The endogenous antiseptic N-chlorotaurine irreversibly inactivates Chlamydia pneumoniae and Chlamydia trachomatis.

PURPOSE: The antimicrobial activity of N-chlorotaurine (NCT), an endogenous long-lived oxidant applied topically, was tested against Chlamydiae in vitro. METHODOLOGY: Elementary bodies of Chlamydia pneumoniae strain CV-6 and Chlamydia trachomatis serovars A and D were incubated in 0.01, 0.1 and 1 % (w/v) NCT solution at pH 7.1 and 37 °C. After different incubation times, aliquots were removed and grown in cell culture. The number of inclusion forming units was quantified by immunofluorescence and real-time qPCR.Results/Key findings.Chlamydia pneumoniae and Chlamydia trachomatis were inactivated by 1 and 0.1 % NCT within 1 min. Moreover, 0.025-0.1 % NCT significantly reduced the number of intracellularly growing C. pneumoniae within 30 min. CONCLUSIONS: This is the first study demonstrating the antimicrobial activity of NCT against Chlamydiae. Clinical implications of these findings have to be investigated in further trials.

Reduction of eddy current losses in inductive transmission systems with ferrite sheets.

BACKGROUND: Improvements in eddy current suppression are necessary to meet the demand for increasing miniaturization of inductively driven transmission systems in industrial and biomedical applications. The high magnetic permeability and the simultaneously low electrical conductivity of ferrite materials make them ideal candidates for shielding metallic surfaces. For systems like cochlear implants the transmission of data as well as energy over an inductive link is conducted within a well-defined parameter set. For these systems, the shielding can be of particular importance if the properties of the link can be preserved. RESULTS: In this work, we investigate the effect of single and double-layered substrates consisting of ferrite and/or copper on the inductance and coupling of planar spiral coils. The examined link systems represent realistic configurations for active implantable systems such as cochlear implants. Experimental measurements are complemented with analytical calculations and finite element simulations, which are in good agreement for all measured parameters. The results are then used to study the transfer efficiency of an inductive link in a series-parallel resonant topology as a function of substrate size, the number of coil turns and coil separation. CONCLUSIONS: We find that ferrite sheets can be used to shield the system from unwanted metallic surfaces and to retain the inductive link parameters of the unperturbed system, particularly its transfer efficiency. The required size of the ferrite plates is comparable to the size of the coils, which makes the setup suitable for practical implementations. Since the sizes and geometries chosen for the studied inductive links are comparable to those of cochlear implants, our conclusions apply in particular to these systems.

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis.

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.

Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae.

BACKGROUND: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. RESULTS: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. CONCLUSION: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the ß-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Investigation of bacterial and viral agents and immune status in Behcet's disease patients from Iran.

AIM: Behçet's disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. METHODS: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. RESULTS: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. CONCLUSION: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

Sequence homologies between Mycoplasma and Chlamydia spp. lead to false-positive results in chlamydial cell cultures tested for mycoplasma contamination with a commercial PCR assay.

Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.

Design and synthesis of 2-arylbenzimidazoles and evaluation of their inhibitory effect against Chlamydia pneumoniae.

Chlamydia pneumoniae is an intracellular bacterium that responds poorly to antibiotic treatment. Insufficient antibiotic usage leads to chronic infection, which is linked to disease processes of asthma, atherosclerosis, and Alzheimer's disease. The Chlamydia research lacks genetic tools exploited by other antimicrobial research, and thus other approaches to drug discovery must be applied. A set of 2-arylbenzimidazoles was designed based on our earlier findings, and 33 derivatives were synthesized. Derivatives were assayed against C. pneumoniae strain CWL-029 in an acute infection model using TR-FIA method at a concentration of 10 µM, and the effects of the derivatives on the host cell viability were evaluated at the same concentration. Fourteen compounds showed at least 80% inhibition, with only minor changes in host cell viability. Nine most potential compounds were evaluated using immunofluorescence microscopy on two different strains of C. pneumoniae CWL-029 and CV-6. The N-[3-(1H-benzimidazol-2-yl)phenyl]-3-methylbenzamide (42) had minimal inhibitory concentration (MIC) of 10 µM against CWL-029 and 6.3 µM against the clinical strain CV-6. This study shows the high antichlamydial potential of 2-arylbenzimidazoles, which also seem to have good characteristics for lead compounds.

Inhibitory effect of the natural product betulin and its derivatives against the intracellular bacterium Chlamydia pneumoniae.

