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.

Effect ofAmblyomma maculatum(Acari: Ixodidae) Saliva on the Acute Cutaneous Immune Response toRickettsia parkeriInfection in a Murine Model

“…Furthermore, biopsies of human eschars caused by R. parkeri rickettsiosis collected later in the disease course are characterized by an influx of mononuclear cells including macrophages (Paddock et al 2004, Paddock et al 2008, Cragun et al 2010, Kaskas et al 2014). Also, similar to previous reports of natural cases of R. parkeri rickettsiosis in humans and experimental inoculation of SFG Rickettsia in experimental models, intact rickettsiae were microscopically detected within macrophages (Paddock et al 2004, Whitman et al 2007, Paddock et al 2008, Cragun et al 2010, Banajee et al 2015, Riley et al 2016). While it was not definitively determined if these intracellular rickettsiae were alive or dead with the current assays, they were found whole with morphology similar to viable bacteria.…”

GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8 + T Cell Immunology in R. typhi Infection

“…monacensis (5.5) (15), while plasmid copy numbers in R. conorii (1–3) (16) and R. prowazekii (0.86) were relatively low (17).…”

“…However, C3H/HeN mice infected with transformed R. conorii died very early after infection, between day 3 and day 5 (16). To analyze the pathogenicity of R.…”

“…montanensis, and R. conorii), has been reported (15–17). In TG rickettsiae, plasmids have not yet been identified, and it is still unclear whether these bacteria carry naturally occurring extrachromosomal DNA or might at all support its presence after experimental introduction.…”

Engineering of obligate intracellular bacteria: progress, challenges and paradigms

“…Both R. montanensis and R. conorii were transformed with this shuttle vector and acquired red fluorescence, which demonstrates the functionality of this construct 29,33 . Interestingly, R. conorii transformed with pRAM18dRGA[AmTrCh] was maintained in cell culture and within an experimentally infected animal without the administration of antibiotics 34 , which indicates that the plasmids were stable for the duration of these experiments in the absence of antibiotic selection. Furthermore, rickA from R. monacensis was overexpressed in R. bellii from the multiple cloning site of the shuttle vector, which resulted in significant changes in adhesion to cells and intracellular motility 35 .…”