Biology of Rickettsia

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species cause diseases of lesser severity. Still other species in the spotted fever group are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. These latter strains are considered simple commensals of ticks. R. rickettsii is a small obligate intracellular Gram-negative organism maintained in its tick host through transovarial transmission. Infection with R. rickettsii occurs through the bite of an infected tick. Once the organism gains access to the host it is able to replicate within the host vascular endothelial cells and spread from cell to cell by polymerizing host cell actin. Damage to vascular endothelial cells by R. rickettsii leads to increased vascular permeability and leakage of fluid into the interstices causing the characteristic rash observed in RMSF. Infection with R. rickettsii results in a severe and potentially life threatening disease if not diagnosed and treated properly. While much is known about the progression of disease, the molecular mechanisms involved in the pathogenesis of RMSF are poorly understood. Strains of Rickettsia rickettsii vary dramatically in their virulence in animal model systems and severity of human disease. The obligate intracellular lifestyle of rickettsiae and, until recently, a dearth of tractable genetic systems made it difficult to identify genes involved in virulence. With the completed sequences of multiple rickettsial species and advances in genetic technologies, it has become possible to investigate differences between virulent and avirulent strains of rickettsiae through comparative genomics. Additional genomic sequencing is being done on two new isolates of R. rickettsii that have current clinical relevance and were isolated directly from ticks. Taiacu is a virulent strain isolated from Amblyomma aureolatum that reflect current cases in Brazil and is likely to be favored in many future published works on Rickettsia, however its genome has not yet been sequenced. AZ-5 is a strain isolated from Rhipicephalus sanguineus in Arizona, which may be representative of the outbreak in Arizona and Mexico which has been ongoing over the last ten years. Comparisons of these new genomes with other existing R. rickettsii genomes may show evolutionary divergence based on the tick vector. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia Ankyrin Repeat Protein 2 (RARP-2). RARP-2 in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith co-localizes with multi-lamellar membranous structures bearing markers of the endoplasmic reticulum whereas the Iowa protein shows no co-localization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith-RARP-2 in R. rickettsii Iowa converts this avirulent strains typically non-lytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith-RARP-2 did not, however, restore the virulence of the Iowa strain in a Guinea pig model; likely due to the multifactorial nature of rickettsial virulence. Highly virulent strains of Rickettsia rickettsii induce selective fragmentation of the trans-Golgi network (TGN) soon after infection of host cells by secretion of RARP2. Remarkably, this fragmentation is pronounced for the trans-Golgi network but the cis-Golgi remains largely intact and appropriately localized. Thus R. rickettsii targets specifically the TGN and not the entire Golgi apparatus. Site-directed mutagenesis of the predicted cysteine protease active site in RARP2 prevents TGN disruption. General protein transport to the cell surface is severely impacted in cells infected with virulent strains of R. rickettsii. These findings suggest a novel manipulation of cellular organization by an obligate intracellular bacterium to determine interactions with the host cell. R. rickettsii coopts host actin to polymerize actin filaments to propel the bacterium through the cytoplasm and allow it to spread to new host cells. Cell-to-cell spread via actin-based motility is considered a key virulence factor, however little is known about how or if this motility is regulated. We isolated a variant of R. rickettsii Sheila Smith that generates larger plaques on Vero cell monolayers and identified a truncation in roAM (regulator of actin-based motility) that correlates with the change in plaque size. By generating a recombinant knockout and strains ectopically expressing full length and truncated versions of roAM, we determined that the ablation of this gene results in a hyper-spreading phenotype. Serially passage of the wild type strain in Vero cells repeatedly led to truncations of roAM that resulted in hyper-spreading mutants, suggesting strong negative selection for this gene in cell culture. The loss of genes important for Rickettsial biology in cell culture due to repeated passaging calls into question the findings of some previous studies regarding actin-based motility, virulence, and dissemination within the tick vector. Current works aims to address these questions. In addition, experimentally generated, highly passaged strains are being genomically sequenced to determine if there are other genes that are lost due to passage in cell culture. Nitric oxide (NO) synthesized by the inducible nitric oxide synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. We examined the antibacterial role of NO on R. rickettsii. Results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent anti-rickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.