Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP UV ) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP UV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP UV fluorescence.Rickettsia prowazekii is the etiologic agent of epidemic typhus, a historically significant human disease that continues to threaten populations subjected to nonhygienic, vector-laden conditions. Tragically, due to the continuing human legacy of war and extreme poverty, such conditions are not rare and recent outbreaks of this disease have been reported (13). In addition, R. prowazekii is a category B select agent and thus is classified as a potential agent of bioterrorism.R. prowazekii is an obligate intracellular bacterium that grows directly within the cytoplasm of eukaryotic host cells, unbounded by a vacuolar membrane. The pathogenicity of R. prowazekii is dependent on its ability to grow to high numbers within the rich cytoplasmic environment, leading to lysis of the host cell and the release of rickettsiae that can then invade and destroy additional host cells. It accomplishes this feat, in organisms as diverse as humans and arthropods, with a small genome of approximately 10 6 base pairs predicted to encode 835 proteins (1,18).Defining the importance of specific rickettsial genes in intracellular invasion and growth has been hampered by the lack of genetic tools. However, progress in developing such tools has been made. Rickettsial genome manipulation has been performed using both homologous recombination mechanisms and transposon-based systems (3, 10-12, 14, 17). Recently, Felsheim et al. (7) described the use of a Himar1-based system to generate transposon insertions in the genome of another obligate intracellular pathogen, Anaplasma phagocytophilum. In this study, we report the use of the mari...

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K T of 2.3 M. Transport was inhibited by S-adenosylethionine and Sadenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly in the cytoplasm of its eukaryotic host cell rather than within a host cell vacuole. To exploit this intracellular niche, R. prowazekii has evolved specialized transporters for the complex metabolic intermediates found in the host's cytoplasm (4,5,14,16,17). The ability to transport the end products of biosynthetic pathways has permitted the rickettsiae to undergo the reductive evolution of biosynthetic genes, resulting in genomes that contain numerous pseudogenes and a high proportion of noncoding DNA (3, 10, 11).The product of the metK gene, methionine adenosyltransferase (MAT), synthesizes S-adenosylmethionine (SAM). SAM is one of the most versatile compounds found in living cells. Not only does it function as the primary methyl donor in a number of biosynthetic reactions, but it also serves as a substrate in the polyamine biosynthetic pathway (8, 9, 13). Thus, SAM is an essential compound in the growth of bacteria, including R. prowazekii (12). The essential nature of SAM is supported by the fact that a deletion of the E. coli metK gene cannot be obtained unless a second functional metK gene is present (15). Examination of the genome sequence of the Madrid E strain of R. prowazekii revealed that the metK gene is a pseudogene in that there is an interruption of the coding sequence (2, 3). However, in contrast to the more disrupted metK pseudogenes of the spotted fever group rickettsiae, the R. prowazekii Madrid E strain contains a single nonsense mutation ne...

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracytoplasmic bacterium with a genome annotated to encode 835 proteins (6, 12). The small rickettsial genome reflects the evolution of specialized transporters that exploit complex metabolic intermediates found in the host cell cytoplasm (7,8,29,32,35), as well as a reductive evolutionary process resulting in the loss of biosynthetic pathway components (3,5,6). This reductive evolution has resulted in a pseudogene-riddled genome with a high proportion of noncoding DNA (4). Characterizing rickettsial protein expression under various conditions and environments is critical to our understanding of how R. prowazekii, with its relatively small protein repertoire, exploits the intracellular niche in host cells as diverse as those of the louse vector and the human host.Several groups have evaluated rickettsial protein expression using both gel-based and mass spectrometry (MS)-based methods (9,10,19,24,26,27). Although gel-based methods offer certain advantages, the use of mass spectrometry offers the opportunity to evaluate comprehensive differential protein expression in complex samples. Differential analysis of proteomes has been performed using methods that employ protein labeling (20), isotope coded affinity tagging (ICAT) (14), and isobaric tags for relative and absolute ...

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.