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4-Chloroisocoumarin compounds have broad inhibitory properties against serine proteases. Here, we show that selected 3-alkoxy-4-chloroisocoumarins preferentially inhibit the activity of the conserved serine protease High-temperature requirement A of Chlamydia trachomatis. The synthesis of a new series of isocoumarin-based scaffolds has been developed, and their anti-chlamydial properties were investigated. The structure of the alkoxy substituent was found to influence the potency of the compounds against High-temperature requirement A, and modifications to the C-7 position of the 3-alkoxy-4-chloroisocoumarin structure attenuate anti-chlamydial properties.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn−/− mice and observed that shedding kinetics of Chlamydia were only affected in FcRn−/− mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn−/− mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

[This corrects the article DOI: 10.3389/fcimb.2023.1281823.].

INTRODUCTION: There is currently no test for pelvic inflammatory disease (PID) that is non-invasive and sufficiently sensitive and specific. Clinicians must therefore diagnose PID clinically, ruling out medical emergencies and conducting pelvic examinations where possible. While guidelines state that clinicians should be prepared to over-diagnose PID, it remains an under-diagnosed condition, with severe reproductive health impacts when left untreated. This research is the first to consider the perspectives of end-users on the development of a diagnostic test for PID. METHODS: Semi-structured live video feed online (Zoom) interviews were conducted with 11 clinicians and nine women (aged 18-30 years) in Australia to understand how a diagnostic test might be used, and what characteristics a test would need for it to be acceptable to clinicians and young women. Participants were recruited via researcher and university student networks. Reflexive thematic analysis was used to identify key themes relating to the acceptability and characteristics of a diagnostic test for PID. RESULTS: Seven general practitioners, four clinicians working in sexual health clinics, and nine young women (aged 21-27 years) were interviewed. Clinicians were aged between 31-58 years and were predominantly female. Clinicians recognised that the development of an accurate test to diagnose PID would be valuable to themselves and other clinicians, particularly those who lack experience diagnosing PID, and those working in certain settings, including emergency departments. They discussed how they might use a test to enhance their clinical assessment but highlighted that it would not replace clinical judgement. Clinicians also considered how a test would impact the patient experience and time to treatment, emphasising that it should be minimally invasive and have a quick turnaround time. Young women said a test would be acceptable if endorsed by a trustworthy clinician. CONCLUSIONS: PID remains a chall

Chlamydia trachomatis, the most common bacterial sexually transmitted infection worldwide, is responsible for considerable health burden due to its significant sequelae. There are growing concerns about chlamydial treatment and management due to widely documented increasing burden of repeat infections. In the current study, a cohort study design of 305 women with urogenital chlamydial infections demonstrated that 11.8% of women experienced repeat infections after treatment with azithromycin. The chlamydial DNA load measured by quantitative PCR was higher in women who experienced a repeat infection (p = 0.0097) and repeat infection was associated with sexual contact. There was no genomic or phenotypic evidence of azithromycin resistance within the chlamydial isolates. During repeat infection, or repeat positive tests during follow up, vaginal chlamydial gene expression (ompA, euo, omcB, htrA, trpAB) was markedly higher compared to baseline, and two of the selected immune genes analyzed had significantly lower expression at the time of repeat infection. Overall, there are two implications of these results. The results could be generalized to all recent infections, or repeat positive events, and indicate that chlamydial infections are have higher transcriptional activity of select genes early in the infection in women. Alternatively, after azithromycin treatment, repeat infections of Chlamydia may be more transcriptionally active at certain genes, and there may be post-treatment immunological alterations that interplay into repeat exposures establishing an active infection. The potential that recent infections may involve a higher level of activity from the organism may have implications for management by more regular testing of the most at risk women to reduce the risk of sequelae.

