse host systems. Supplemental Abstract in Spanish (File S1) is provided.

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.

Wolbachia and Cardinium are bacterial endosymbionts that are widely distributed amongst arthropods. Both cause reproductive alterations, such as cytoplasmic incompatibility, parthenogenesis and feminization. Here we studied differentially expressed genes in Wolbachia- and Cardinium-infected Bm-aff3 silkworm cells using a silkworm microarray. Wolbachia infection did not alter gene expression or induce or suppress immune responses. In contrast, Cardinium infection induced many immune-related genes, including antimicrobial peptides, pattern recognition receptors and a serine protease. Host immune responses differed, possibly because of the different cell wall structures of Wolbachia and Cardinium because the former lacks genes encoding lipopolysaccharide components and two racemases for peptidoglycan formation. A few possibly non-immune-related genes were differentially expressed, but their involvement in host reproductive alteration was unclear., (© 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.)

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae., (© 2011 Burkhardt et al.)

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

In spite of the global medical and veterinary importance of Ixodid ticks, relatively little is known about their genome organization. To address this, we developed the first fluorescence in situ hybridization (FISH)-based chromosome markers in the Lyme disease vector, Ixodes scapularis. Shotgun genomic DNA (gDNA) sequences were used to identify three major tandem repeat families which were localized to specific heterochromatic regions of I. scapularis chromosomes prepared from the mitotic cell line ISE18. Together, these repeats were estimated to contribute approximately 159 Mb (8%) of the 2.1 Gb (haploid) I. scapularis genome. The relative arrangement of each tandem repeat family and the nucleolar organizing regions was determined by rehybridization to individual chromosome spreads, which was useful to distinguish different chromosomes in the ISE18 karyotype. Long stretches (>20 kb) of tandem repeat-containing gDNA were resistant to digestion by the methylation-sensitive restriction enzyme HpaII and localized to the presumed peri-centromeric regions of the chromosomes. A telomeric probe based on the arthropod-conserved (TTAGG)(n) tandemly repetitive motif was localized to the termini of each I. scapularis chromosome. Localization of these markers produced the first link between DNA sequences and major structural features of I. scapularis chromosomes and thereby provided the framework for a FISH-based physical map.

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, "Candidatus Rickettsia amblyommii," R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two "Ca. Rickettsia amblyommii" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of "Ca. Rickettsia amblyommii," R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, < or =1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80-90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.

Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts. Genetic manipulation of rickettsiae should facilitate a better understanding of their interactions with hosts., Methodology/principal Findings: We transformed a species never associated with human disease, Rickettsia montanensis, by electroporation with a TN5 transposon (pMOD700) containing green fluorescent protein (GFPuv) and chloramphenicol acetyltransferase (CAT) genes under regulation of promoters cloned from the Rickettsia rickettsii ompB gene, and isolated a Chloramphenicol-resistant GFP-fluorescent rickettsiae population (Rmontanensis700). The Rmontanensis700 rickettsiae contained a single transposon integrated near an acetyl-CoA acetyltransferase gene in the rickettsial chromosome. Northern blots showed that GFPuv and CAT mRNAs were both expressed as two transcripts of larger and smaller than predicted length. Western immunoblots showed that Rmontanensis700 and E. coli transformed with a plasmid containing the pMOD700 transposon both expressed GFPuv proteins of the predicted molecular weight., Conclusions/significance: Long-standing barriers to transformation of rickettsiae have been overcome by development of transposon-based rickettsial transformation vectors. The ompB promoter may be the most problematic of the four promoters so far employed in those vectors.

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F1 larvae by individual ticks at efficiencies of 10-40 and 95-100%, respectively. The FLEs were transovarially transmitted to F2 larvae obtained as progeny of adults from F1 larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission.

The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.

Background: Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1)., Results: Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6) and the human (HL-60 and HMEC-1) cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system) showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins) paralogs (of 114 total), through elevated signal produced to the unique hypervariable region of each - 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6., Conclusion: Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

