Comparative genome sequencing of Rickettsia rickettsii strains that differ in virulence

Abstract

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii, the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

FIG 1

Fever curves for R. rickettsii strains. Female Hartley strain guinea pigs were inoculated intradermally with 100 PFU of R. rickettsii strains Sheila Smith, R, Morgan, Sao Paulo, HLP7421, and Iowa. Controls received an equivalent mass of formalin-killed Sheila Smith (SSfixed) in K36 diluent. Temperatures were monitored for 14 days.

FIG 2

SDS-PAGE and Western blotting of major outer membrane proteins. All strains examined in the animal model were separated by SDS-PAGE. SS, Sheila Smith; SP, Sao Paulo; M, Morgan; I, Iowa; HLP, HLP7421. (A) Coomassie staining showing the presence of rOmpA in all strains except Iowa and the 120-kDa rOmpB protein in which the larger unprocessed form is abundant in the Iowa strain. (B) Immunoblot of rOmpA with 13-3 monoclonal antibody. (C) Immunoblot of the 120-kDa rOmpB protein using a rabbit polyclonal anti-R. rickettsii rOmpB antiserum. In the Iowa strain, both the processed and larger, unprocessed forms are detected.

FIG 3

Sequencing of the rOmpA repeat region and alignment of rOmpA. The transposon insertion kit EZ-Tn5 (DHFR-1) (Epicentre) was used to sequence and arrange the rOmpA series of repeats. This method generated randomly inserted primer binding sites that allowed for extended sequence reads and assembly of the repeats. (A and B) Sequence coverage from E. coli clones for the R (A) and Morgan (B) strains. Transposon insertion sites from which sequencing was primed are shown as gaps (H) in the sequence. (C) Sequence alignment of R and Morgan strains to Sheila Smith reveals seven shared SNPs, resulting in three nonsynonymous amino acid changes at the beginning of the type II G repeat. Clear boxed letters represent type I repeats, and shaded boxes represent type II. The area of shared changes is expanded to show hydrophobic A-to-hydrophilic S residue substitution. Morgan also contains 17 other SNPs, 9 of which result in 8 amino acid changes (two SNPs in one codon).

FIG 4

Comparison of rOmpB sequences. Alignment of each strain's sequenced rOmpB to Sheila Smith shows that the R and Sao Paulo sequences are identical to the reference sequence. Morgan and Iowa are identical to each other, with 4 amino acid substitutions compared to the sequence of Sheila Smith. HLP7421 has these same changes plus 9 more compared to the Sheila Smith sequence.

FIG 5

Phylogenetic analysis of closely related Rickettsia rickettsii strains. The evolutionary history was inferred by using a maximum likelihood method based on the Tamura-Nei model (45). The tree with the highest log likelihood (−58,350.3501) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The initial tree(s) for the heuristic search was obtained automatically by applying neighbor joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with the superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 0.0500]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 10-nucleotide sequences. Codon positions included were 1st, 2nd, 3rd, and noncoding. There were a total of 42,243 positions in the final data set. Evolutionary analyses were conducted in MEGA, version 5 (46).