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Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

BackgroundGenetic systems have been developed forChlamydiabut the extremely low transformation frequency remains a significant bottleneck. Our goal is to develop a self-replicating transposon delivery vector forC. trachomatiswhich can be expanded prior to transposase induction.MethodsWe madeE. coli/C. trachomatisshuttle vectors bearing theHimar1C9 transposase under control of thetetpromoter and a novel rearrangement of theHimar1transposon with the β-lactamase gene. Activity of the transposase was monitored by immunoblot and by DNA sequencing.ResultsWe constructed pSW2-mCh-C9, aC. trachomatisplasmid designed to act as a self-replicating vector carrying both theHimar1C9 transposase undertetpromoter control and its transposon. However, we were unable to recover this plasmid inC. trachomatisfollowing multiple attempts at transformation.Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only theHimar1C9 transposase (undertetpromoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon. We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active inE. coli. Both these plasmids could be independently recovered inC. trachomatis.We attempted to perform lateral gene transfer by transformation and mixed infection withC. trachomatisstrains bearingpSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon(a green fluorescent version ofpSW2-mCh-C9-ΔTpase). Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids.ConclusionsWe have designed a self-replicating plasmid vector pSW2-mCh-C9 forC. trachomatiscarrying theHimar1C9 transposase undertetpromoter control. Whilst this can be transformed intoE. coliit cannot be recovered inC. trachomatis. Based on selected deletions and phenotypic analyses we conclude that low level expression from thetetinducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

Chlamydia trachomatis is an obligate intracellular pathogen of humans, causing both the sexually transmitted infection, chlamydia, and the most common cause of infectious blindness, trachoma. The majority of sequenced C. trachomatis clinical isolates carry a 7.5-Kb plasmid, and it is becoming increasingly evident that this is a key determinant of pathogenicity. The discovery of the Swedish New Variant and the more recent Finnish variant highlight the importance of understanding the natural extent of variation in the plasmid. In this study we analysed 524 plasmid sequences from publicly available whole-genome sequence data. Single nucleotide polymorphisms (SNP) in each of the eight coding sequences (CDS) were identified and analysed. There were 224 base positions out of a total 7550 bp that carried a SNP, which equates to a SNP rate of 2.97%, nearly three times what was previously calculated. After normalising for CDS size, CDS8 had the highest SNP rate at 3.97% (i.e., number of SNPs per total number of nucleotides), whilst CDS6 had the lowest at 1.94%. CDS5 had the highest total number of SNPs across the 524 sequences analysed (2267 SNPs), whereas CDS6 had the least SNPs with only 85 SNPs. Calculation of the genetic distances identified CDS6 as the least variable gene at the nucleotide level (d = 0.001), and CDS5 as the most variable (d = 0.007), however, at the amino acid level CDS2 was the least variable (d = 0.001), whilst CDS5 remained the most variable (d = 0.013). This study describes the largest in-depth analysis of the C. trachomatis plasmid to date, through the analysis of plasmid sequence data mined from whole genome sequences spanning 50 years and from a worldwide distribution, providing insights into the nature and extent of existing variation within the plasmid as well as guidance for the design of future diagnostic assays. This is crucial at a time when single-target diagnostic assays are failing to detect natural mutants, putting those infected at risk of a serious long-term and life-changing illness.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Background Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison has been performed on the plasmid. Therefore, there is a gap in the current knowledge on Chlamydia plasmid diversity. Aims This project is aimed to investigate and establish the nature and extent of diversity across the entire genus of Chlamydia, by comparing the sequences of all currently available plasmid carrying strains. Methods The PUBMED database was used to identify plasmid sequences from all available strains that met the set quality criteria for their inclusion in the study. Alignments were performed on the 51 strains that fulfilled the criteria using MEGA X software. Following that Maximum Likelihood estimation was used to construct 11 phylogenetic trees of the whole plasmid sequence, the individual 8 coding sequences, the iteron and a chromosomal gene ompA as a comparator. Results The genus-wide plasmid phylogeny produced three distinct lineages labelled as alpha, beta and gamma. Nineteen genotypes were found in the initial whole plasmid analysis. Their distribution was allocated as six C. pecorum, two C. pneumoniae, one C. gallinacea, one C. avium, one C. caviae, one C. felis, two C. psittaci, one C. trachomatis, one C. muridarum, and two C. suis. The chromosomal comparative gene ompA supported this distribution, with the same number of primary clades with the same species distribution. However, ompA sequence comparison resulted in fewer genotypes due to a reduced amount of available sequences (33 out of 51). All results were statistically significant. Conclusion The results of this study indicate that the common bacterial ancestor of all the species had a plasmid, which has diverged over time. Moreover, it suggests that there is a strong evolutionary selection towards these species retaining their plasmids due to its high level of conservation across the genus, with the notable exception of C. pneumoniae. Furthermore, the evolutionary analysis showed that the plasmid and the chromosome have co-evolved.

Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections worldwide and has been associated with male infertility. Recently, it was hypothesized that Ct may infect the epithelium of the seminiferous tubule, formed by Sertoli cells, thus leading to impaired spermatogenesis. To date, there is a lack of data on Ct infection of the seminiferous epithelium; therefore, we aimed to characterize, for the first time, an in vitro infection model of primary human Sertoli cells. We compared Ct inclusion size, morphology and growth kinetics with those in McCoy cells and we studied F-actin fibres, Vimentin-based intermediate filaments and α-tubulin microtubules in Sertoli and McCoy cells. Our main finding highlighted the ability of Ct to infect Sertoli cells, although with a unique growth profile and the inability to exit host cells. Furthermore, we observed alterations in the cytoskeletal fibres of infected Sertoli cells. Our results suggest that Ct struggles to generate a productive infection in Sertoli cells, limiting its dissemination in the host. Nevertheless, the adverse effect on the cytoskeleton supports the notion that Ct may compromise the blood-testis barrier, impairing spermatogenesis.

Chlamydia trachomatis causes sexually transmitted infections and the blinding disease trachoma. Current data on C. trachomatis phylogeny show that there is only a single trachoma-causing clade, which is distinct from the lineages causing urogenital tract (UGT) and lymphogranuloma venerum diseases. Here we report the whole-genome sequences of ocular C. trachomatis isolates obtained from young children with clinical signs of trachoma in a trachoma endemic region of northern Australia. The isolates form two lineages that fall outside the classical trachoma lineage, instead being placed within UGT clades of the C. trachomatis phylogenetic tree. The Australian trachoma isolates appear to be recombinants with UGT C. trachomatis genome backbones, in which loci that encode immunodominant surface proteins (ompA and pmpEFGH) have been replaced by those characteristic of classical ocular isolates. This suggests that ocular tropism and association with trachoma are functionally associated with some sequence variants of ompA and pmpEFGH., Chlamydia trachomatis isolates causing a blinding disease (trachoma) form a single lineage that is different from the lineages causing urogenital infections. Here, Andersson et al. show however that trachoma isolates from Australia are more closely related to urogenital strains than to other trachoma isolates.

The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae. C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species., We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis, C. muridarum, C. caviae, C. pecorum, and C. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid. IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae. C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

Background:Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle.In vivosystems exist that allow studies of different aspects of basic biology of chlamydiae, the murineChlamydia muridarummodel is one of great importance and thus an essential research tool.C. muridarumcarries a plasmid that has a role in virulence. Our aim was to compare and contrast theC. muridarumplasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle.Methods:We measured infectivity for plasmid bearing and plasmid-curedC. muridarumby inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A newE.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transformC. muridarumfor further phenotypic studies.Results:We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined theC. muridarumplasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-curedC. muridarumchallenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-curedC. muridarumrestored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication.Conclusions:Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-freeC. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence. Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.

