title: Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection.
abstract: Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Riley
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Abigail I
authors.initials: AI
authors.lastname: Fish
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Garza
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Kaikhushroo H
authors.initials: KH
authors.lastname: Banajee
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Emma K
authors.initials: EK
authors.lastname: Harris
authors.affiliation: Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
authors.forename: Fabio
authors.initials: F
authors.lastname: del Piero
authors.affiliation: Vector Borne Disease Laboratories, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA jmartinez@lsu.edu.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Martinez
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001907:Boutonneuse Fever / Q000382:microbiology*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000382:microbiology; D006801:Humans; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D020114:Rickettsia conorii / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D014170:Transformation, Genetic; D014774:Virulence
chemical_list: D001426:Bacterial Proteins