Dataset Information

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.

SUBMITTER: Liao HM

PROVIDER: S-EPMC4991697 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Genomic Sequencing of Orientia tsutsugamushi Strain Karp, an Assembly Comparable to the Genome Size of the Strain Ikeda.

Liao Hsiao-Mei HM Chao Chien-Chung CC Lei Haiyan H Li Bingjie B Tsai Shien S Hung Guo-Chiuan GC Ching Wei-Mei WM Lo Shyh-Ching SC

Genome announcements 20160818 4

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. ...[more]

PMID: 27540052

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Project description:UnlabelledA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins.ImportanceAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets. Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates that O. tsutsugamushi strain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that an Orientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fide Orientia effector and the first example of an endogenous F-box-containing Ank-mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

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:Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

Dataset Information

Genomic Sequencing of Orientia tsutsugamushi Strain Karp, an Assembly Comparable to the Genome Size of the Strain Ikeda.

Publications

Genomic Sequencing of Orientia tsutsugamushi Strain Karp, an Assembly Comparable to the Genome Size of the Strain Ikeda.

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