BACKGROUND: Urogenital infection with Chlamydia trachomatis is the most commonly diagnosed sexually transmitted infection in the developed world. Accurate measurement and therefore understanding the seroprevalence of urogenital C. trachomatis infections requires a rigorously optimised and validated ELISA. Previous ELISAs based on the C. trachomatis plasmid-encoded protein, PGP3, have been described but lack standardisation and critical controls or use a less common PGP3 as the capture antigen.METHODOLOGY/PRINCIPAL FINDINGS: A sensitive and specific indirect ELISA was developed based on recombinant PGP3 derived from a urogenital strain of C. trachomatis, serovar E (pSW2), using a rigorous validation protocol. Serum samples were collected from 166 genitourinary medicine (GUM) clinic patients diagnosed as positive or negative for urogenital C. trachomatis infection by nucleic acid amplification testing (NAATs). Overall sensitivity and specificity compared to NAATs was 68.18% and 98.0%, respectively. Sensitivities for female and male samples were 71.93% and 64.15%, respectively. Comparison of samples from these patients diagnosed positive for C. trachomatis by NAAT and patients diagnosed negative by NAAT revealed statistical significance (p≤0.0001).CONCLUSIONS: We have developed and validated a sensitive and specific ELISA to detect anti-PGP3 antibodies as an indicator of past and current infection to C. trachomatis using PGP3 from a common urogenital strain. It is anticipated that this assay will be used for seroepidemiological analysis of urogenital C. trachomatis in populations.

Chlamydia trachomatis is the most common sexually transmitted infection (STI) in England. Our objective was to perform a detailed survey of the molecular epidemiology of C. trachomatis in the population of Southampton UK attending the genitourinary medicine clinic (GUM) to seek evidence of sexual network activity. Our hypothesis was that certain genotypes can be associated with specific demographic determinants. 380 positive samples were collected from 375 C. trachomatis positive GUM attendees out of the 3118 who consented to be part of the survey. 302 of the positive samples were fully genotyped. All six of the predominant genotypes possessed ompA locus type E. One ward of Southampton known to contain a large proportion of students had a different profile of genotypes compared to other areas of the city. Some genotypes appeared embedded in the city population whilst others appeared transient. Predominant circulating genotypes remain stable within a city population whereas others are sporadic. Sexual networks could be inferred but not conclusively identified using the data from this survey.

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis., Funding agencies:Sanger Institute through the Wellcome Trust 098051 Australian National Health and Medical Research Council 1060768

The study of bacterial populations using whole-genome sequencing is of considerable scientific and clinical interest. However, obtaining bacterial genomic information is not always trivial: the target bacteria may be difficult to culture or uncultured, and they may be found within samples containing complex mixtures of other contaminating microbes and/or host cells, from which it is very difficult to derive robust sequencing data. Here we describe our procedure to generate sufficient DNA for whole-genome sequencing from clinical samples and without the need for culture, as successfully used on the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis. Our protocol combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification (WGA), which is followed by high-throughput sequencing. Compared with other techniques that might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process that provides amplified genomic DNA for sequencing from target bacteria in under 5 h, with little hands-on time.

Chlamydia trachomatis is the most common bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness in developing countries. Tetracycline is commonly the drug of choice for treating C. trachomatis infections, but cases of antibiotic resistance in clinical isolates have previously been reported. Here, we used antibiotic resistance assays and whole-genome sequencing to interrogate the hypothesis that two clinical isolates (IU824 and IU888) have acquired mechanisms of antibiotic resistance. Immunofluorescence staining was used to identify C. trachomatis inclusions in cell cultures grown in the presence of tetracycline; however, only antibiotic-free control cultures yielded the strong fluorescence associated with the presence of chlamydial inclusions. Infectivity was lost upon passage of harvested cultures grown in the presence of tetracycline into antibiotic-free medium, so we conclude that these isolates were phenotypically sensitive to tetracycline. Comparisons of the genome and plasmid sequences for the two isolates with tetracycline-sensitive strains did not identify regions of low sequence identity that could accommodate horizontally acquired resistance genes, and the tetracycline binding region of the 16S rRNA gene was identical to that of the sensitive control strains. The porB gene of strain IU824, however, was found to contain a premature stop codon not previously identified, which is noteworthy but unlikely to be related to tetracycline resistance. In conclusion, we found no evidence of tetracycline resistance in the two strains investigated, and it seems most likely that the small, aberrant inclusions previously identified resulted from the high chlamydial load used in the original antibiotic resistance assays.

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.

