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Targeted Knockout of the Rickettsia Rickettsii OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System

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Targeted Knockout of the Rickettsia Rickettsii OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System

Nicholas F Noriea et al. mBio.

Abstract

Strains of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), differ dramatically in virulence despite >99% genetic homology. Spotted fever group (SFG) rickettsiae produce two immunodominant outer membrane proteins, rickettsial OmpA (rOmpA) and rOmpB, which are conserved throughout the SFG and thought to be fundamental to pathogenesis. rOmpA is present in all virulent strains of R. rickettsii but is not produced in the only documented avirulent strain, Iowa, due to a premature stop codon. Here we report the creation of an isogenic ompA mutant in the highly virulent strain Sheila Smith by insertion of intronic RNA to create a premature stop codon 312 bp downstream of the 6,747-bp open reading frame initiation site (int312). Targeted insertion was accomplished using an LtrA group II intron retrohoming system. Growth and entry rates of Sheila Smith ompA::int312 in Vero cells remained comparable to those of the wild type. Virulence was assessed in a guinea pig model by challenge with 100 PFU of either ompA::int312 Sheila Smith or the wild type, but no significant difference in either fever peak (40.5°C) or duration (8 days) were shown between the wild type and the knockout. The ability to disrupt genes in a site-specific manner using an LtrA group II intron system provides an important new tool for evaluation of potential virulence determinants in rickettsial disease research.

Importance: R. rickettsii rOmpA is an immunodominant outer membrane autotransporter conserved in the spotted fever group. Previous studies and genomic comparisons suggest that rOmpA is involved in adhesion and may be critical for virulence. Little information is available for rickettsial virulence factors in an isogenic background, as limited systems for targeted gene disruption are currently available. Here we describe the creation of an rOmpA knockout by insertion of a premature stop codon into the 5' end of the open reading frame using a group II intron system. An isogenic rOmpA knockout mutation in the highly virulent Sheila Smith strain did not cause attenuation in a guinea pig model of infection, and no altered phenotype was observed in cell culture. We conclude that rOmpA is not critical for virulence in a guinea pig model but may play a role in survival or transmission from the tick vector.

Figures

FIG 1
FIG 1
Construction of the pARR plasmid. Sigma’s TargeTron vector pACDK4-C was altered for experimentation in Rickettsia. The selectable marker cat and the intron kanamycin RAM marker were removed and replaced with bla and Rparr-2, respectively. The T7 promoter was removed and replaced with a multiple cloning site (MCS) to create pARL-C. The strong rickettsial rpsL promoter was cloned into the MCS to create pARR.
FIG 2
FIG 2
Intron insertion into ompA. (A) The insertion target for the group II intron was 312 bp downstream of the ompA start site. DNA primers flanking ompA312 were used for identification and sequencing of R. rickettsii transformant plaques. Sequence data of the intronic disruption of ompA revealed a stop codon in the open reading frame 354 bp downstream of the ompA start site, predicting a truncated 116-amino-acid rOmpA protein lacking the passenger and autotransporter domains. (B) PCR amplification was performed to identify initial ompA mutants. With the correctly targeted insertion present, ompA produces a band 1,400 bp larger than the wild-type band.
FIG 3
FIG 3
Characterization of R. rickettsii Sheila Smith and Sheila Smith ompA::int312. (A) SDS-PAGE gel stained with Coomassie brilliant blue. Protein lysates were equalized to 1 × 107 rickettsiae per lane. No rOmpA (210 kDa) was detected in Sheila Smith ompA::int312 or Iowa (open arrow) but was detected in Sheila Smith. rOmpB was identified in all three samples. Sheila Smith and Sheila Smith ompA::int312 displayed the postprocessed 120-kDa and 32-kDa rOmpB subunits (black arrows), while Iowa displayed the characteristic defective processing of rOmpB, with the predominant form as the unprocessed 168-kDa precursor. Molecular masses (in kilodaltons) are noted at the left. (B) Immunofluorescence of R. rickettsii using a species-specific polyclonal antibody and a monoclonal antibody specific to rOmpA. Vero cell monolayers on coverslips were infected with R. rickettsii Iowa, Sheila Smith, or Sheila Smith ompA::int312 and probed with MAb 13-3 to rOmpA. No rOmpA was detected in any sample of Iowa or Sheila Smith ompA::int312. (C) Protein lysates normalized to 1 × 107 rickettsiae per lane were resolved on a 10% polyacrylamide gel, transferred to nylon membranes, and blotted with MAb 13-3 specific to rOmpA. No rOmpA was detected in any sample except Sheila Smith. Molecular masses (in kilodaltons) are noted at the right. (D) Southern hybridization of 5 µg of purified genomic DNA from the ompA::int312 mutant and Sheila Smith and linearized pARR plasmid DNA using α-P32-labeled probe specific to the intron marker Rparr-2. No bands were detectable in the Sheila Smith lane, while a single band was detected in Sheila Smith ompA::int312 and linearized-pARR lanes.
FIG 4
FIG 4
Growth rate and infectivity of the ompA::int312 mutant. (A) R. rickettsii Sheila Smith and its ompA::int312 mutant were assessed for growth in Vero cell culture using a plaque assay. Vero monolayers were infected at an MOI of 0.25 in serial dilutions. No difference in growth was observed between the rOmpA knockout and the wild type, with both strains reaching peak densities at 3 to 4 days. (B) An in/out assay was used to assess internalization. Approximately 50% of the rickettsiae were internalized by 30 min. Each time point was tested in triplicate; no significant difference was detected between Sheila Smith and its ompA::int312 mutant. (C) Guinea pig fever responses to challenges with 100 PFU of Sheila Smith, Sheila Smith ompA::int312, and fixed Sheila Smith. No significant difference was detected between Sheila Smith and Sheila Smith ompA::int312, while fixed Sheila Smith did not produce any observable fever response. Peak fever was observed between days 5 and 7 for both strains, and all guinea pigs were afebrile by day 12.
FIG 5
FIG 5
Immunofluorescence microscopy and PCR of guinea pig spleens recovered 8 days after infection with R. rickettsii Sheila Smith (SS) and Sheila Smith ompA::int312. (A) Immunofluorescence microscopy of R. rickettsii isolated and purified from guinea pig spleen removed at peak fever. Slides were incubated with MAb 13-3, specific to rOmpA, as well as polyclonal antibody αRr. No rOmpA fluorescence was observed from spleens isolated from guinea pigs infected with Sheila Smith ompA::int312, confirming that no wild-type reversion or mixed-population infections had occurred. Bar = 5 µm. (B) PCR amplification of the ompA region of R. rickettsii from spleen lysates (taken from guinea pigs during peak fever). No wild-type amplicon was detected in Sheila Smith ompA::int312-challenged guinea pigs. Sizes (in bp) are noted at the left.

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