Chlamydial Plasmid Research Articles

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance.

Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone.

Chlamydia, comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis, a close phylogenetic relative of human C. trachomatis, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin (inv), located within a chromosomal region between the rRNA operon (rrn) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis. These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication (ori) and selection markers but lacked the native chlamydial plasmids or its ori. We first recovered allele-replacement mutants using a vector that targets the tryptophan (trp) operon of C. suis. The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori. Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene.IMPORTANCEThe obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

Plasmid-mediated virulence in Chlamydia.

Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmid-associated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease.

Molecular characterisation of the Australian and New Zealand livestock Chlamydia pecorum strains confirms novel but clonal ST23 in association with ovine foetal loss

Chlamydia pecorum is a veterinary pathogen associated with abortions and perinatal mortality in sheep. Recent studies investigating foetal and perinatal lamb mortality in sheep from Australia and New Zealand identified C. pecorum clonal sequence type (ST)23 strains in aborted and stillborn lambs. Presently, there is limited genotypic information on C. pecorum strains associated with reproductive disease, although whole genome sequencing (WGS) of one abortigenic ST23 C. pecorum strain identified unique features, including a deletion in the CDS1 locus of the chlamydial plasmid. We applied WGS on two ST23 strains detected in aborted and stillborn lambs from Australia and used phylogenetic and comparative analyses to compare these to the other available C. pecorum genomes. To re-evaluate the genetic diversity of contemporary strains, we applied C. pecorum genotyping, and chlamydial plasmid sequencing to a range of C. pecorum positive samples and isolates from ewes, aborted foetuses and stillborn lambs, cattle and a goat from diverse geographical regions across Australia and New Zealand.The two new C. pecorum genomes are nearly identical to the genome of the Australian abortigenic strain including the unique deletion in the chlamydial plasmid. Genotyping revealed that these novel C. pecorum ST23 strains are widespread and associated with sheep abortions on Australian and New Zealand farms. In addition, a goat C. pecorum strain (denoted ST 304) from New Zealand was also characterised. This study expands the C. pecorum genome catalogue and describes a comprehensive molecular characterisation of the novel livestock ST23 strains associated with foetal and lamb mortality.

Transformation and Mutagenesis of Chlamydia trachomatis and C. muridarum Utilizing pKW Vector

A gene deletion by allelic exchange via homologous recombination from a bacterial genome represents a valuable genetic tool for studying a role(s) of determinants involved in various aspects of pathogenesis. Due to chlamydial obligate intracellular lifestyle and comparatively low transformation rate, the mutagenesis of Chlamydia utilizes types of suicide vectors that have to be maintained and propagated by the bacteria throughout several rounds of their intracellular developmental cycle. These deletion constructs must be lost by chlamydiae once null mutant formation is achieved. pKW is a small, 5.45 bp, pUC19-derived vector, which has been recently successfully employed for the generation of deletion mutants in C. trachomatis, serovariant D, and C. muridarum. This vector contains both, E. coli as well as chlamydial species-specific plasmid origins of replication, allowing for its propagation by both bacterial genera under a selective pressure. However, once the selective antibiotic is removed from culture, chlamydiae rapidly lose pKW, and the subsequent reintroduction of the selective antibiotic back to chlamydiae-infected cells results efficiently in the selection of generated deletion mutants. Protocols provided here describe in detail the preparation of pKW deletion constructs for C. trachomatis and C. muridarum applicable for chlamydial transformation and the production of null mutant in non-essential genes. Protocols provided here, are describing in detail methods for assembly of the pKW shuttle vector and generation of deletion mutants in C. trachomatis and C. muridarum. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of pKW shuttle vector Basic Protocol 2: Generation of a deletion mutant in C. trachomatis, serovars D and L2 and, Chlamydia muridarum Support Protocol: Transformation of C. trachomatis, serovars B.

Association between serum Chlamydia trachomatis antibody levels and infertility among reproductive-aged women in the U.S.