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

The experimental infection of newborn calves with bovine norovirus was used as a homologous large animal model to study the pathogenesis of norovirus infection and to determine target cells for viral replication. Six newborn calves were inoculated orally with Jena virus (JV), a bovine norovirus GIII.1 strain, and six calves served as mock-inoculated controls. Following infection, calves were euthanized before the onset of diarrhea (12 h postinoculation [hpi]), shortly after the onset of diarrhea (18 to 21 hpi), and postconvalescence (4 days pi [dpi]). Calves inoculated with JV developed severe watery diarrhea at 14 to 16 hpi, and this symptom lasted for 53.5 to 67.0 h. Intestinal lesions were characterized by severe villus atrophy together with loss and attenuation of villus epithelium. Viral capsid antigen (JV antigen) was detected by immunohistochemistry in the cytoplasm of epithelial cells on villi. In addition, granular material positive for JV antigen was detected in the lamina propria of villi. Lesions first appeared at 12 hpi and were most extensive at 18 to 19 hpi, extending from midjejunum to ileum. The intestinal mucosa had completely recovered at 4 dpi. There was no indication of systemic infection as described for norovirus infection in mice. JV was found in intestinal contents by reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) as early as 12 hpi. Fecal shedding of the virus started at 13 hpi and stopped at 23 hpi or at necropsy (4 dpi), respectively. Throughout the trial, none of the control calves tested positive for JV by ELISA or RT-PCR.

The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?

We know surprisingly little about the evolutionary origins of Chlamydia trachomatis. It causes both ocular (trachoma) and sexually transmitted infections in humans, it is an obligate intracellular pathogen, and there are only a few "isolates" that have been well characterized. From the first few genomes analyzed, it seems that the C. trachomatis genome is highly conserved. The genomes possess high synteny and, in some cases, the sequence variation between genomes is as little as 20 SNPs. Recent indications from partial genome analyses suggest that recombination is the mechanism for generating diversity. There is no accurate molecular clock by which to measure the evolution of C. trachomatis. The origins of both sexually transmitted and ocular C. trachomatis are unclear, but it seems likely that they evolved with humans and shared a common ancestor with environmental chlamydiae some 700 million years ago. Subsequently, evolution within mammalian cells has been accompanied by radical reduction in the C. trachomatis genome.

This review focuses on the anatomy of the Swedish new variant of Chlamydia trachomatis (nvCT). This information provides an interesting insight into the emergence of new strains (how, where, and when), and the important lessons learned are discussed.In late 2006, the nvCT was first reported in Sweden; it carries a 377 bp deletion within its plasmid which covers the single targets originally used by Roche and Abbott diagnostic systems. The nvCT spread rapidly with thousands of falsely negative diagnoses. Genome sequencing and phenotypic characterization showed that the biological fitness of nvCT when compared with wild-type CT in vitro is unaltered. Therefore, the rapid transmission of nvCT was due to the selective advantage gained from failed diagnosis and the introduction of nvCT into a high-frequency transmitting population. The proportions of nvCT cases are now converging toward equilibrium with the wild-type CT strains. Interestingly, the nvCT remains rarely reported beyond the Nordic countries.The spread of nvCT had a substantial impact on C. trachomatis identification, epidemiology, and public health in Sweden. Lessons learned from this experience include the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the careful design of diagnostic assays. The nvCT presents a unique opportunity to study the spread of a single C. trachomatis strain within both the human and bacterial populations; this may substantially increase our knowledge of epidemiology and transmission of chlamydial infections, and other sexually transmitted infections.

Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.

Chlamydia trachomatis is the most common cause of sexually transmitted infections in the UK, a statistic that is also reflected globally. There are three biovariants of C. trachomatis: trachoma (serotypes A–C) and two sexually transmitted pathovars; serotypes D–K and lyphogranuloma venereum (LGV). Trachoma isolates and the sexually transmitted serotypes D–K are noninvasive, whereas the LGV strains are invasive, causing a disseminating infection of the local draining lymph nodes. Genome sequences are available for single isolates from the trachoma (serotype A) and sexually transmitted (serotype D) biotypes. We sequenced two isolates from the remaining biotype, LGV, a long-term laboratory passaged strain and the recent “epidemic” LGV isolate-causing proctitis. Although the genome of the LGV strain shows no additional genes that could account for the differences in disease outcome, we found evidence of functional gene loss and identified regions of heightened sequence variation that have previously been shown to be important sites for interstrain recombination. We have used new sequencing technologies to show that the recent clinical LGV isolate causing proctitis is unlikely to be a newly emerged strain but is most probably an old strain with relatively new clinical manifestations.

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. These viruses have remained refractory to detailed molecular studies because of the lack of a reverse genetics system coupled to a permissive cell line for targeted genetic manipulation. There is no permissive cell line in which to grow infectious human noroviruses nor an authentic animal model that supports their replication. In contrast, murine norovirus (MNV) offers a tractable system for the study of noroviruses with the recent discovery of permissive cells and a mouse model. The lack of a reverse genetic system for MNV has been a significant block to understanding the biology of noroviruses. We report recovery of infectious MNV after baculovirus delivery of viral cDNA to human hepatoma cells under the control of an inducible DNA polymerase (pol) II promoter. Recovered virus replicated in murine macrophage (RAW264.7) cells, and the recovery of MNV from DNA was confirmed through recovery of virus containing a marker mutation. This pol II promoter driven expression of viral cDNA also generated infectious virus after transfection of HEK293T cells, thus providing both transduction and transfection systems for norovirus reverse genetics. We used norovirus reverse genetics to demonstrate by mutagenesis of the protease–polymerase (pro–pol) cleavage site that processing of pro–pol is essential for the recovery of infectious MNV. This represents the first infectious reverse genetics system for a norovirus, and should provide approaches to address fundamental questions in norovirus molecular biology and replication.

Saccharomyces boulardii , a yeast that was isolated from fruit in Indochina, has been used as a remedy for diarrhea since 1950 and is now a commercially available treatment throughout Europe, Africa, and South America. Though initially classified as a separate species of Saccharomyces , recent publications have shown that the genome of S. boulardii is so similar to Saccharomyces cerevisiae that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in S. boulardii that make it perform more effectively as a probiotic organism compared to other strains of S. cerevisiae . This investigation reports some of these distinguishing characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX, altered copy number of a number of individual genes, and sporulation deficiency have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of S. boulardii to its probiotic nature are discussed.

Objectives: In this prospective study, we aimed to determine the distribution of genotypes by multilocus variable number tandem repeat (VNTR) analysis plus analysis of the ompA gene (MLVA-ompA) of rectal Chlamydia trachomatis among men who have sex with men (MSM) attending Brighton Genitourinary Medicine (GUM) Clinic and to examine any correlations with clinical variables, including HIV status, and to isolate rectal C. trachomatis cultures maximising the possibility of obtaining complete genotyping data.Methods: Samples were assigned genotypes by PCR and sequencing of the markers of the MLVA-ompA genotyping system. Rectal C. trachomatis was isolated in cell culture using McCoy cells. Data regarding demographics, HIV status, rectal symptoms and history of sexually transmitted infections, including C. trachomatis, were collected.Results: 1809 MSM attending the clinic between October 2011 and January 2013 took part in the study, 112 (6.2%) of whom had rectal samples that tested positive for C. trachomatis. 85/112 (75.9%) C. trachomatis-positive rectal samples were assigned 66 different genotypes. Two distinct genotype subclusters were identified: subcluster 1 consisted of more HIV-negative men than subcluster 2 (p=0.025), and the MLVA-ompA genotypes in these subclusters reflected this. Isolates were successfully cultured from 37 of the 112 specimens, from which 27 otherwise unobtainable (from direct PCR) MLVA-ompA genotypes were gained.Conclusions: The most prevalent genotypes were G, E and D representing some overlap with the heterosexual distribution in UK. Subcluster 1 consisted of more ‘heterosexual genotypes’ and significantly more HIV-negative men than subcluster 2, associated with ‘MSM genotypes’. There was a higher diversity of C. trachomatis strains among MSM in Brighton than observed in other cities.

An immunogenic loop within the diphtheria toxin has been deleted from the B-subunit by a modification of the inverse polymerase chain reaction (IPCR) and replaced by a unique restriction endonuclease site. An oligonucleotide encoding an identified epitope sequence from the major outer membrane protein of Neisseria meningitidis of similar size and structure to that deleted has been introduced into the restriction site. Expression of the resulting chimeric B-subunit from Escherichia coli yielded a protein that was recognised by a panel of antibodies specific for the meningococcal epitope. Initial immunisation data suggest that this protein could elicit an antibody response against both diphtheria toxin and meningococcal proteins.

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.