title: Targeted knockout of the Rickettsia rickettsii OmpA surface antigen does not diminish virulence in a mammalian model system.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Noriea
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Tina R
authors.initials: TR
authors.lastname: Clark
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA Ted_Hackstadt@NIH.gov.
authors.forename: Ted
authors.initials: T
authors.lastname: Hackstadt
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense; D004195:Disease Models, Animal; D005334:Fever; D055786:Gene Knockout Techniques; D006168:Guinea Pigs; D012284:Rickettsia rickettsii / Q000235:genetics* / Q000254:growth & development*; D012373:Rocky Mountain Spotted Fever / Q000382:microbiology / Q000473:pathology; D013696:Temperature; D014709:Vero Cells; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D018389:Codon, Nonsense; D037521:Virulence Factors; C078167:OMPA outer membrane proteins
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