Rachel J. Skilton

Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.

Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing

Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections, causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed phylogeny based on whole-genome sequencing of representative strains of C. trachomatis from both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis shows that predicting phylogenetic structure using ompA, which is traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks any true relationships present. We show that in many instances, ompA is a chimera that can be exchanged in part or as a whole both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, which is another key diagnostic target. We used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.

Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization.

Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms - the genes for central metabolism, development cycle and virulence are conserved - or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population.

Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

Background Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence. Principal Findings Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome. Conclusions We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype

The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP–). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP– transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a ‘hole’ in the centre. These green fluorescent inclusions appear ‘doughnut-shaped’ with an empty centre when examined under blue light, giving rise to a characteristic ‘black hole’ phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.

Evaluation of a High Resolution Genotyping Method for Chlamydia trachomatis Using Routine Clinical Samples

Background Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D–K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing. Principal Findings We evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or “MLVA-ompA”) to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types. Conclusions Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

ABSTRACT The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.

Behind the chlamydial cloak: The replication cycle of chlamydiaphage Chp2, revealed

Studying the replication of the chlamydiaphages presents significant challenges. Their host bacteria, chlamydiae, have a unique obligate intracellular developmental cycle. Using qPCR, immunochemistry, and electron microscopy, the life cycle of chlamydiaphage Chp2 was characterised. Chp2 infection has a dramatic inhibitory effect on bacterial cell division. The RB to EB transition is arrested and RBs enlarge without further division. There is a phase of rapid Chp2 genome replication 36 to 48 h post infection that is coincident with the expression of viral proteins and the replication of the host chromosome. The end stage of Chp2 replication is characterised by the appearance of paracrystalline structures followed by bacterial cell lysis. These data indicate that the Chp2 life cycle is closely coordinated with the developmental cycle of its bacterial host. This is a remarkable adaptation by a microvirus to infect and replicate in a bacterial host that has an obligate intracellular developmental cycle.