Urogenital Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection throughout the world. While progress has been made to better understand how type strains develop and respond to environmental stress in vitro, very few studies have examined how clinical isolates behave under similar conditions. Here, we examined the development and persistence phenotypes of several clinical isolates, to determine how similar they are to each other, and the type strain C. trachomatis D/UW-3/Cx. The type strain was shown to produce infectious progeny at a higher magnitude than each of the clinical isolates, in each of the six tested cell lines. All chlamydial strains produced the highest number of infectious progeny at 44 h post-infection in the McCoy B murine fibroblast cell line, yet showed higher levels of infectivity in the MCF-7 human epithelial cell line. The clinical isolates were shown to be more susceptible than the type strain to the effects of penicillin and iron deprivation persistence models in the MCF-7 cell line. While subtle differences between clinical isolates were observed throughout the experiments conducted, no significant differences were identified. This study reinforces the importance of examining clinical isolates when trying to relate in vitro data to clinical outcomes, as well as the importance of considering the adaptations many type strains have to being cultured in vitro.

Devastating fires in Australia over 2019-20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/bloo

Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells. IMPORTANCEChlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. In

Adverse reproductive health outcomes, such as pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility, have been associated with Chlamydia trachomatis and Neisseria gonorrhoea infections. These reproductive health outcomes could be complemented by measuring subsequent pregnancies to assess impact on fertility. The study design was a cohort study of women in Queensland (QLD), Australia, using data linkage methods to link chlamydia and/or gonorrhea testing records (including an unexposed group undergoing full blood count tests) (2000 and 2005) with the QLD Perinatal Registry (2000 to 2013). The cohort included 132 962 women, with 69 533 records of pregnancies. Women in the exposed group, with no prior pregnancy, had a reduced odds of a pregnancy during the follow up of the study (20 year old (at 2005) aOR 0.91 95% CI 0.87-0.95, and 25 year old aOR 0.71 95% CI 0.68-0.75). Women in the exposed group with a prior pregnancy had increased odds of pregnancy during the follow up of the study (20 year old (at 2005) aOR 1.72 95% CI 1.59-1.86, and 25 year old aOR 1.35 95% CI 1.26-1.45). Our data provides further evidence at a population level of the significant impact on reproductive outcomes associated with chlamydia and gonorrhea.

© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. There are numerous structural and cultural barriers to the progression of women and marginalized groups to leadership in academia, especially in Science, Technology, Engineering, Mathematics and Medicine (STEMM). A range of interventions have been described to address this inequity, with varying success. Here, we suggest that sponsorship could be one effective intervention and propose an institutional action plan to implement a sponsorship program in academia. We outline why sponsorship could be an effective strategy, especially if implemented through a deliberate program by an institution. We then detail the three components of an action plan to be considered in implementation: the elements of the program, the activities that sponsorship in academia likely encompasses, and the selection of sponsors and protégés. The plan could also be enacted by academic leadership in the absence of an institutional program and could serve as a guide to individuals in academia aspiring to address diversity and inclusion in STEMM.

© FEMS 2019. Serological assays can be used to investigate the population burden of infection and potentially sequelae from Chlamydia. We investigated the PGP3 ELISA as a sero-epidemiological tool for infection or sub-fertility in Australian and Samoan women. The PGP3 ELISA absorbance levels were compared between groups of women with infertility, fertile, and current chlamydial infections. In the Australian groups, women with chlamydial tubal factor infertility had significantly higher absorbance levels in the PGP3 ELISA compared to fertile women (P < 0.0001), but not when compared to women with current chlamydial infection (P = 0.44). In the Samoan study, where the prevalence of chlamydial infections is much higher there were significant differences in the PGP3 ELISA absorbance levels between chlamydial sub-fertile women and fertile women (P = 0.003). There was no difference between chlamydial sub-fertile women and women with a current infection (P = 0.829). The results support that the PGP3 assay is effective for sero-epidemiological analysis of burden of infection, but not for evaluation of chlamydial pathological sequelae such as infertility.

