Characterization of Dermacentor variabilis molecules associated with Rickettsial infection.

To ultimately define the virulence factors of rickettsiae, an understanding of the biology of the organism is essential. Comprehension of the pathogen-human interaction is critical to the development of control measures; and, in the case of vector-borne diseases, the role of the vector in maintaining and transmitting pathogens to vertebrate hosts is crucial to ultimate control. Recent studies have identified tick molecules that are likely involved in the tick-rickettsiae interchange, including tick response to infection and possible molecules exploited by rickettsiae during transmission events. We have further characterized several tick-derived molecules, including a histamine release factor, serine proteases, and lysozymes.

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae).

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.

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.

Functional analysis of secA homologues from rickettsiae.

The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 degrees C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 degrees C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.

Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis and an embryonic cell line of the Rocky Mountain wood tick, D. andersoni.

Immune-responsive lysozyme encoding cDNAs were identified from two medically important tick species by an expressed sequence tag approach of D. variabilis hemocytes (Dv Lys) and a D. andersoni embryonic derived cell line, DAE100. Comparative sequence analyses indicated the Dermacentor molecules to be products of orthologous genes and to be most similar to arthropod c-type lysozymes. Northern blotting analyses demonstrated that Dv Lys expression levels were most abundant in tick hemocytes and to a much lesser degree in the midgut while barely detectable in ovary, salivary gland, and Malpighian tubule tissues. Involvement of the Dermacentor c-type lysozymes in innate immunity was demonstrated by Escherichia coli challenges of D. variabilis ticks by injection resulting in a temporal profile of significantly elevated transcript abundances above those of naive controls that was similarly observed of the D. andersoni cells co-cultured with E. coli. In contrast to that reported of the digestive gut lysozyme of the soft tick Ornithodoros moubata, Dv Lys levels were not statistically differentially regulated by blood meal digestion. Additionally, given the differences in tissue distribution, sequence characteristics and phylogenetic placements between the Dermacentor and Ornithodoros lysozymes demonstrates that ticks possess differently adapted c-type lysozymes that are spatially and temporally differentially expressed.

Sequence and expression analysis of the ompA gene of Rickettsia peacockii, an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni.

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.

Differential expression of genes in uninfected and rickettsia-infected Dermacentor variabilis ticks as assessed by differential-display PCR.

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, alpha-catenin, tubulin alpha-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.

Interactions between rickettsiae and Dermacentor variabilis ticks: analysis of gene expression.

Tick-borne spotted fever group rickettsiae are maintained in nature primarily through transstadial and transovarial transmission in their vector. In the tick, Dermacentor variabilis, the infection of, and persistence within, the ovaries are critical steps in the transmission cycle of rickettsiae from one generation to the next. Although ixodid ticks can experimentally maintain several species of rickettsiae, often only a single species is maintained transovarially. The molecular mechanisms underlying exclusion processes are unknown. In this study, an attempt to identify the molecules that may be involved in rickettsial adhesion to tick cells was carried out. Monoclonal antibodies to mammalian alpha and beta subunits of integrins were used to screen a lambda phage library, constructed from uninfected tick ovarian tissues. Positive plaques for both alpha and beta integrins were identified. For one integrin, beta(3), the plaque was isolated and the insert sequenced. The role of these molecules, identified by library screening, in rickettsial infection and survival are discussed.

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.

Rickettsia monacensis sp. nov., a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park.

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) 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.