Chlamydia pneumoniae is a universal pathogen that has been indicated to play a part in the development of asthma, atherosclerosis and lung cancer. The complete eradication of this intracellular bacterium is in practice impossible with the antibiotics that are currently in use and studies on new antichlamydial compounds is challenging because Chlamydia research lacks the tools required for the genetic modification of this bacterium. Betulin is a natural lupane-class triterpene derived from plants with a wide variety of biological activities. This compound group thus has wide medical potentials, and in fact has been shown to be active against intracellular pathogens. For this reason, betulin and its derivatives were selected to be assayed against C. pneumoniae in the present study. Thirty-two betulin derivatives were assayed against C. pneumoniae using an acute infection model in vitro. Five promising compounds with potential lead compound characteristics were identified. Compound 24 (betulin dioxime) gave a minimal inhibitory concentration (MIC) of 1 microM against strain CWL-029 and showed activity in nanomolar concentrations, as 50% inhibition was achieved at 290 nM. The antichlamydial effect of 24 was confirmed with a clinical isolate CV-6, showing a MIC of 2.2 microM. Previous research on betulin and its derivatives has not identified such a remarkable inhibition of Gram-negative bacterial growth. Furthermore, we also demonstrated that this antichlamydial activity was not due to PLA(2) (EC 3.1.1.4) inhibition caused by the betulin derivatives.

Chlamydia pneumoniae adversely modulates vascular cell properties by direct interaction with signalling cascades.

Due to its dependence on intracellular development Chlamydia pneumoniae has developed numerous strategies to create an adequate environment within its host cells ensuring both chlamydial reproduction and target cell survival. The bacterium that has been related to atherogenesis due to its presence in vascular tissue is able to enter a persistent state of chronic infection in the vasculature that escapes antibiotic targeting. Ingestion of the bacterium results in severe modifications and reprogramming of signalling pathways and the metabolism of the host cell. Processes range from the prevention of direct lysosomal destruction of chlamydial inclusions to the inhibition of host cell apoptosis and an enhanced cellular glucose uptake to maintain energy-consuming mechanisms. Furthermore, infection regularly causes the development of a proinflammatory and proproliferative phenotype in the host cell in vitro, ex vivo and in vivo and own new findings suggest a detrimental proliferative loop within vascular cells upon a modified endothelin-1 axis demonstrating a potential for proatherosclerotic processes in early and progressed atherosclerosis. This review displays crucial mechanisms of Chlamydia pneumoniae-induced interactions with vascular host cell signalling cascades with an emphasis on mitogenic and inflammatory processes as well as target cell activation.

Proliferative stimulation of the vascular Endothelin-1 axis in vitro and ex vivo by infection with Chlamydia pneumoniae.

Endothelin-1 (ET-1) is a vasoactive peptide that modifies vascular function via the G-protein coupled transmembrane receptors, Endothelin-A receptor (ETAR) and Endothelin-B receptor (ETBR). Dysregulation of the ET-1 axis plays a role in atherosclerotic development as it triggers cell proliferation, inflammation, and vasoconstriction. The respiratory pathogen Chlamydia pneumoniae (Cp) has been recovered from atherosclerotic lesions, and related to atherogenesis, via activation of vascular small GTPases and leukocyte recruitment. Cp effectively reprograms host cell signalling and is able to enter an intracellular persistent state in vascular cells that is refractory to antibiotics. Upon chlamydial infection, vascular smooth muscle cells, which do not produce significant ET-1 under physiological conditions were switched into a fundamental source of ET-1 mRNA and protein in a p38-MAP-kinase-dependent pathway. Endothelial cells did not overproduce ET-1 but showed upregulation of mitogenic ETAR mRNA and protein while the counterbalancing ETBR, which regulates ET-1 clearance, remained unaffected. This disruption of the ET-1 axis was confirmed in an ex vivo mouse aortic ring model, and resulted in endothelial cell proliferation that could be abrogated by ETAR-siRNA and the selective ETAR-antagonist BQ-123. Chronic chlamydial infection of the vascular wall might represent a permanent noxious stimulus linked to the endothelial cell proliferation characteristic of early atherosclerosis. Suppression of this deleterious paracrine loop by ETAR antagonism opens up a new option of preventing possible vascular sequelae of otherwise untreatable chronic chlamydial infection. In conclusion, this is the first study to demonstrate infection to dysregulate the ET-1 axis towards inducing a proatherogenic proliferative phenotype.

Chlamydia pneumoniae-induced pathological signaling in the vasculature.

