Genomic Sequencing of Orientia tsutsugamushi Strain Karp, an Assembly Comparable to the Genome Size of the Strain Ikeda.
ABSTRACT: Orientia tsutsugamushi, an intracellular bacterium, belongs to the family Rickettsiaceae This study presents the draft genome sequence of strain Karp, with 2.0 Mb as the size of the completed genome. This nearly finished draft genome sequence was annotated with the RAST server and the contents compared to those of the other strains.
Project description:We recently reported the genome of Orientia tsutsugamushi (OT) strain Karp (GenBank Accession #: NZ_LYMA00000000.2, https://www.ncbi.nlm.nih.gov/nuccore/NZ_LYMA00000000.2) with > 2 Mb in size through clone-based sequencing and high throughput genomic shotgun sequencing (HTS). The genomes of OT strains AFSC4 and AFSC7 were similarly sequenced by HTS Since strains AFSC4 (GenBank Accession #: NZ_LYMT00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035784408) and AFSC7 (GenBank Accession #: NZ_LYMB00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035854767) were more resistant to antibiotics than strain Karp, we conducted comparative analysis of the three draft genomes annotated by RAST server aimed to identify possible genetic bases of difference in microbial antibiotic sensitivity. Intraspecies comparative genomics analysis of the three OT strains revealed that two ORFs encoding hypothetical proteins in both strains AFSC4 and AFSC7 are absent in strain Karp.
Project description:Scrub typhus is an infectious disease caused by Orientia tsutsugamushi. The differences in virulence of O. tsutsugamushi prototypes in humans are still unknown. We investigated whether there are any differences in the clinical features of the Boryoung and Karp genotypes.Patients infected with O. tsutsugamushi, as Boryoung and Karp clusters, who had visited 6 different hospitals in southwestern Korea were prospectively compared for clinical features, complications, laboratory parameters, and treatment responses. Infected patients in the Boryoung cluster had significantly more generalized weakness, eschars, skin rashes, conjunctival injection, high albumin levels, and greater ESR and fibrinogen levels compared to the Karp cluster. The treatment response to current antibiotics was significantly slower in the Karp cluster as compared to the Boryoung cluster.The frequency of occurrence of eschars and rashes may depend on the genotype of O. tsutsugamushi.
Project description:BACKGROUND:Orientia tsutsugamushi is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of Orientia have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species. RESULTS:We report new high-quality genomes of O. tsutsugamushi, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of Orientia. CONCLUSIONS:Complete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have larger genomes compared with most other Rickettsiaceae, with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, Orientia has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation.
Project description:In a recent case of scrub typhus in Australia, Orientia tsutsugamushi isolated from the patient's blood was tested by sequence analysis of the 16S rDNA gene. The sequence showed a strain of O. tsutsugamushi that was quite different from the classic Karp, Kato, and Gilliam strains. The new strain has been designated Litchfield.
Project description:Scrub typhus also known as bush typhus is a disease with symptoms similar to Chikungunya infection. It is caused by a gram-negative bacterium Orientia tsutsugamushi which resides in its vertebrate host, Mites. The genome of Orientia tsutsugamushi str. Karp encodes for 1,563 proteins, of which 344 are characterized as hypothetical ones. In the present study, we tried to identify the probable functions of these 344 hypothetical proteins (HPs). All the characterized hypothetical proteins (HPs) belong to the various protein classes like enzymes, transporters, binding proteins, metabolic process and catalytic activity and kinase activity. These hypothetical proteins (HPs) were further analyzed for virulence factors with 62 proteins identified as the most virulent proteins among these hypothetical proteins (HPs). In addition, we studied the protein sequence similarity network for visualizing functional trends across protein superfamilies from the context of sequence similarity and it shows great potential for generating testable hypotheses about protein structure-function relationships. Furthermore, we calculated toplogical properties of the network and found them to obey network power law distributions showing a fractal nature. We also identifed two highly interconnected modules in the main network which contained five hub proteins (KJV55465, KJV56211, KJV57212, KJV57203 and KJV57216) having 1.0 clustering coefficient. The structural modeling (2D and 3D structure) of these five hub proteins was carried out and the catalytic site essential for its functioning was analyzed. The outcome of the present study may facilitate a better understanding of the mechanism of virulence, pathogenesis, adaptability to host and up-to-date annotations will make unknown genes easy to identify and target for experimentation. The information on the functional attributes and virulence characteristic of these hypothetical proteins (HPs) are envisaged to facilitate effective development of novel antibacterial drug targets of Orientia tsutsugamushi.
Project description:Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between 'classic' and 'new' types of Tsutsugamushi disease in Japan.
Project description:Rickettsia monacensis and Orientia tsutsugamushi are bacteria of the family Rickettsiaceae, which causes fever, rash, and eschar formation; outdoor activities are a risk factor for Rickettsiaceae infection. A 75-year-old woman presented with fever, rash, and eschar and was confirmed as being scrub typhus based on a nested-polymerase chain reaction (N-PCR) test for a 56-kDa gene of O. tsutsugamushi; the genome was identified as the Boryong genotype. In addition, a pan-Rickettsia real-time PCR test was positive and a N-PCR test using a Rickettsia-specific partial outer membrane protein A (rOmpA) confirmed R. monacensis. This is the first case wherein a patient suspected of having scrub typhus owing to the presence of rash and eschar was also found to be coinfected with O. tsutsugamushi and R. monacensis based on molecular testing.
Project description:Scrub typhus, a chigger-borne febrile illness, occurs primarily in countries of the Asia-Pacific rim and islands of the Western Pacific. The etiologic agent is the obligate intracellular rickettsial bacterium Orientia tsutsugamushi. Research on O. tsutsugamushi has relied on the availability of several prototype strains, which were isolated from human cases of scrub typhus in the 1940s and 1950s. We review the history of the three original, and most important, prototype strains, Gilliam, Karp and Kato, including information on their isolation, their culture history, their clinical characteristics, their importance within the research literature on scrub typhus, and recent advances in elucidating their molecular genomics. The importance of these strains to the research and development of clinical tools related to scrub typhus is also considered. Finally, we examine whether the strains have been genetically stable since their isolation, and whether prototype strains maintained in separate laboratories are identical, based on pairwise comparisons of several sequences from four genes. By using genetic information archived in international DNA databases, we show that the prototype strains used by different laboratories are essentially identical, and that the strains have retained their genetic integrity at least since the 1950s. The three original prototype strains should remain a standard by which new diagnostic procedures are measured. Given their fundamental position in any comparative studies, they are likely to endure as a critical part of present and future research on scrub typhus and Orientia.