Chlamydia trachomatis infection, the most prevalent sexually transmitted bacterial infection worldwide, is a significant cause of infertility. Many countries have introduced the widespread use of serologic assays for IgG seropositivity to chlamydial plasmid gene product 3 (Pgp3). However, data on the association between the level of Pgp3-IgG in the multiplex bead array assay (Pgp3AbMBA) and female infertility are still scarce. This cross-sectional analysis included 1,425 women from the National Health and Nutrition Examination Survey (NHANES) from 2013 to 2016. In the fully adjusted logistic regression model, each standard deviation increments of Pgp3AbMBA (SD = 17,079.63) led to a 28% increase in the risk of infertility. The relationship remained consistent in women who had been pregnant and women who gave birth. Smooth curve fitting revealed that the association was linear across the entire range of Pgp3AbMBA. Subgroup analysis suggested that the association was significantly stronger in women who had ever used marijuana and lived in poverty. This study revealed a linear and independent association between the level of Pgp3AbMBA and self-reported infertility in U.S. women. Furthermore, we found that women who had ever used marijuana and lived in poverty were at the highest risk of infertility upon chlamydial infection.

Virulence Protein Pgp3 Is Insufficient To Mediate Plasmid-Dependent Infectivity of Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of infectious blindness and sexually transmitted bacterial infection globally. C. trachomatis contains a conserved chlamydial plasmid with eight coding sequences. Plasmid-cured Chlamydia strains are attenuated and display reduced infectivity in cell culture and in vivo genital infection of female mice. Mutants that do not express the plasmid-encoded proteins Pgp3, a secreted protein with unknown function, or Pgp4, a putative regulator of pgp3 and other chromosomal loci, display an infectivity defect similar to plasmid-deficient strains. Our objective was to determine the combined and individual contributions of Pgp3 and Pgp4 to this phenotype. Deletion of pgp3 and pgp4 resulted in an infectivity defect detected by competition assay in cell culture and in mice. The pgp3 locus was placed under the control of an anhydrotetracycline-inducible promoter to examine the individual contributions of Pgp3 and Pgp4 to infectivity. Expression of pgp3 was induced 100- to 1,000-fold after anhydrotetracycline administration, regardless of the presence or absence of pgp4. However, secreted Pgp3 was not detected when pgp4 was deleted, confirming a role for Pgp4 in Pgp3 secretion. We discovered that expression of pgp3 or pgp4 alone was insufficient to restore normal infectivity, which required expression of both Pgp3 and Pgp4. These results suggest Pgp3 and Pgp4 are both required for infectivity during C. trachomatis infection. Future studies are required to determine the mechanism by which Pgp3 and Pgp4 influence chlamydial infectivity as well as the potential roles of Pgp4-regulated loci.

TargeTron inactivation of plasmid-regulated Chlamydia trachomatis CT084 results in a nonlytic phenotype.

Chlamydia trachomatis is an obligate intracellular bacterium that causes blinding trachoma and sexually transmitted disease. The chlamydial plasmid is a critical virulence factor in the pathogenesis of these diseases. Plasmid gene protein 4 (Pgp4) plays a major role in chlamydial virulence by regulating the expression of both chromosomal genes and Pgp3. Despite the importance of Pgp4 in mediating lytic exit from host cells the pathogenic mechanism by which it functions is unknown. CT084 is a highly conserved chromosomal gene with homology to phospholipase D. We showed CT084 expression is regulated by Pgp4 and expressed late in the chlamydial developmental cycle. To investigate the function of CT084 in chlamydial lytic exit from infected cells, we made a CT084 null strain (ct084::bla) by using Targetron. The ct084::bla strain grew normally in vitro compared to wild-type strain; however, the strain did not lyse infected cells and produced significantly less and smaller plaques. Collectively, our finding shows Pgp4-regulated CT084-mediated chlamydia lytic exit from infected host cells.

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.

Occurrence of Chlamydiae in Corvids in Northeast Italy.

