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.

FIG. 1.

PCR amplification of the rickettsial ompA gene. The 7,088-bp R. rickettsii R strain ompA sequence (GenBank accession no. M31277) (2) is represented by the solid line, with a numerical nucleotide scale. The protein-encoding portion of the gene is represented by the open rectangle, with the tandem repeat region indicated by vertical bars. Overlapping PCR fragments (primer positions in Table 1) are indicated by lines above the open rectangle. The left- and right-most fragments contain the promoter and transcription terminator regions, respectively. Amplified sequences were assembled into a 4,032-bp composite sequence excluding the primer binding sites at the termini.

FIG. 2.

RFLP analysis of the rickettsial ompA gene 532-bp PCR product amplified from purified rickettsiae (DAE100R, Rustic) and D. andersoni tick extracts. PCR products were left uncut (U) or were digested with RsaI (R) or PstI (P). A 100-bp DNA size marker ladder is shown on the left of each panel. (A) R. peacockii strain DaE100R (Rpea) and Crestone tick 5 and 6 extracts (DaC5 and 6). (B) R. peacockii (Rpea) and Montana Bitterroot Valley east side tick extracts (DaEs 6, 8, and 10). (C) R. rickettsii Hlp#2 (HlP#2), R. peacockii (Rpea), and Bitterroot Valley west side tick 9 extract (DaWs9). (D) Bitterroot Valley west side tick 12 extract (DaWs12) and R. peacockii (Rpea).

FIG. 3.

Phylogenetic tree (neighbor-joining phylogram) of SFGR (Table 2) inferred from comparison of rickettsial ompA sequences. Numbers are the proportion of 1,000 bootstrap resamplings that supported the topology. Phylograms constructed using maximum parsimony tree-building analyses were similar. The inset phylogram in the figure is an expanded view of the fourth branch, using R. felis as an outgroup. A monophyletic group containing R. peacockii and R. rickettsii is shown in bold.

FIG. 4.

RT-PCR detection of R. peacockii DaE100R and R. rickettsii Hlp#2 ompA, citrate synthase (gltA), and 17-kDa surface antigen gene transcripts. Control reactions were performed on RNA extracts without reverse transcriptase, and PCR-only reactions were performed with DaE100R DNA for comparison of product sizes with those of RT-PCRs. The relative gel migration positions of 400- and 600-bp markers (Life Technologies) are indicated on the left.

FIG. 5.

SDS-PAGE and immunoblot analyses of R. peacockii DaE100R, R. ricketsii Hlp#2, and Rickettsia sp. MOAa proteins. (A) Coomassie blue-stained SDS-PAGE gel. (B) Western blot probed with MAb 13-5 against rickettsial OmpA. (C) Western blot probed with polyclonal sera against R. monacensis to show absence of OmpA but presence of OmpB in R. peacockii DAE100R. Large arrows indicate the position of the 190-kDa OmpA, and arrowheads indicate the position of the 120-kDa OmpB. Small arrows indicate the relative gel migration positions of SeeBlue protein molecular mass markers (Invitrogen).