© Copyright © 2019 Hayward, Marsh, Humphrys, Huston and Myers. Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. Chlamydia trachomatis causes trachoma in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility, pitfalls and challenges of single cell approaches applied to chlamydial biology, and to potentially identify early host cell biomarkers of chlamydial infection. Two hundred sixty-four time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-h cells from 6- and 12-h. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. We find that changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host res

© 2019 Christensen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and—using recombinant purified proteins—demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein’s thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB dif

© 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.

Chlamydia trachomatis infects the urogenital tract (UGT) and eyes. Anatomical tropism is correlated with variation in the major outer membrane protein encoded by ompA. Strains possessing the ocular ompA variants A, B, Ba and C are typically found within the phylogenetically coherent "classical ocular lineage". However, variants B, Ba and C have also been found within three distinct strains in Australia, all associated with ocular disease in children and outside the classical ocular lineage. CtGEM genotyping is a method for detecting and discriminating ocular strains and also the major phylogenetic lineages. The rationale was facilitation of surveillance to inform responses to C. trachomatis detection in UGT specimens from young children. CtGEM typing is based on high resolution melting analysis (HRMA) of two PCR amplified fragments with high combinatorial resolving power, as defined by computerised comparison of 65 whole genomes. One fragment is from the hypothetical gene defined by Jali-1891 in the C. trachomatis B_Jali20 genome, while the other is from ompA. Twenty combinatorial CtGEM types have been shown to exist, and these encompass unique genotypes for all known ocular strains, and also delineate the TI and T2 major phylogenetic lineages, identify LGV strains and provide additional resolution beyond this. CtGEM typing and Sanger sequencing were compared with 42 C. trachomatis positive clinical specimens, and there were no disjunctions. CtGEM typing is a highly efficient method designed and tested using large scale comparative genomics. It divides C. trachomatis into clinically and biologically meaningful groups, and may have broad application in surveillance.

© 2018 Elsevier Ltd Genital Chlamydia trachomatis infection is the most commonly diagnosed sexually transmitted infection. Trachoma is caused by ocular infection with C trachomatis and is the leading infectious cause of blindness worldwide. New serological assays for C trachomatis could facilitate improved understanding of C trachomatis epidemiology and prevention. C trachomatis serology offers a means of investigating the incidence of chlamydia infection and might be developed as a biomarker of scarring sequelae, such as pelvic inflammatory disease. Therefore, serological assays have potential as epidemiological tools to quantify unmet need, inform service planning, evaluate interventions including screening and treatment, and to assess new vaccine candidates. However, questions about the performance characteristics and interpretation of C trachomatis serological assays remain, which must be addressed to advance development within this field. In this Personal View, we explore the available information about C trachomatis serology and propose several priority actions. These actions involve development of target product profiles to guide assay selection and assessment across multiple applications and populations, establishment of a serum bank to facilitate assay development and evaluation, and development of technical and statistical methods for assay evaluation and analysis of serological findings. The field of C trachomatis serology will benefit from collaboration across the public health community to align technological developments with their potential applications.

© 2017 Elsevier Ltd JO146, a mixture of two diastereomers of a peptidic phosphonate inhibitor for Chlamydial HtrA (CtHtrA), has reported activity against Chlamydia species in both human and koala. In this study we isolated the individual diastereomers JO146-D1 and JO146-D2 (in ≥90% purity) and assessed their individual inhibitory activity against the serine protease human neutrophil elastase (HNE) which is structurally and functionally related to CtHtrA, as well as in Chlamydia trachomatis cell culture. JO146-D2 [S,S,R-Boc-Val-Pro-ValP(OPh)2], the isomer with the physiologically relevant valine at P1, had an approximate 2.5 – fold increase in in vitro HNE inhibition potency over JO146-D1 [S,S,S-Boc-Val-Pro-ValP(OPh)2] and greater than 100 – fold increase in cellular anti-chlamydial activity compared to JO146-D1 which possesses the unnatural valine at P1. JO146 and the individual diastereomers had excellent selectivity for the serine protease HNE over the potential off-target serine proteases trypsin and chymotrypsin. Docking studies supported the biological data with a geometrically unfavoured interaction observed between the P1 valine residue of JO146-D1 and the enzyme S1 sub-pocket.