Simple SummaryThe continuous improvement of next-generation sequencing techniques has led to an expansion of the number of Chlamydia species, as well as their host range. Recent studies performed on wild birds have detected Chlamydia strains with characteristics intermediate between Chlamydia psittaci and Chlamydia abortus. In this study, 12/108 corvids tested positive for Chlamydia by real-time PCR. Molecular characterisation at the species level was possible for eight samples, with one positive for C. psittaci and seven for C. abortus. Considering the well-known zoonotic role of C. psittaci and that a potential zoonotic role of avian C. abortus strains cannot be excluded, people who may have professional or other contact with wild birds should take appropriate preventive measures.Chlamydiaceae occurrence has been largely evaluated in wildlife, showing that wild birds are efficient reservoirs for avian chlamydiosis. In this study, DNA extracted from cloacal swabs of 108 corvids from Northeast Italy was screened for Chlamydiaceae by 23S real-time (rt)PCR. The positive samples were characterised by specific rtPCRs for Chlamydia psittaci, Chlamydia abortus, Chlamydia gallinacea, Chlamydia avium, Chlamydia pecorum and Chlamydia suis. Cloacal shedding of Chlamydiaceae was detected in 12 out of 108 (11.1%, 5.9%–18.6% 95% CI) corvids sampled. Molecular characterisation at the species level was possible in 8/12 samples, showing C. psittaci positivity in only one sample from a hooded crow and C. abortus positivity in seven samples, two from Eurasian magpies and five from hooded crows. Genotyping of the C. psittaci-positive sample was undertaken via PCR/high-resolution melting, clustering it in group III_pigeon, corresponding to the B genotype based on former ompA analysis. For C. abortus genotyping, multilocus sequence typing was successfully performed on the two samples with high DNA load from Eurasian magpies, highlighting 100% identity with the recently reported Polish avian C. abortus genotype 1V strain 15-58d44. To confirm the intermediate characteristics between C. psittaci and C. abortus, both samples, as well as two samples from hooded crows, showed the chlamydial plasmid inherent in most C. psittaci and avian C. abortus, but not in ruminant C. abortus strains. The plasmid sequences were highly similar (≥99%) to those of the Polish avian C. abortus genotype 1V strain 15-58d44. To our knowledge, this is the first report of avian C. abortus strains in Italy, specifically genotype 1V, confirming that they are actively circulating in corvids in the Italian region tested.

Completing the Genome Sequence of Chlamydia pecorum Strains MC/MarsBar and DBDeUG: New Insights into This Enigmatic Koala (Phascolarctos cinereus) Pathogen.

Chlamydia pecorum, an obligate intracellular pathogen, causes significant morbidity and mortality in livestock and the koala (Phascolarctos cinereus). A variety of C. pecorum gene-centric molecular studies have revealed important observations about infection dynamics and genetic diversity in both koala and livestock hosts. In contrast to a variety of C. pecorum molecular studies, to date, only four complete and 16 draft genomes have been published. Of those, only five draft genomes are from koalas. Here, using whole-genome sequencing and a comparative genomics approach, we describe the first two complete C. pecorum genomes collected from diseased koalas. A de novo assembly of DBDeUG_2018 and MC/MarsBar_2018 resolved the chromosomes and chlamydial plasmids each as single, circular contigs. Robust phylogenomic analyses indicate biogeographical separation between strains from northern and southern koala populations, and between strains infecting koala and livestock hosts. Comparative genomics between koala strains identified new, unique, and shared loci that accumulate single-nucleotide polymorphisms and separate between northern and southern, and within northern koala strains. Furthermore, we predicted novel type III secretion system effectors. This investigation constitutes a comprehensive genome-wide comparison between C. pecorum from koalas and provides improvements to annotations of a C. pecorum reference genome. These findings lay the foundations for identifying and understanding host specificity and adaptation behind chlamydial infections affecting koalas.

Chlamydia psittaci Plasmid-Encoded CPSIT_P7 Elicits Inflammatory Response in Human Monocytes via TLR4/Mal/MyD88/NF-κB Signaling Pathway.

The chlamydial plasmid, an essential virulence factor, encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. However, the virulence factors and the molecular mechanisms of Chlamydia psittaci are not well understood. In the present study, we investigated the roles and mechanisms of the plasmid-encoded protein CPSIT_P7 of C. psittaci in regulating the inflammatory response in THP-1 cells (human monocytic leukemia cell line). Based on cytokine arrays, CPSIT_P7 induces the expression of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in THP-1 cells. Moreover, the expression levels of IL-6, IL-8, and MCP-1 stimulated by CPSIT_P7 declined after silencing of the Toll-like receptor 4 (TLR4) gene using small interfering RNA and transfection of a dominant negative plasmid encoding TLR4 (pZERO-hTLR4). We further demonstrated that transfection with the dominant negative plasmid encoding MyD88 (pDeNy-hMyD88) and the dominant negative plasmid encoding Mal (pDeNy-hMal) could also abrogate the expression of the corresponding proteins. Western blot and immunofluorescence assay results showed that CPSIT_P7 could activate nuclear factor κB (NF-κB) signaling pathways in THP-1 cells. Altogether, our results indicate that the CPSIT_P7 induces the TLR4/Mal/MyD88/NF-κB signaling axis and therefore contributes to the inflammatory cytokine response.