Several fungal pathogens are able to enter and persist within eukaryotic cells as part of their infectious life cycle. Metarhizium anisopliae is a saprophytic entomopathogenic fungus virulent towards numerous tick species, including those within the genera Ixodes and Amblyomma. Infection of the target organism by this fungus proceeds via several steps, including adhesion and penetration of the host cuticle, proliferation within tissues and the haemolymph, and eventual eruption through the host cadaver. To determine whether M. anisopliae could enter and persist within tick cells, we examined the uptake of wild-type and green fluorescent protein (GFP)-expressing fungal strains into two different tick cells lines, IDE12 and AAE2, derived from Ixodes scapularis and Amblyomma americanum, respectively. Uptake by tick cells was monitored by confocal fluorescent microscopy, as well as by scanning and transmission electron microscopy. M. anisopliae-specific antibodies were used to discriminate between extracellular and internalized fungal cells and to quantify the rate of uptake. Both cell lines internalized fungal conidia, and quantitative studies using the AAE2 cell line indicated that almost 70% of the AAE2 cells contained internalized conidia after 6 h incubation. Internalization of conidia by AAE2 cells was time and temperature dependent and could be inhibited by 80% with 1 mM cytochalasin D. Internalized conidia remained viable within the AAE2 cells, where they were able to germinate and grow, eventually erupting from the host cells. These data provide evidence that M. anisopliae conidia can be internalized, survive and grow within phagocytic cells in vitro and indicate that phagocytosis may serve as an alternative invasion route facilitating fungal nutrient acquisition, immune system evasion and dissemination throughout the host.

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors, antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens.

The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.

Soft ticks are medically important ectoparasites of birds and mammals that are found throughout the world. This report describes isolation and partial characterization of two embryonic cell lines, CCE2 and CCE3, from the seabird soft tick Carios capensis (Neumann). Sequencing of the mitochondrial 16S rRNA gene and karyology confirmed the lines were derived from C. capensis. CCE3 cells were diploid with a modal chromosome number of 20. The population doubling time for cell lines CCE2 and 3 in passage 40 was 6-9 d. A rickettsial endosymbiont, RCCE3, was co-isolated along with line CCE3. Nucleotide sequences of polymerase chain reaction (PCR) products generated using primers specific for rickettsial 17-kDa antigen, outer membrane protein (omp) A, ompB, and citrate synthase genes along with phylogenetic analyses demonstrated that RCCE3 is a previously uncultured endosymbiont. The rickettsia was identified as a symbiont of C. capensis, closely related to rickettsiae previously detected by PCR in C. capensis, Ornithodoros moubata (Murray) and Hemaphysalis sulcata Canestrini & Fanzago, a hard tick. RCCE3 caused a cytopathic effect in C. capensis host cells, and it was transferred to Ixodes scapularis Say cell line ISE6 for maintenance. The rickettsial endosymbiont was eliminated from CCE3 by treatment with oxytetracycline. Cell lines from C. capensis will be useful to researchers investigating interactions between soft ticks and microorganisms, soft tick physiology, and molecular biology. The rickettsia adds to the growing number of Rickettsia species that have been isolated in tick cell culture, and it is available for characterization.

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms.

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.

Tick cell lines were used to model the effects of endosymbiont infection on phagocytic immune responses. The lines tested for their ability to phagocytose the Lyme disease spirochete, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), were ISE6 and IDE12 from the black-legged tick, Ixodes scapularis Say (Acari: Ixodidae) and DAE15 from the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of D. andersoni, was used as a representative tick endosymbiont. 70-80% of uninfected or R. peacocciz-infected IDE12 and DAE15 cells phagocytosed heat-killed borreliae and 80-90% of IDE12 and DAE15 cells phagocytosed viable spirochetes. ISE6 cells were permissive of spirochetes; less than 1% of these cells phagocytosed borreliae, and spirochetes remained adherent to the cells seven days after inoculation. Cytochalasin B blocked phagocytosis of killed and viable borreliae by IDE12 cells, and prevented phagocytosis of killed spirochetes by DAE15 cells, whereas viable spirochetes successfully invaded cytochalasin-treated DAE15. IDE12 and DAE15 cells degraded borreliae within phagolysosome-like compartments. Time-lapse microscopy showed that DAE15 cells phagocytosed borreliae more rapidly than IDE12 cells. IDE12 and DAE15 cells eliminated most adherent spirochetes within 7 days of inoculation. Thus, endosymbiont infection does not significantly interfere with the phagocytic activity of immunocompetent tick cells.

Ixodes scapularis and Dermacentor andersoni cell lines were stimulated with heat-killed Escherichia coli and Micrococcus luteus to investigate whether infection by Rickettsia peacockii, an endosymbiont of D. andersoni, modifies humoral immune responses. Radial diffusion assays, western blotting, flow cytometry, and quantitative reverse-transcription PCR were used to determine if expression of bacteriolytic peptides, including lysozyme and defensin, was upregulated by bacterial stimulation or infection with R. peacockii. The I. scapularis line IDE12 upregulated expression of lysozyme and defensin following stimulation. The D. andersoni cell line DAE15 also expressed defensin and lysozyme, but only lysozyme was upregulated by bacterial stimulation. R. peacockii infection alone, or in cells stimulated with bacteria, did not modify defensin or lysozyme expression in either cell line. These results suggest tick endosymbionts may avoid recognition by the tick immune system, and infection may not affect humoral immune responses to bacteria not normally associated with ticks.