HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Bioinformatic analysis of bacteria and host cell dual RNA-sequencing experiments.

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

Evaluation and diagnosis of the traumatized dentition.

Traumatic dental injuries comprise a number of the dental emergency patients who are often seen after hours or on an unscheduled basis in a dental practice environment. Although there are a variety of traumatic dental injuries that can occur, each with their own recommended treatment protocols, the initial evaluation and diagnosis of the traumatized dentition make up a critical aspect of the management of these cases. This article will highlight the key components of a thorough and efficient examination process of the traumatized dentition to include (a) documenting an accurate history of the events causing the injury, (b) performing a systematic clinical examination to include the use of clinical photographs and pulp sensibility tests, (c) obtaining appropriate radiographic images and scans, (d) understanding some considerations unique to evaluating young patients with traumatic injuries, and (e) recognizing the importance of having accurate and thorough documentation of these types of cases. Once the evaluation and diagnosis phase has been completed, the necessary treatment protocols can be initiated in an appropriate manner.

A retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women.

BACKGROUND: We know very little about the microbiota inhabiting the upper female reproductive tract and how it impacts on fertility. AIMS: This pilot study aimed to examine the vaginal, cervical and endometrial microbiota for women with a history of infertility compared to women with a history of fertility. MATERIALS AND METHODS: Using a retrospective case-control study design, women were recruited for collection of vaginal, cervical and endometrial samples. The microbiota composition was analysed by 16S ribosomal RNA (rRNA) gene amplification and endometrial expression of selected human genes by quantitative reverse transcription polymerase chain reaction. RESULTS: Sixty-five specimens from the reproductive tract of 31 women were successfully analysed using 16S rRNA gene amplicon sequencing (16 controls and 15 cases). The dominant microbial community members were consistent in the vagina and cervix, and generally consistent with the endometrium although the relative proportions varied. We detected three major microbiota clusters that did not group by tissue location or case-control status. There was a trend that infertile women more often had Ureaplasma in the vagina and Gardnerella in the cervix. Testing for the expression of selected genes in the endometrium did not show evidence of correlation with case-control status, or with microbial community composition, although Tenascin-C expression correlated with a history of miscarriage. CONCLUSIONS: There is a need for further exploration of the endometrial microbiota, and how the microbiota members or profile interplays with fertility or assisted reproductive technologies.

Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues.

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent.

BACKGROUND: The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. RESULTS: In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. CONCLUSIONS: Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BACKGROUND: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts. METHODS: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts. RESULTS: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec. CONCLUSIONS: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

Culture-independent genome sequencing of clinical samples reveals an unexpected heterogeneity of infections by Chlamydia pecorum.

Chlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probe-based genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains.

Genetic variation in vitro and in vivo of an attenuated Lassa vaccine candidate.

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

Circleator: flexible circular visualization of genome-associated data with BioPerl and SVG.

SUMMARY: Circleator is a Perl application that generates circular figures of genome-associated data. It leverages BioPerl to support standard annotation and sequence file formats and produces publication-quality SVG output. It is designed to be both flexible and easy to use. It includes a library of circular track types and predefined configuration files for common use-cases, including. (i) visualizing gene annotation and DNA sequence data from a GenBank flat file, (ii) displaying patterns of gene conservation in related microbial strains, (iii) showing Single Nucleotide Polymorphisms (SNPs) and indels relative to a reference genome and gene set and (iv) viewing RNA-Seq plots. AVAILABILITY AND IMPLEMENTATION: Circleator is freely available under the Artistic License 2.0 from http://jonathancrabtree.github.io/Circleator/ and is integrated with the CloVR cloud-based sequence analysis Virtual Machine (VM), which can be downloaded from http://clovr.org or run on Amazon EC2.

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum.

BACKGROUND: Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced. RESULTS: Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations. CONCLUSIONS: The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes.

BACKGROUND: Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing. RESULTS: Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection. CONCLUSIONS: Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding of chlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Full genome sequences of all nine Chlamydia psittaci genotype reference strains.

Chlamydia psittaci primarily infects birds, but zoonotic transmission occurs in people in close contact with infected birds. The clinical outcome ranges from inapparent disease to pneumonia. Here we report the genome sequences of all 9 Chlamydia psittaci genotype reference strains.

Unity in variety--the pan-genome of the Chlamydiae.

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Chlamydia pneumoniae is genetically diverse in animals and appears to have crossed the host barrier to humans on (at least) two occasions.

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non-Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.

Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58.

Chlamydia pecorum is an obligate intracellular bacterial pathogen that causes diverse disease in a wide variety of economically important mammals. We report the finished complete genome sequence of C. pecorum E58, the type strain for the species.

Genome sequences of the zoonotic pathogens Chlamydia psittaci 6BC and Cal10.

Chlamydia psittaci is a highly prevalent avian pathogen and the cause of a potentially lethal zoonosis, causing life-threatening pneumonia in humans. We report the genome sequences of C. psittaci 6BC, the prototype strain of the species, and C. psittaci Cal10, a widely used laboratory strain.

Denosumab Use as a Predictor Variable for External Cervical Resorption: A Case-Control Study.

INTRODUCTION: The objective of this case-control study was to investigate the association between denosumab use and the risk of developing external cervical resorption (ECR). METHODS: Thirty-three patients ≥45 years old who were diagnosed with ECR were selected. Controls were matched to the cases based on sex and age (±5 years) in a 1:1 ratio. Confounders were classified into systemic factors, including a history of systemic sclerosis, hepatitis B, denosumab use, and bisphosphonate use, or local factors, including a history of traumatic occlusion, periodontal procedures (scaling and root planing and periodontal surgeries), and tooth extraction (excluding third molar extraction). Additionally, the number of remaining teeth in each subject was recorded using panoramic radiographs. The baseline characteristics of the 2 groups, including age, sex, and the number of remaining teeth, were compared using the chi-square and Mann-Whitney U tests. Binary logistic regression was used to determine the possible association between denosumab use and the risk of developing ECR (α < 0.05). RESULTS: No significant differences in baseline characteristics were observed between the case and control groups (P > .05). After adjusting for systemic and local cofounders, denosumab use was significantly associated with the occurrence of ECR (odds ratio = 7.317; 95% confidence interval, 1.410-37.966; P < .05). CONCLUSIONS: Based on the binary logistic regression model, denosumab use could significantly predict the risk of developing ECR.