Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> 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, whic...

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Autores principales:	Nicholas F. Noriea, Tina R. Clark, Ted Hackstadt
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/350a310c223c41e8963c69942a4305a3
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spelling	oai:doaj.org-article:350a310c223c41e8963c69942a4305a32021-11-15T15:41:34ZTargeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System10.1128/mBio.00323-152150-7511https://doaj.org/article/350a310c223c41e8963c69942a4305a32015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00323-15https://doaj.org/toc/2150-7511ABSTRACT 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.Nicholas F. NorieaTina R. ClarkTed HackstadtAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Nicholas F. Noriea Tina R. Clark Ted Hackstadt Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
description	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.
format	article
author	Nicholas F. Noriea Tina R. Clark Ted Hackstadt
author_facet	Nicholas F. Noriea Tina R. Clark Ted Hackstadt
author_sort	Nicholas F. Noriea
title	Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
title_short	Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
title_full	Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
title_fullStr	Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
title_full_unstemmed	Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
title_sort	targeted knockout of the <named-content content-type="genus-species">rickettsia rickettsii</named-content> ompa surface antigen does not diminish virulence in a mammalian model system
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/350a310c223c41e8963c69942a4305a3
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Targeted Knockout of the <named-content content-type="genus-species">Rickettsia rickettsii</named-content> OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System

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