Transcriptional analysis of in vitro expression patterns of chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle.

Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in theloci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peakexpression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 hp.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may beindicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis

Experimental challenge of pregnant cattle with the putative abortifacient Waddlia chondrophila

Waddlia chondrophila is a Gram-negative intracellular bacterial organism that is related to classical chlamydial species and has been implicated as a cause of abortion in cattle. Despite an increasing number of observational studies linking W. chondrophila infection to cattle abortion, little direct experimental evidence exists. Given this paucity of direct evidence the current study was carried out to investigate whether experimental challenge of pregnant cattle with W. chondrophila would result in infection and abortion. Nine pregnant Friesian-Holstein heifers received 2 × 108 inclusion forming units (IFU) W. chondrophila intravenously on day 105–110 of pregnancy, while four negative-control animals underwent mock challenge. Only one of the challenged animals showed pathogen-associated lesions, with the organism being detected in the diseased placenta. Importantly, the organism was re-isolated and its identity confirmed by whole genome sequencing, confirming Koch’s third and fourth postulates. However, while infection of the placenta was observed, the experimental challenge in this study did not confirm the abortifacient potential of the organism

Proteolysis-inducing factor core peptide mediates dermcidin-induced proliferation of hepatic cells through multiple signalling networks

Dermcidin is a candidate oncogene capable of increasing the number of cultured neuronal, breast cancer and prostate cancer cells and improving the survival of hepatic cells. The dermcidin gene encodes the proteolysis-inducing factor core peptide (PIF-CP) and the skin antimicrobial peptide DCD-1. The peptide responsible for inducing proliferation of cells and the mechanisms involved are unknown. In this study, we confirmed a proliferative effect of dermcidin over-expression of 20% (p<0.02) in the HuH7 human hepatic cell line. Proliferation was abrogated by prevention of PIF-CP translation or inactivation of its calcineurin-like phosphatase domain by site-directed mutagenesis. Prevention of DCD-1 translation had no effect. Treatment of cells with a 30 amino acid synthetic PIF-CP induced an analogous increase in proliferation of 14%. Microarray analysis of PIF-CP-treated cells revealed low but significant changes in 111 potential mediator genes. Pathway analysis revealed several gene networks involved in the cellular response to the peptide, one with VEGFB as a hub and two other networks converging on FOS and MYC. Quantitative PCR confirmed direct upregula-tion of VEGFB. These data reveal PIF-CP as the key mediator of dermcidin-induced proliferation and demonstrate induction of key oncogenic pathways

A mechanistic investigation of the N-hydroxyphthalimide catalyzed benzylic oxidation mediated by sodium chlorite

A detailed investigation into the mechanistic course of N-hydroxyphthalimide catalyzed oxidation of benzylic centers using sodium chlorite as the stoichiometric oxidant is reported. Through a combination of experimental, spectroscopic, and computational techniques, the transformation is interrogated, providing improved reaction conditions and an enhanced understanding of the mechanism. Performing the transformation in the presence of acetic acid or a pH 4.5 buffer leads to extended reaction times but improves the catalyst lifetime, leading to the complete consumption of the starting material. Chlorine dioxide is identified as the active oxidant that is able to oxidize the N-hydroxyphthalimide anion to the phthalimide-N-oxyl radical, the proposed catalytically active species, which is able to abstract a hydrogen atom from the substrate. A second molecule of chlorine dioxide reacts with the resultant radical and, after loss of hypochlorous acid, leads to the observed product. Through a broad variety of techniques including UV/vis, EPR and Raman spectroscopy, isotopic labeling, and the use of radical traps, evidence for the mechanism is presented that is supported through electronic structural calculations

A mechanistic investigation of the N-hydroxyphthalimide catalyzed benzylic oxidation mediated by sodium chlorite

A detailed investigation into the mechanistic course of a N-hydroxyphthalimide catalyzed oxidation of benzylic centers using sodium chlorite as the stoichiometric oxidant is reported. Through a combination of experimental, spectroscopic and computational techniques the transformation is interrogated providing improved reaction conditions and an enhanced understanding of the mechanism. Performing the transformation in the presence of acetic acid or a pH 4.5 buffer leads to extended reaction times but improves the catalyst lifetime leading to complete consumption of starting material. Chlorine dioxide is identified as the active oxidant which is able to oxidize the N-hydroxyphthalimide anion to the phthalimide-N-oxyl radical, the proposed catalytically active species, which is able to abstract a hydrogen atom from the substrate. A second molecule of chlorine dioxide reacts with the resultant radical, and after loss of hypochlorous acid, leads to the observed product. Through a broad variety of techniques including UV/vis, EPR and Raman spectroscopy, isotopic labelling and the use of radical traps evidence for the mechanism is presented which is supported through electronic structural calculations

Efficacy of two Chlamydia abortus subcellular vaccines in a pregnant ewe challenge model for ovine enzootic abortion

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required

Intranasal infection with Chlamydia abortus induces dose-dependent latency and abortion in sheep

BACKGROUND: Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5x10(3), 5x10(5) and 5x10(7) inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2x10(6) IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50-67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium. CONCLUSIONS/SIGNIFICANCE: The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection

Pathogenic outcome following experimental infection of sheep with Chlamydia abortus variant strains LLG and POS

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50±60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines

Chlamydia in cases of bovine abortion in Ireland

We report the findings of a preliminary investigation into chlamydial involvement in cases of bovine abortion in Ireland. Chlamydia abortus has long been recognised as a known major cause of ruminant abortion. However, several newly recognised species of Chlamydia-related organisms have emerged as putative cattle abortifacients