Scrub typhus is caused by the obligate intracellular rickettsiaOrientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host-cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host-cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.bacterial genome ͉ duplication ͉ repeats

The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein ofR. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm. Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with O. tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.Orientia tsutsugamushi, the causative agent of scrub typhus, has been reported to attach to the susceptible host cells in a heparan sulfate glycosaminoglycan-mediated manner (25). After attachment to the host plasma membrane, O. tsutsugamushi induces its own uptake by a process termed induced phagocytosis (40). After entry into the cells, O. tsutsugamushi escapes from the phagocytic vacuole by an unknown mechanism. This probably occurs shortly after phagocytosis, since phagocytic vacuoles containing O. tsutsugamushi have never been seen far from the cell surface (12). Once free in the host cytoplasm, the bacteria replicate in the perinuclear area. Listeriae, shigellae, and rickettsiae also escape from the phagocytic vacuole and replicate in the cytoplasm. These bacteria have independently developed an apparently similar host cell actin-based mechanism that is essential for their intercellular spread (16,18,33,39). In the case of viruses, such as the common type 2 or 5 adenoviruses, they enter a new host cell by receptor-mediated endocytosis and reach the cytosol by acid-dependent disruption of the endosomal membrane (37). It is known that microtubules (MTs) and dyneins are required for directional transport of cytosolic adenovirus to the nucleus (36). Herpes simplex virus 1 (HSV-1) also overcomes the cytosolic barrier by packaging its genome into a capsid structure that is capable of using the minus-end-directed MT-dependent dynein motor (35). Some bacteria, such as Chlamydia trachomatis and Campylobacter jejuni, utilize the host MTs and dyneins during the early events of infection (9, 24). However, it is not clear whether O. tsutsugamushi uses the host cell cytoskeleton for its intracellular movement and intercellular spread.The MT network of an interphase cell is a dyna...

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes ofRicketasia tsutsugamushi, Gifliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia conl as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were * Corresponding author. epitopes are present within the protein (6, 9, 16, 17, 19, 23), it may be useful in the development of tools for serologic diagnosis.

Scrub typhus, caused by