Biology of Rickettsia

National Institute Of Allergy And Infectious Diseases

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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. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain (Iowa) with an extensive laboaratory passage history. Among the genes differing from all sequence virulent strain of R. rickettsii is one denoted as roaM for Regulator Of Actin-based Moility. 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. 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. Disruption of RoaM significantly increased the number of actin tails compared to the wild type strain. In contrast, overexpression of RoaM dramatically decreased the presence of actin tails and moderated fever response. Localization experiments suggest RoaM is not secreted, while RT-qPCR data show that varying levels of RoaM do not significantly affect the expression of the known rickettsial actin regulating proteins sca2, sca4, or rickA. Taken together, the data suggest a previously unrecognized level of regulation of actin-based motility in spotted fever group rickettsiae. Serial 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. RoaM is not surface exposed; thus, its mechanism of regulating actin-based motility is unclear. Using R. rickettsii strains derived from the virulent Sheila Smith strain that express varying levels of roaM, an RNA-seq experiment was performed. We found that roaM-overexpressing strains downregulate expression of at least six genes which may link the regulatory effects of RoaM to the phenotypic effect on motility. Genes regulated by RoaM were confirmed by RT-qPCR. Among the genes regulated is the secreted effector RarP2, which disrupts the trans-Golgi network. Two of the hypothetical proteins were shown to be secreted via fusion to a glycogen synthase kinase tag, which when phosphorylated reveals exposure to the host-cell cytosol. Taken together, these data support the hypothesis that RoaM affects transcription, downregulating rickettsial genes important for pathogenicity in the mammalian host but which are perhaps otherwise detrimental within the tick vector. To determine how RoaM activity may itself be regulated, we investigated a role of temperature in roaM transcription. RoaM expression itself is not temperature dependent, but many other rickettsial genes are, including some also regulated by RoaM. This suggests that rickettsiae utilize multiple mechanisms to control gene expression in response to environmental signals. RoaM appears to be part of a larger biological program encompassing active spreading in mammalian cells and may be a critical component for R. rickettsii to transition from arthropod to mammalian host. We compared growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa), and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24 hr period while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an Interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-beta secretion correlated with increased host cell lysis and treatment with anti-IFNAR2 antibody decreased lysis from Iowa infected, but not Sheila Smith infected cells. Both Sheila Smith and Iowa infected cells eventually lysed although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We therefore examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-beta secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-beta production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication. Current efforts are focused on transcriptional analyses to define unique responses to the different strains and to specific recombinant rickettsial virulence determinants. The demanding nature of cultivating these obligate intracellular bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples. This work expands upon the genetic toolbox available for R. rickettsii.