Coevolving Plasmids Drive Gene Flow and Genome Plasticity in Host-Associated Intracellular Bacteria

SummaryPlasmids are important in microbial evolution and adaptation to new environments. Yet, carrying a plasmid can be costly, and long-term association of plasmids with their hosts is poorly understood. Here, we provide evidence that the Chlamydiae, a phylum of strictly host-associated intracellular bacteria, have coevolved with their plasmids since their last common ancestor. Current chlamydial plasmids are amalgamations of at least one ancestral plasmid and a bacteriophage. We show that the majority of plasmid genes are also found on chromosomes of extant chlamydiae. The most conserved plasmid gene families are predominantly vertically inherited, while accessory plasmid gene families show significantly increased mobility. We reconstructed the evolutionary history of plasmid gene content of an entire bacterial phylum over a period of around one billion years. Frequent horizontal gene transfer and chromosomal integration events illustrate the pronounced impact of coevolution with these extrachromosomal elements on bacterial genome dynamics in host-dependent microbes.

Diversity in Chlamydial plasmids.

BackgroundEvolutionary 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.AimsThis 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.MethodsThe 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.ResultsThe 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.ConclusionThe 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.

Impact of bacterial traces belonging to the Enterobacteriaceae on the prevalence of Chlamydia trachomatis in women visiting a community hospital in Japan.

We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n=257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n=27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n=20) and -negative samples (n=27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

Activation of neutrophils by Chlamydia trachomatis-infected epithelial cells is modulated by the chlamydial plasmid

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation.

Culture-independent metagenomics supports discovery of uncultivable bacteria within the genus Chlamydia

Advances in culture-independent methods have meant that we can more readily detect and diagnose emerging infectious disease threats in humans and animals. Metagenomics is fast becoming a popular tool for detection and characterisation of novel bacterial pathogens in their environment, and is particularly useful for obligate intracellular bacteria such as Chlamydiae that require labour-intensive culturing. We have used this tool to investigate the microbial metagenomes of Chlamydia-positive cloaca and choana samples from snakes. The microbial complexity within these anatomical sites meant that despite previous detection of chlamydial 16S rRNA sequences by single-gene broad-range PCR, only a chlamydial plasmid could be detected in all samples, and a chlamydial chromosome in one sample. Comparative genomic analysis of the latter revealed it represented a novel taxon, Ca. Chlamydia corallus, with genetic differences in regards to purine and pyrimidine metabolism. Utilising statistical methods to relate plasmid phylogeny to the phylogeny of chromosomal sequences showed that the samples also contain additional novel strains of Ca. C. corallus and two putative novel species in the genus Chlamydia. This study highlights the value of metagenomics methods for rapid novel bacterial discovery and the insights it can provide into the biology of uncultivable intracellular bacteria such as Chlamydiae.

Protective immunity induced by recombinant protein CPSIT_p8 of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen with a broad host range that can lead to severe respiratory and systemic disease in humans. Currently, an effective commercial vaccine against C. psittaci infection is not available. The chlamydial plasmid is an important virulence factor and encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. In this study, we assessed the efficacy of vaccination with plasmid proteins at preventing C. psittaci lung infection in a murine model. BALB/c mice were immunized intraperitoneally, three times at 2-week intervals, with purified recombinant CPSIT_p8 protein and then infected with C. psittaci. Immunization significantly decreased chlamydial load in the lungs of infected mice, resulted in a lower level of IFN-γ, and reduced the extent of inflammation. In vivo or in vitro neutralization of C. psittaci with sera collected from immunized mice did not reduce the amount of viable C. psittaci in the lungs of mice, indicating that CPSIT_p8-specific antibodies do not have neutralizing capacity. Furthermore, confocal fluorescence microscopy using a mouse anti-CPSIT_p8 antibody revealed that CPSIT_p8 was localized inside the inclusion of C. psittaci 6BC-infected cells. Our results demonstrate that CPSIT_p8 protein induces significant protective immunity against challenge with C. psittaci in mice and represents a promising new vaccine candidate for the prevention of C. psittaci infection.

Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated.

ABSTRACTChlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene.