“Rickettsia amblyommii” Induces Cross Protection Against Lethal Rocky Mountain Spotted Fever in a Guinea Pig Model

ABSTRACTStrains ofRickettsia 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 ofR. rickettsiibut is not produced in the only documented avirulent strain, Iowa, due to a premature stop codon. Here we report the creation of an isogenicompAmutant 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 SmithompA::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 eitherompA::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.IMPORTANCER. rickettsiirOmpA 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.

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The guinea pig model for tick-borne spotted fever rickettsioses: A second look

Ticks and Tick-borne Diseases ◽

10.1016/j.ttbdis.2020.101538 ◽

2020 ◽

Vol 11 (6) ◽

pp. 101538

Author(s):

John V. Stokes ◽

David H. Walker ◽

Andrea S. Varela-Stokes

Keyword(s):

Guinea Pig ◽

Spotted Fever ◽

Pig Model ◽

Second Look ◽

Guinea Pig Model

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Cross-protection among Rickettsia species and subspecies in a guinea pig model of cutaneous infection

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2012.06.002 ◽

2012 ◽

Vol 35 (6) ◽

pp. 551-556 ◽

Cited By ~ 1

Author(s):

Yassina Bechah ◽

Jean-Louis Mege ◽

Didier Raoult

Keyword(s):

Guinea Pig ◽

Cross Protection ◽

Pig Model ◽

Cutaneous Infection ◽

Rickettsia Species ◽

Guinea Pig Model

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Disruption of the Rickettsia rickettsii Sca2 Autotransporter Inhibits Actin-Based Motility

Infection and Immunity ◽

10.1128/iai.00100-10 ◽

2010 ◽

Vol 78 (5) ◽

pp. 2240-2247 ◽

Cited By ~ 87

Author(s):

Betsy Kleba ◽

Tina R. Clark ◽

Erika I. Lutter ◽

Damon W. Ellison ◽

Ted Hackstadt

Keyword(s):

Spotted Fever ◽

Insertion Site ◽

Rocky Mountain ◽

Etiologic Agent ◽

Spotted Fever Group ◽

Rocky Mountain Spotted Fever ◽

Spotted Fever Group Rickettsiae ◽

Rickettsia Rickettsii ◽

Transposon Insertion ◽

Guinea Pig Model

ABSTRACT Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

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