Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations

Putman, Tim; Suchland, Robert J.; Ivanovitch, John D.; Rockey, Daniel D.

doi:10.1099/mic.0.070029-0

Cited by 32 publications

(33 citation statements)

References 26 publications

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“…In addition, it has been shown that extended in vitro culturing of other chlamydial species may alter the genotype of these isolates (15). Due to these issues, culture-independent methods have been devised to sequence Chlamydia genomes directly from clinical swabs (17,18). In this study, a probe hybridization sequence capture method was used to extract C. pecorum DNA from swab samples collected from koalas and Australian sheep and a cell-cultured Australian bovine isolate (20).…”

Section: Discussionmentioning

confidence: 99%

“…In a major breakthrough in the Chlamydia research field, cultureindependent sequencing methods have been developed, with one of the first approaches involving immunomagnetic separation (IMS) of chlamydial cells using antibodies specific for the chlamydial lipopolysaccharide (16). Using IMS in conjunction with multiple displacement amplification (MDA) produced whole-genome sequences for C. trachomatis strains from low-volume archival samples and swab samples collected from patients (17,18).Sequence capture by hybridization also can be used to sequence chlamydial DNA without the need for cell culturing (19). The sequence capture method involves designing customized biotin-labeled RNA probes that can hybridize to a complete target genome sequence so that magnetic beads coated with the biotinbinding protein streptavidin can be used to extract the captured sequences (20).…”

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Culture-Independent Genome Sequencing of Clinical Samples Reveals an Unexpected Heterogeneity of Infections by Chlamydia pecorum

et al. 2015

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bChlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probebased genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains. Chlamydia pecorum is a widespread pathogen of economically important livestock species, such as cattle and sheep. In cattle, C. pecorum is associated with sporadic bovine encephalomyelitis (SBE), which presents as a fever followed by limb stiffness and staggering (1). In sheep, C. pecorum infections commonly are linked to polyarthritis and conjunctivitis, which can spread rapidly in a flock (2, 3). While these infections are economically relevant to producers, most C. pecorum infections in ruminants appear to be asymptomatic or subclinical, characterized by a consistent presence in the gastrointestinal tract (4, 5). While questions remain over the impact of these infections in livestock globally, the best example of the pathogenic potential of this obligate intracellular bacterium actually is found in koalas, a native Australian marsupial that continues to experience localized extinctions. C. pecorum infections in koalas can cause debilitating ocular and urogenital tract diseases (6, 7). Epidemiological questions have been raised about the relationships between C. pecorum strains infecting domesticated animals and the koala, with a recent C. pecorum multilocus sequence typing (MLST) study revealing the presence of identical sequence types in samples collected from each host (8). As a follow-up to these studies, we recently sequenced the genomes of several cultured koala C. pecorum isolates, revealing a high degree of synteny and sequence identity (98.5 to 98.8%) with C. pecorum genomes from European and U.S. cattle and sheep (9).High-throughput comparative genome sequencin...

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Culture-Independent Genome Sequencing of Clinical Samples Reveals an Unexpected Heterogeneity of Infections by Chlamydia pecorum

et al. 2015

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“…Single-end 100-bp sequencing was performed on an Illumina HiSeq 2000 sequencing system at the Center for Genomic Research and Biocomputing Core, Oregon State University, Corvallis, OR. Genome sequences were assembled and analyzed as described previously (45). Mutations were confirmed by PCR amplification of the corresponding genomic regions and Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…HeLa cells infected with various mutants were harvested by bead agitation in SPG, and the lysate was clarified by centrifugation at 500 ϫ g for 20 min. Host cell genomes were depleted by DNase treatment (45), and the remaining DNA was amplified with REPLI-g (Qiagen) according to the manufacturer's instructions. Whole-genome sequencing (WGS) libraries were prepared using a Nextera XT DNA library preparation kit according to the manufacturer's instructions.…”

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Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

et al. 2016

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cChlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts L-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-␥)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-␥-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells. Chlamydia trachomatis has a characteristic biphasic developmental cycle in cell culture (1). The two dominant chlamydial forms are elementary bodies (EBs) and reticulate bodies (RBs) (1). EBs represent the infectious, nonreplicative form capable of invading host cells (1). Following entry, EBs differentiate into RBs that replicate inside parasitophorous vacuoles termed inclusions (1). Maturing RBs then redifferentiate back to EBs and exit the host cell via lysis or extrusion (2). Morphologically aberrant RBs have been observed in clinical specimens (3). This abnormal developmental cycle can be recapitulated in a laboratory setting by exposing C. trachomatis to various stressors such as host cytokines (4-6), excess amino acids (7), iron deficiency (8, 9), virus coinfections (10), and antibiotics (11, 12) (reviewed in reference 13). Under these conditions, normal C. trachomatis RBs can transition into persistent forms that do not divide, are enlarged, and have aberrant morphology (13). Upon removal of the persistence-inducing stressor, RBs transition back into normal RBs and continue normal development (13). Aberrant RB morphology is a shared feature of multiple C. trachomatis persistence models and may reflect activation of a common stress response program (13). Thus, characterizing how C. trachomatis enters, survives, and reactivates from persistence could reveal new insights into chlamydial pathogenesis.The Th1 cytokine interferon gamma (IFN-␥) can induce dif...

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The Hidden Genomics of Chlamydia trachomatis

Hadfield

Bènard²,

Domman³

et al. 2017

Current Topics in Microbiology and Immunology

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The application of whole-genome sequencing has moved us on from sequencing single genomes to defining unravelling population structures in different niches, and at the -species, -serotype or even -genus level, and in local, national and global settings. This has been instrumental in cataloguing and revealing a huge a range of diversity in this bacterium, when at first we thought there was little. Genomics has challenged assumptions, added insight, as well as confusion and glimpses of truths. What is clear is that at a time when we start to realise the extent and nature of the diversity contained within a genus or a species like this, the huge depth of knowledge communities have developed, through cell biology, as well as the new found molecular approaches will be more precious than ever to link genotype to phenotype. Here we detail the technological developments and insights we have seen during the relatively short time since we began to see the hidden genome of Chlamydia trachomatis.

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Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations

Cited by 32 publications

References 26 publications

Culture-Independent Genome Sequencing of Clinical Samples Reveals an Unexpected Heterogeneity of Infections by Chlamydia pecorum

Culture-Independent Genome Sequencing of Clinical Samples Reveals an Unexpected Heterogeneity of Infections by Chlamydia pecorum

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

The Hidden Genomics of Chlamydia trachomatis

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