Jason A. Simser

Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

ABSTRACT We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/MunichT) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line.

ABSTRACT An embryonic cell line (DAE100) of the Rocky Mountain wood tick,Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals.R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

Sequence and Expression Analysis of the ompA Gene of Rickettsia peacockii, an Endosymbiont of the Rocky Mountain Wood Tick, Dermacentor andersoni

ABSTRACT The transmission dynamics of Rocky Mountain spotted fever in Montana appears to be regulated by Rickettsia peacockii, a tick symbiotic rickettsia that interferes with transmission of virulent Rickettsia rickettsii. To elucidate the molecular relationships between the two rickettsiae and glean information on how to possibly exploit this interference phenomenon, we studied a major rickettsial outer membrane protein gene, ompA, presumed to be involved in infection and pathogenesis of spotted fever group rickettsiae (SFGR) but which is not expressed in the symbiont. Based on PCR amplification and DNA sequence analysis of the SFGR ompA gene, we demonstrate that R. peacockii is the most closely related of all known SFGR to R. rickettsii. We show that R. peacockii, originally described as East Side agent in Dermacentor andersoni ticks from the east side of the Bitterroot Valley in Montana, is still present in that tick population as well as in D. andersoni ticks collected at two widely separated locations in Colorado. The ompA genes of R. peacockii from these locations share three identical premature stop codons and a weakened ribosome binding site consensus sequence relative to ompA of R. rickettsii. The R. peacockii ompA promoter closely resembles that of R. rickettsii and is functional based on reverse transcription-PCR results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that OmpA translation products were not detected in cultured tick cells infected with R. peacockii. Double immunolabeling studies revealed actin tail structures in tick cells infected with R. rickettsii strain Hlp#2 but not in cells infected with R. peacockii.

A novel and naturally occurring transposon, ISRpe1 in the Rickettsia peacockii genome disrupting the rickA gene involved in actin‐based motility

While examining the molecular basis for the lack of actin‐based motility for the non‐pathogenic spotted fever group (SFG) R. peacockii, we identified a novel insertion sequence (IS) element, ISRpe1, disrupting the coding sequence of rickA, demonstrated to induce actin‐tail polymerization for the SFG rickettsiae. This rickettsial IS element appears to be active in that complete terminal inverted repeat and recombinase/transposase open reading frame sequences are present and the transposase is transcriptionally expressed. Phylogenetically, ISRpe1 belongs to a new IS family that is most closely related to those transposable elements of other intracellular bacteria like Wolbachia spp. ISRpe1 was demonstrated to be present in at least 10 locations throughout the R. peacockii genome, including one that disrupted the putative cell surface antigen encoding gene, sca1 considered to be involved in adhesion and virulence of the rickettsiae. Additionally, three IS sites demonstrated rearrangements/relocations of the R. peacockii genome when compared to those of other SFG rickettsiae. Our findings of the disruptions of rickA and sca1 along with the comparative genomic reassortments associated with ISRpe1 in the non‐virulent R. peacockii provides opportunities to uncover molecular mechanisms underlying the pathogenesis and evolution of rickettsiae as well as its potential to be used in rickettsial transposon‐based mutagenesis.

Differential Expression of Genes in Uninfected and Rickettsia-Infected Dermacentor variabilis Ticks as Assessed by Differential-Display PCR

ABSTRACT Ticks serve as both the vector and the reservoir for members of the spotted fever group rickettsiae. The molecular interaction(s) that results from this close relationship is largely unknown. To identify genetic factors associated with the tick response to rickettsial infection, we utilized differential-display PCR. The majority of upregulation appeared in the infected tissue. We cloned and sequenced 54 differentially expressed transcripts and compared the sequences to those in the GenBank database. Nine of the 54 clones were assigned putative identities and included a clathrin-coated vesicle ATPase, peroxisomal farnesylated protein, Ena/vasodilator-stimulated phosphoprotein-like protein, α-catenin, tubulin α-chain, copper-transporting ATPase, salivary gland protein SGS-3 precursor, glycine-rich protein, and Dreg-2 protein. Confirmation of the rickettsial influence on the differential expression in the ovaries for a number of these clones was demonstrated by semiquantitative reverse transcription-PCR and Northern blot analyses, resulting in confirmation of six out of nine and three out of four assessed clones, respectively. Further characterization of the clones identified tissue-dependent expression in the midguts and salivary glands. The potential roles of these molecules in the maintenance and transmission of rickettsiae are discussed.

