scholarly journals Long-read whole genome sequencing and comparative analysis of six strains of the human pathogenOrientia tsutsugamushi

2018 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Suwittra Chaemchuen ◽  
Stuart D. Blacksell ◽  
Daniel Paris ◽  
Rory Bowden ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Single Molecule ◽  
Genomic Variation ◽  
Intracellular Bacteria ◽  
Whole Genome ◽  
Orientia Tsutsugamushi ◽  
Bacterial Genomes ◽  
Obligate Intracellular ◽  
Long Read ◽  
Obligate Intracellular Bacteria

AbstractBackgroundOrientia tsutsugamushiis 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 ofOrientiahave 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.ResultsWe report new high-quality genomes ofOrientia 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 ofOrientia.ConclusionsComplete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have large genomes 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.

Rickettsioses

2020 ◽  
pp. 1230-1251
Author(s):  
Karolina Griffiths ◽  
Carole Eldin ◽  
Didier Raoult ◽  
Philippe Parola
Keyword(s):  
Scrub Typhus ◽  
Spotted Fever ◽  
Intracellular Bacteria ◽  
Spotted Fever Group ◽  
Orientia Tsutsugamushi ◽  
Obligate Intracellular ◽  
Metabolic Characteristics ◽  
The Past ◽  
Life Threatening ◽  
Obligate Intracellular Bacteria

Rickettsioses are mild to life-threatening zoonoses caused by obligate intracellular bacteria of the order Rickettsiales (family Rickettsiaceae). Arthropods, including ticks, fleas, and mites, are implicated as their vectors, reservoirs, or amplifiers. With an increasing number of new pathogens and recognition of new pathogenicity and affected geographical areas over the past few decades, there is a better understanding of the scope and importance of these pathogens, particularly as a paradigm to understanding emerging and remerging infections. The taxonomy has undergone numerous changes, with now three main groups classified as rickettsioses according to morphological, antigenic and metabolic characteristics: (1) Rickettsioses due to the bacteria of the genus Rickettsia, including the spotted fever group, typhus groups (Rickettsiaceae), (2) Ehrlichioses and Anaplasmoses due to bacteria of the Anaplasmataceae and (3) scrub typhus due to Orientia tsutsugamushi.

scholarly journals The growing repertoire of genetic tools for dissecting chlamydial pathogenesis

2021 ◽  
Author(s):  
Arkaprabha Banerjee ◽  
David E Nelson
Keyword(s):  
Genetic Engineering ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Pathogenic Potential ◽  
Host Preferences ◽  
Genetic Tools ◽  
Obligate Intracellular ◽  
Obligate Intracellular Bacteria ◽  
Multiple Species

Abstract Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last ten years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens.

scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Y. Chung ◽  
Amanda M. Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Clp Protease ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Developmental Form ◽  
Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi

2018 ◽  
Vol 12 (6) ◽  
pp. e0006566 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Suwittra Chaemchuen ◽  
Stuart Blacksell ◽  
Allen L. Richards ◽  
Daniel Paris ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Orientia Tsutsugamushi ◽  
Human Pathogen ◽  
Long Read

Infections caused by obligate intracellular bacteria

2017 ◽  
Author(s):  
Philippa C. Matthews
Keyword(s):  
Clinical Significance ◽  
Classification System ◽  
Q Fever ◽  
Intracellular Bacteria ◽  
Rickettsial Infection ◽  
Obligate Intracellular ◽  
Treatment And Prevention ◽  
Clinical Syndromes ◽  
Obligate Intracellular Bacteria ◽  
The Tropics

This chapter consists of short notes, diagrams, and tables to summarize infections caused by obligate intracellular bacteria. The chapter begins with a classification system to divide these organisms into Rickettsia, Anaplasma, Chlamydia, Coxiella, and Bartonella species. Separate sections then follow on the infections of most clinical significance for the tropics and subtropics, including the typhus group (caused by rickettsial infection) and Q fever. For ease of reference, each topic is broken down into sections, including classification, epidemiology, microbiology, pathophysiology, clinical syndromes, diagnosis, treatment, and prevention.

scholarly journals Whole genome sequencing of Borrelia miyamotoi isolate Izh-4: reference for a complex bacterial genome

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Konstantin V. Kuleshov ◽  
Gabriele Margos ◽  
Volker Fingerle ◽  
Joris Koetsveld ◽  
Irina A. Goptar ◽  
...  
Keyword(s):  
Single Molecule ◽  
Bacterial Genome ◽  
Borrelia Miyamotoi ◽  
Whole Genome ◽  
Relapsing Fever ◽  
Linear Chromosome ◽  
The North ◽  
Long Read ◽  
Variable Major Proteins ◽  
Tick Vectors

Abstract Background The genus Borrelia comprises spirochaetal bacteria maintained in natural transmission cycles by tick vectors and vertebrate reservoir hosts. The main groups are represented by a species complex including the causative agents of Lyme borreliosis and relapsing fever group Borrelia. Borrelia miyamotoi belongs to the relapsing fever group of spirochetes and forms distinct populations in North America, Asia, and Europe. As all Borrelia species B. miyamotoi possess an unusual and complex genome consisting of a linear chromosome and a number of linear and circular plasmids. The species is considered an emerging human pathogen and an increasing number of human cases are being described in the Northern hemisphere. The aim of this study was to produce a high quality reference genome that will facilitate future studies into genetic differences between different populations and the genome plasticity of B. miyamotoi. Results We used multiple available sequencing methods, including Pacific Bioscience single-molecule real-time technology (SMRT) and Oxford Nanopore technology (ONT) supplemented with highly accurate Illumina sequences, to explore the suitability for whole genome assembly of the Russian B. miyamotoi isolate, Izh-4. Plasmids were typed according to their potential plasmid partitioning genes (PF32, 49, 50, 57/62). Comparing and combining results of both long-read (SMRT and ONT) and short-read methods (Illumina), we determined that the genome of the isolate Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding contigs in the Asian B. miyamotoi isolate FR64b, there were only four that matched plasmids of the North American isolate CT13–2396, indicating differences between B. miyamotoi populations. Several plasmids, e.g. lp41, lp29, lp23, and lp24, were found to carry variable major proteins. Amongst those were variable large proteins (Vlp) subtype Vlp-α, Vlp-γ, Vlp-δ and also Vlp-β. Phylogenetic analysis of common plasmids types showed the uniqueness in Russian/Asian isolates of B. miyamotoi compared to other isolates. Conclusions We here describe the genome of a Russian B. miyamotoi clinical isolate, providing a solid basis for future comparative genomics of B. miyamotoi isolates. This will be a great impetus for further basic, molecular and epidemiological research on this emerging tick-borne pathogen.

scholarly journals Genome Assembly of the A-Group Wolbachia in Nasonia oneida Using Linked-Reads Technology

2019 ◽  
Vol 11 (10) ◽  
pp. 3008-3013 ◽  
Author(s):  
Xiaozhu Wang ◽  
Xiao Xiong ◽  
Wenqi Cao ◽  
Chao Zhang ◽  
John H Werren ◽  
...  
Keyword(s):  
Read Depth ◽  
Intracellular Bacteria ◽  
Bacterial Genomes ◽  
Wasp Species ◽  
Draft Assembly ◽  
Arthropod Species ◽  
Low Concentrations ◽  
Wolbachia Genome ◽  
Obligate Intracellular Bacteria ◽  
Laboratory Culturing

Abstract Wolbachia are obligate intracellular bacteria which commonly infect various nematode and arthropod species. Genome sequences have been generated from arthropod samples following enrichment for the intracellular bacteria, and genomes have also been assembled from arthropod whole-genome sequencing projects. However, these methods remain challenging for infections that occur at low titers in hosts. Here we report the first Wolbachia genome assembled from host sequences using 10× Genomics linked-reads technology. The high read depth attainable by this method allows for recovery of intracellular bacteria that are at low concentrations. Based on the depth differences (714× for the insect and 59× for the bacterium), we assembled the genome of a Wolbachia in the parasitoid jewel wasp species Nasonia oneida. The final draft assembly consists of 1,293, 06 bp in 47 scaffolds with 1,114 coding genes and 97.01% genome completeness assessed by checkM. Comparisons of the five Multi Locus Sequence Typing genes revealed that the sequenced Wolbachia genome is the A1 strain (henceforth wOneA1) previously reported in N. oneida. Pyrosequencing confirms that the wasp strain lacks A2 and B types previously detected in this insect, which were likely lost during laboratory culturing. Assembling bacterial genomes from host genome projects can provide an effective method for sequencing bacterial genomes, even when the infections occur at low density in sampled tissues.

scholarly journals Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

2017 ◽  
Author(s):  
Tslil Gabrieli ◽  
Hila Sharim ◽  
Yael Michaeli ◽  
Yuval Ebenstein
Keyword(s):  
Single Molecule ◽  
Genome Mapping ◽  
Single Point ◽  
Read Length ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Whole Genome Analysis ◽  
Long Read

ABSTRACTVariations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, single-molecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

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