© FEMS 2018. All rights reserved. The American Society for Microbiology's curricular guidelines for Introductory Microbiology highlighted key laboratory skills in the isolation, visualization and identification of microorganisms as core learning objectives in the discipline. Since the publication of these guidelines in 2012, there has been a paucity of diagnostic assessment tools in the literature that can be used to assess competencies in the microbiology laboratory. This project aimed to establish a laboratory competency examination for introductory microbiology, with tasks specifically aligned to laboratory skills and learning outcomes outlined in curricular guidelines for microbiology. A Laboratory Competency Examination assessing student skills in light microscopy, Gram-staining, pure culture, aseptic technique, serial dilution, dilution calculations and pipetting was developed at The University of Queensland, Australia. The Laboratory Competency Examination was field-tested in a large introductory microbiology subject (~400 students), and student performance and learning gains data were collected from 2016 to 2017 to evaluate the validity of the assessment. The resulting laboratory assessment is presented as an endpoint diagnostic tool for assessing laboratory competency that can be readily adapted towards different educational contexts.

© 2018 Oxford University Press. All rights reserved. Microplastics (plastics <5 mm diameter) are at the forefront of current environmental pollution research, however, little is known about the degradation of microplastics through ingestion. Here, by exposing Antarctic krill (Euphausia superba) to microplastics under acute static renewal conditions, we present evidence of physical size alteration of microplastics ingested by a planktonic crustacean. Ingested microplastics (31.5 µm) are fragmented into pieces less than 1 µm in diameter. Previous feeding studies have shown spherical microplastics either; pass unaffected through an organism and are excreted, or are sufficiently small for translocation to occur. We identify a new pathway; microplastics are fragmented into sizes small enough to cross physical barriers, or are egested as a mixture of triturated particles. These findings suggest that current laboratory-based feeding studies may be oversimplifying interactions between zooplankton and microplastics but also introduces a new role of Antarctic krill, and potentially other species, in the biogeochemical cycling and fate of plastic.

© 2018 Giffard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis infects the urogenital tract (UGT) and eyes. Anatomical tropism is correlated with variation in the major outer membrane protein encoded by ompA. Strains possessing the ocular ompA variants A, B, Ba and C are typically found within the phyloge-netically coherent “classical ocular lineage”. However, variants B, Ba and C have also been found within three distinct strains in Australia, all associated with ocular disease in children and outside the classical ocular lineage. CtGEM genotyping is a method for detecting and discriminating ocular strains and also the major phylogenetic lineages. The rationale was facilitation of surveillance to inform responses to C. trachomatis detection in UGT specimens from young children. CtGEM typing is based on high resolution melting analysis (HRMA) of two PCR amplified fragments with high combinatorial resolving power, as defined by computerised comparison of 65 whole genomes. One fragment is from the hypothetical gene defined by Jali-1891 in the C. trachomatis B_Jali20 genome, while the other is from ompA. Twenty combinatorial CtGEM types have been shown to exist, and these encompass unique genotypes for all known ocular strains, and also delineate the TI and T2 major phylogenetic lineages, identify LGV strains and provide additional resolution beyond this. CtGEM typing and Sanger sequencing were compared with 42 C. trachomatis positive clinical specimens, and there were no disjunctions. CtGEM typing is a highly efficient method designed and tested using large scale comparative genomics. It divides C. trachomatis into clinically and biologically meaningful groups, and may have broad application in surveillance.

© 2018 American Society for Reproductive Medicine The human microbiome project has shown a remarkable diversity of microbial ecology within the human body. The vaginal microbiota is unique in that in many women it is most often dominated by Lactobacillus species. However, in some women it lacks Lactobacillus spp. and is comprised of a wide array of strict and facultative anaerobes, a state that broadly correlates with increased risk for infection, disease, and poor reproductive and obstetric outcomes. Interestingly, the level of protection against infection can also vary by species and strains of Lactobacillus, and some species although dominant are not always optimal. This factors into the risk of contracting sexually transmitted infections and possibly influences the occurrence of resultant adverse reproductive outcomes such as tubal factor infertility. The composition and function of the vaginal microbiota appear to play an important role in pregnancy and fertility treatment outcomes and future research in this field will shed further translational mechanistic understanding onto the interplay of the vaginal microbiota with women's health and reproduction.

