The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

AbstractChlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum. C. trachomatis is an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient populations globally. While Agilent SureSelectXT target-enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival and rectal samples, efficiencies are only 60-80% for ≥95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection for C. trachomatis genome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.ImportanceChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectal C. trachomatis transmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis. We show the new system is sensitive for near identical genomes of C. trachomatis from two body sites in four women. Further, we provide a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital and rectal infections, and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

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Whole-genome sequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome evolution

Nature Communications ◽

10.1038/ncomms2596 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 109

Author(s):

Jinfeng Chen ◽

Quanfei Huang ◽

Dongying Gao ◽

Junyi Wang ◽

Yongshan Lang ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Evolution ◽

Genome Sequencing ◽

Whole Genome ◽

Oryza Brachyantha

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Emergence of dominant multidrug-resistant bacterial clades: Lessons from history and whole-genome sequencing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717162115 ◽

2018 ◽

Vol 115 (51) ◽

pp. 12872-12877 ◽

Cited By ~ 18

Author(s):

Elizabeth J. Klemm ◽

Vanessa K. Wong ◽

Gordon Dougan

Keyword(s):

Antibiotic Resistance ◽

Whole Genome Sequencing ◽

Genome Evolution ◽

Genome Sequencing ◽

Bacterial Genome ◽

Multidrug Resistant ◽

Whole Genome ◽

Bacterial Populations ◽

Global Challenge ◽

The Past

Antibiotic resistance in bacteria has emerged as a global challenge over the past 90 years, compromising our ability to effectively treat infections. There has been a dramatic increase in antibiotic resistance-associated determinants in bacterial populations, driven by the mobility and infectious nature of such determinants. Bacterial genome flexibility and antibiotic-driven selection are at the root of the problem. Genome evolution and the emergence of highly successful multidrug-resistant clades in different pathogens have made this a global challenge. Here, we describe some of the factors driving the origin, evolution, and spread of the antibiotic resistance genotype.

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Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants

Frontiers in Microbiology ◽

10.3389/fmicb.2021.630293 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hanna Marti ◽

Sankhya Bommana ◽

Timothy D. Read ◽

Theresa Pesch ◽

Barbara Prähauser ◽

...

Keyword(s):

Antibiotic Resistance ◽

Homologous Recombination ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Field Isolate ◽

Tetracycline Resistance ◽

Whole Genome ◽

Gene Encoding ◽

Obligate Intracellular

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

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Exploitation of the Endocytic Pathway by Orientia tsutsugamushi in Nonprofessional Phagocytes

Infection and Immunity ◽

10.1128/iai.01620-05 ◽

2006 ◽

Vol 74 (7) ◽

pp. 4246-4253 ◽

Cited By ~ 33

Author(s):

Hyuk Chu ◽

Jung-Hee Lee ◽

Seung-Hoon Han ◽

Se-Yoon Kim ◽

Nam-Hyuk Cho ◽

...

Keyword(s):

Host Cell ◽

Causative Agent ◽

Scrub Typhus ◽

Adaptor Protein ◽

Endocytic Pathway ◽

Intracellular Bacterium ◽

Orientia Tsutsugamushi ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular ◽

Endocytic Vesicles

ABSTRACT Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium that requires the exploitation of the endocytic pathway in the host cell. We observed the localization of O. tsutsugamushi with clathrin or adaptor protein 2 within 30 min after the infection of nonprofessional phagocytes. We have further confirmed that the infectivity of O. tsutsugamushi is significantly reduced by drugs that block clathrin-mediated endocytosis but not by filipin III, an inhibitor that blocks caveola-mediated endocytosis. In the present study, with a confocal microscope, O. tsutsugamushi was sequentially colocalized with the early and late endosomal markers EEA1 and LAMP2, respectively, within 1 h after infection. The colocalization of O. tsutsugamushi organisms with EEA1 and LAMP2 gradually disappeared until 2 h postinfection, and then free O. tsutsugamushi organisms were found in the cytoplasm. When the acidification of endocytic vesicles was blocked by treating the cells with NH4Cl or bafilomycin A, the escape of O. tsutsugamushi organisms from the endocytic pathway was severely impaired, and the infectivity of O. tsutsugamushi was drastically reduced. To our knowledge, this is the first report that the invasion of O. tsutsugamushi is dependent on the clathrin-dependent endocytic pathway and the acidification process of the endocytic vesicles in nonprofessional phagocytes.

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Developmental differentiation in a cytoplasm-dwelling obligate intracellular bacterium

10.1101/2020.03.03.975011 ◽

2020 ◽

Author(s):

Suparat Giengkam ◽

Jantana Wongsantichon ◽

Sharanjeet Atwal ◽

Yanin Jaiyen ◽

Wah Ing Goh ◽

...

Keyword(s):

Host Cell ◽

Metabolic Activity ◽

Model System ◽

Intracellular Bacterium ◽

Host Cells ◽

Orientia Tsutsugamushi ◽

Obligate Intracellular Bacterium ◽

New Model ◽

Obligate Intracellular ◽

Developmental Differentiation

AbstractDevelopmental differentiation has been described for several vacuole-dwelling obligate intracellular bacteria but never for an obligate intracellular bacterium that resides in the cytoplasm. Here, we show that the cytoplasm-dwelling obligate intracellular bacterium Orientia tsutsugamushi (Ot) exists in five distinct subpopulations. We show that Ot differentiates into a distinct, metabolically inactive, extracellular state upon budding from the surface of host cells and that this stage is preceded by a surface-associated maturation stage. We identify proteins that are differentially expressed in intracellular, replicative bacteria and extracellular, metabolically inactive bacteria. Metabolic activity resumes rapidly upon entry into the cytoplasm and is triggered by the host cell reducing environment. This example of developmental differentiation in a species of Rickettsiaceae provides a new model system for studying synchronized differentiation in a bacterium that has a minimal genome and where the interactions between bacterium and host cell are more direct than they are for bacteria separated from the eukaryote cell host by a vacuolar membrane.Author SummaryScrub typhus is a life-threatening human infection that is caused by the bacterium Orientia tsutsugamushi and spread by mites. Although the disease is estimated to affect at least one million people annually and is often fatal, the infectious agent is much less well understood than many other pathogens. O. tsutsugamushi is an intracellular bacterium that can only grow and divide within eukaryotic cells. During infection, it is found primarily in the cells that make up the lining of blood vessels and in certain immune cell types. O. tsutsugamushi bacteria can remain inside a single infected cell for seven days or more before budding out. The ways in which the bacterium itself changes during the course of an intracellular infection cycle have not been studied. In the current work, we used a range of techniques to show that O. tsutsugamushi differentiates into five distinct subpopulations, and that these are associated with measurable differences in metabolic activity, replication and infectivity. This work opens new avenues of research into the regulation and mechanisms of differentiation of O. tsutsugamushi, which could lead to improved diagnosis and treatments. It also provides a new model system for studying fundamental questions about bacterial development.

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