Nicole Y. Burkhardt

Transformation of Anaplasma phagocytophilum

BackgroundTick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available.ResultsTo facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin.ConclusionThese transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.

Transovarial Transmission of Francisella-Like Endosymbionts and Anaplasma phagocytophilum Variants in Dermacentor albipictus (Acari: Ixodidae)

ABSTRACT 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.

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.

Analysis of fluorescent protein expression in transformants of Rickettsia monacensis, an obligate intracellular tick symbiont

ABSTRACT We developed and applied transposon-based transformation vectors for molecular manipulation and analysis of spotted fever group rickettsiae, which are obligate intracellular bacteria that infect ticks and, in some cases, mammals. Using the Epicentre EZ::TN transposon system, we designed transposons for simultaneous expression of a reporter gene and a chloramphenicol acetyltransferase (CAT) resistance marker. Transposomes (transposon-transposase complexes) were electroporated into Rickettsia monacensis, a rickettsial symbiont isolated from the tick Ixodes ricinus. Each transposon contained an expression cassette consisting of the rickettsial ompA promoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or the ompB promoter and a red fluorescent protein reporter gene (DsRed2), followed by the ompA transcription terminator and a second ompA promoter CAT gene cassette. Selection with chloramphenicol gave rise to rickettsial populations with chromosomally integrated single-copy transposons as determined by PCR, Southern blotting, and sequence analysis. Reverse transcription-PCR and Northern blots demonstrated transcription of all three genes. GFPuv transformant rickettsiae exhibited strong fluorescence in individual cells, but DsRed2 transformants did not. Western blots confirmed expression of GFPuv in R. monacensis and in Escherichia coli, but DsRed2 was expressed only in E. coli. The DsRed2 gene, but not the GFPuv gene, contains many GC-rich amino acid codons that are rare in the preferred codon suite of rickettsiae, possibly explaining the failure to express DsRed2 protein in R. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescent R. monacensis associated with actin tails in tick host cells.

Plasmids of the pRM/pRF Family Occur in Diverse Rickettsia Species

ABSTRACT 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.

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

ABSTRACT 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.

Isolation of Cell Lines and a Rickettsial Endosymbiont from the Soft Tick Carios capensis (Acari: Argasidae: Ornithodorinae)

Abstract 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.

Development of shuttle vectors for transformation of diverse rickettsia species

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.

Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis

ABSTRACT 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.

Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a Heterologous rickA Gene

ABSTRACT 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.