© 2017 Wiley Periodicals, Inc. There are numerous proteases of pathogenic organisms that are currently targeted for therapeutic intervention along with many that are seen as potential drug targets. This review discusses the chemical and biological makeup of some key druggable proteases expressed by the five major classes of disease causing agents, namely bacteria, viruses, fungi, eukaryotes, and prions. While a few of these enzymes including HIV protease and HCV NS3-4A protease have been targeted to a clinically useful level, a number are yet to yield any clinical outcomes in terms of antimicrobial therapy. A significant aspect of this review discusses the chemical and pharmacological characteristics of inhibitors of the various proteases discussed. A total of 25 inhibitors have been considered potent and safe enough to be trialed in humans and are at different levels of clinical application. We assess the mechanism of action and clinical performance of the protease inhibitors against infectious agents with their developmental strategies and look to the next frontiers in the use of protease inhibitors as anti-infective agents.

© 2017 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists 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.

BACKGROUND: Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure). One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment. METHODS: Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11) or over 9 days (N = 10). Azithromycin concentrations were normalised to an internal standard (leucine enkephalin), and the bulk lipid species phosphatidylcholine [PC(34:1)], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1), and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens. RESULTS: Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg). In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg), and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg) on day 9. CONCLUSION: Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting that poor antibiotic absorption is unlikely to be an explanation for treatment failure.

Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.

This text is the only book to provide a comprehensive and state-of-the-art review of issues relevant to STI care in the HIV-infected adult, adolescent, and transgendered populations.

© 2017 Vodstrcil et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure). One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment. Methods Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11) or over 9 days (N = 10). Azithromycin concentrations were normalised to an internal standard (leucine enkephalin), and the bulk lipid species phosphatidylcholine [PC(34:1)], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1), and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens. Results Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg). In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg), and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg) on day 9. Conclusion Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting t

© 2017 The Author(s). A systematic analysis of 240 causes of death in 2013 revealed that parasitic diseases were responsible for more than one million deaths. The vast majority of these fatalities resulted from protozoan infections presenting with neurological sequelae. In the absence of a vaccine, development of effective therapies is essential to improving global public health. In 2015, an intriguing strategy to prevent cerebral malaria was proposed by Gordon et al. 2015 mBio, 6:e00625. Their study suggested that inhibition of the mammalian target of rapamycin prevented experimental cerebral malaria by blocking the damage to the blood brain barrier and stopping the accumulation of parasitized red blood cells and T cells in the brain. Here, we hypothesize that the same therapeutic strategy could be adopted for other protozoan infections with a brain tropism, to prevent cerebral parasitosis by limiting pathogen replication and preventing immune mediated destruction of brain tissue.

© 2017 The Author(s). The order Chlamydiales are biphasic intracellular bacterial pathogens infecting humans and domesticated animals. Wildlife infections have also been reported, with the most studied example being Chlamydia pecorum infections in the koala, an iconic Australian marsupial. In koalas, molecular evidence suggests that spill-over from C. pecorum infected livestock imported into Australia may have had a historical or contemporary role. Despite preliminary evidence that other native Australian marsupials also carry C. pecorum, their potential as reservoirs of this pathogen and other Chlamydia-related bacteria (CRBs) has been understudied. Mucosal epithelial samples collected from over 200 native Australian marsupials of different species and geographic regions across Australia were PCR screened for Chlamydiales. Previously described and genetically distinct C. pecorum genotypes and a range of 16S rRNA genotypes sharing similarity to different CRBs in the broader Chlamydiales order were present. One 16S rRNA Chlamydiales genotype recently described in Australian ticks that parasitise native Australian marsupials was also identified. This study provides further evidence that chlamydial infections are widespread in native fauna and that detailed investigations are required to understand the influence these infections have on host species conservation, but also whether infection spill-over plays a role in their epidemiology.