The American dog tick, Dermacentor variabilis, encodes a functional histamine release factor homolog

We have identified a functional Dermacentor variabilis histamine release factor (DVHRF) homolog and shown that it is a secreted tick saliva protein. The 945 base pair (bp) full-length DVHRF cDNA has a 522 bp open reading frame that encodes a 20 kDa (173 amino acid) polypeptide. Sequence analysis showed that the two HRF signature amino acid sequences were conserved in DVHRF, indicating close structural similarity between DVHRF and other characterized HRF homologs. Northern and Western blotting analyses of partially fed and unfed ticks indicates that neither DVHRF transcriptional nor translational regulation were influenced by tick feeding activity. Like its counterparts from the mammalian system, tick DVHRF is expressed in various tissues, as assessed by both Northern and Western blotting analyses. Furthermore, an Escherichia coli-expressed recombinant DVHRF induced histamine secretion from a rat basophilic leukemic cell line in a dose-dependent manner. Extensive experimental evidence has shown that high levels of histamine at tick attachment sites impede the biological success of feeding ticks and, in response, ticks secrete histamine-binding proteins to minimize the adverse effects of histamine. Our results suggest the existence of a tick-derived multifaceted control mechanism for levels of histamine at tick feeding sites.

Differential expression of two glutathione S‐transferases identified from the American dog tick, Dermacentor variabilis

Reciprocal signalling and gene expression play a cardinal role during pathogen–host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione‐S‐transferases (GSTs) were among the genes found up‐regulated in Rickettsia‐infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full‐length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up‐regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate‐buffered saline (sham) and naïve ticks.

Molecular and Functional Analysis of the lepB Gene, Encoding a Type I Signal Peptidase from Rickettsia rickettsii and Rickettsia typhi

The type I signal peptidase lepB genes from Rickettsia rickettsii and Rickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the cloned lepB genes from R. rickettsii and R. typhi shows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that the lepB gene of R. rickettsii is cotranscribed in a polycistronic message with the putative nuoF (encoding NADH dehydrogenase I chain F), secF (encoding protein export membrane protein), and rnc (encoding RNase III) genes in a secF-nuoF-lepB-rnc cluster. The cloned lepB genes from R. rickettsii and R. typhi have been demonstrated to possess signal peptidase I activity in Escherichia coli preprotein processing in vivo by complementation assay.

Stress and transcriptional regulation of tick ferritin HC

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks ( Mulenga et al., 2003a ). We have used rapid amplification of cDNA ends to clone full‐length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem–loop structure of a putative iron responsive element in the 5′ untranslated region (nucleotide positions, 16–42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (≈ 1.5‐ and ≈ 3.0‐fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.

An immune responsive factor D‐like serine proteinase homologue identified from the American dog tick, Dermacentor variabilis

A Dermacentor variabilis cDNA encoding a clip‐domain serine proteinase homologue with glycine replacing the catalytic serine was identified from tick haemocytes. The D. variabilis product was most similar to Tachypleus tridentatus haemocyte antimicrobial factor D and shared significant homologies with a number of immune‐responsive gene products of arthropods, including insect prophenoloxidase‐activating cofactors. Northern blotting analyses confirmed that the tick serine proteinase homologue expression levels were highest in haemocytes, and to lesser degrees in ovaries and then salivary glands whereas steady‐state levels of expression in whole ticks were found to be slightly higher in fed versus unfed adults or eggs. Challenge of fed adults by Escherichia coli injection demonstrated that transcript abundance was significantly increased above those of naive controls in a temporal fashion. Additionally, an apparent orthologue of the D. variabilis clip‐domain molecule was cloned, and expression detected, from a Dermacentor andersoni cell line indicating cross species conservation.