© 2017 Indian Journal of Medical Microbiology | Published by Wolters Kluwer - Medknow. Introduction: Chlamydia trachomatis is the etiological agent for the most prevalent bacterial sexually transmitted infection in both developed and developing countries. The aim of present study was to characterize the antibody response between two groups of individuals, having either a single C. trachomatis infection and or repeated infections. Material and Methods: Current study consisted of two groups, one with an initial Chlamydia infection and a second with repeated infections. A titre based estimation of specific serum (IgG and IgA) levels using ELISA were performed, which further validated by western blot. In vitro neutralizing ability of each patient's serum against both homologous and heterologous strains was also determined. Results: Individuals infected with one of the C. trachomatis serovars D, E or K exhibited a strong systemic antibody response as characterized by ELISA and western blot. These individuals may have developed at least some level of protection as they only represented single infection. By comparison, individuals infected with serovar D, E or F that exhibited low systemic antibody response often presented repeated C. trachomatis infections, suggesting an association with poor immune response. An in vitro neutralizing level of 60-90% was observed in the human sera against homologous serovar D and two heterologous C. trachomatis serovars E and K, compared to <40% against heterologous serovars F. Conclusion: Individuals infected with serovars D and K showed a potential association between circulating antibody response and re-infection risk. While the patients infected with serovars E showed a disconnection between systemic antibody response and re-infection risk.

© 2017 Future Medicine Ltd. Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection worldwide and the leading cause of preventable blindness. Reports have emerged of treatment failure, suggesting a need to develop new antibiotics to battle Chlamydia infection. One possible candidate for a new treatment is the protease inhibitor JO146, which is an effective anti-Chlamydia agent that targets the CtHtrA protein. CtHtrA is a lynchpin on the chlamydial cell surface due to its essential and multifunctional roles in the bacteria's stress response, replicative phase of development, virulence and outer-membrane protein assembly. This review summarizes the current understanding of CtHtrA function and presents a mechanistic model that highlights CtHtrA as an effective target for anti-Chlamydia drug development.

© FEMS 2017. Lipophilic copper (Cu)-containing complexes have shown promising antibacterial activity against a range of bacterial pathogens. To examine the susceptibility of the intracellular human pathogen Chlamydia trachomatis to copper complexes containing bis(thiosemicarbazone) ligands [Cu(btsc)], we tested the in vitro effect of CuII-diacetyl- and CuII-glyoxal-bis[N(4)-methylthiosemicarbazonato] (Cu(atsm) and Cu(gtsm), respectively) on C. trachomatis. Cu(atsm) and to a greater extent, Cu(gtsm), prevented the formation of infectious chlamydial progeny. Impacts on host cell viability and respiration were also observed in addition to the Chlamydia impacts. This work suggests that copper-based complexes may represent a new lead approach for future development of new therapeutics against chlamydial infections, although host cell impacts need to be fully explored.

© Paul et al. During infection chlamydial pathogens form an intracellular membrane-bound replicative niche termed the inclusion, which is enriched with bacterial transmembrane proteins called Incs. Incs bind and manipulate host cell proteins to promote inclusion expansion and provide camouflage against innate immune responses. Sorting nexin (SNX) proteins that normally function in endosomal membrane trafficking are a major class of inclusion-associated host proteins, and are recruited by IncE/CT116. Crystal structures of the SNX5 phox-homology (PX) domain in complex with IncE define the precise molecular basis for these interactions. The binding site is unique to SNX5 and related family members SNX6 and SNX32. Intriguingly the site is also conserved in SNX5 homologues throughout evolution, suggesting that IncE captures SNX5-related proteins by mimicking a native host protein interaction. These findings thus provide the first mechanistic insights both into how chlamydial Incs hijack host proteins, and how SNX5-related PX domains function as scaffolds in protein complex assembly.