Since its description in 1986, Chlamydia pneumoniae has remained one of the most enigmatic pathogens. This intracellular bacterium is highly seroprevalent, but rarely recovered from cell culture, it can genetically switch between a proliferative and a nonreplicative state and has been linked to a vast number of chronic diseases, most notably to atherosclerosis, as it can be found in the plaques. It has become quite clear that persistent bacteria in atherosclerotic lesions cannot be eradicated by currently available antibiotic treatments and that attempts to do so without a better understanding of the pathobiology of chlamydial persistence are futile. However, there is growing knowledge on how vascular chlamydial infection may lead to the pathological reprogramming of the host cell signaling pathways. Chlamydia pneumoniae is now well known to induce, at least in vitro, the two pathogenetic main events that define atherosclerosis: angiogenesis and inflammation. In vivo a contribution of chlamydial infection to the progression of atherosclerosis remains unproven. This minireview provides a brief overview on the proproliferative and proinflammatory effects of vascular C. pneumoniae infection and their potential link to atherogenesis.

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Genetic diversity of the obligate intracellular bacterium Chlamydophila pneumoniae by genome-wide analysis of single nucleotide polymorphisms: evidence for highly clonal population structure.

BACKGROUND: Chlamydophila pneumoniae is an obligate intracellular bacterium that replicates in a biphasic life cycle within eukaryotic host cells. Four published genomes revealed an identity of > 99 %. This remarkable finding raised questions about the existence of distinguishable genotypes in correlation with geographical and anatomical origin. RESULTS: We studied the genetic diversity of C. pneumoniae by analysing synonymous single nucleotide polymorphisms (sSNPs) that are under reduced selection pressure. We conducted an in silico analysis of the four sequenced genomes, chose 232 representative sSNPs and analysed the loci in 38 C. pneumoniae isolates. We identified 15 different genotypes that were separated in four major clusters. Clusters were not associated with anatomical or geographical origin. However, animal lineages are basal on the C. pneumomiae phylogeny, suggesting a recent transmission to humans through successive bottlenecks some 150,000 years ago. A lack of detectable variation in 17 isolates emphasizes the extraordinary genetic conservation of this species and the high clonality of the population. Moreover, the largest cluster, which encompasses 80% of all analysed strains, is an extremely young clade, that went through an important population expansion some 3,300 years ago. CONCLUSION: sSNPs have proven useful as a sensitive marker to gain new insights into genetic diversity, population structure and evolutionary history of C. pneumoniae.

Chlamydia pneumoniae directly interferes with HIF-1alpha stabilization in human host cells.

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1alpha regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1alpha stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1alpha degradation. HIF-1alpha was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1alpha stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen-host interaction in chlamydial diseases.

Recurrent optic neuritis associated with Chlamydia pneumoniae infection of the central nervous system.

It has been suggested that Chlamydia pneumoniae (C. pneumoniae) is involved in the pathogenesis of diverse diseases of the central nervous system (CNS), including multiple sclerosis. We report the case of a 12-year-old male with isolated recurrent optic neuritis and an associated CNS infection with C. pneumoniae. The patient presented with three attacks of optic neuritis within 5 months. A positive polymerase chain reaction for C. pneumoniae in the cerebrospinal fluid led to the diagnosis of a CNS infection with C. pneumoniae. After treatment with the antibiotic rifampicin, he experienced no further attacks during the follow-up period of 6 years. These findings suggest the possibility of a C. pneumoniae infection as a contributing factor or even causative event for the development of optic neuritis.

Mechanisms of Chlamydophila pneumoniae-mediated GM-CSF release in human bronchial epithelial cells.

Chlamydophila pneumoniae is an important respiratory pathogen. In this study we characterized C. pneumoniae strain TW183-mediated activation of human small airway epithelial cells (SAEC) and the bronchial epithelial cell line BEAS-2B and demonstrated time-dependent secretion of granulocyte macrophage colony-stimulating factor (GM-CSF) upon stimulation. TW183 activated p38 mitogen-activated protein kinase (MAPK) in epithelial cells. Kinase inhibition by SB202190 blocked Chlamydia-mediated GM-CSF release on mRNA and protein levels. In addition, the chemical inhibitor as well as dominant-negative mutants of p38 MAPK isoforms p38alpha, beta2, and gamma inhibited C. pneumoniae-related NF-kappaB activation. In contrast, blocking of MAPK ERK, c-Jun kinase/JNK, or PI-3 Kinase showed no effect on Chlamydia-related epithelial cell GM-CSF release. Ultraviolet-inactivated pathogens as compared with viable bacteria induced a smaller GM-CSF release, suggesting that viable Chlamydiae were only partly required for a full effect. Presence of an antichlamydial outer membrane protein-A (OmpA) antibody reduced and addition of recombinant heat-shock protein 60 from C. pneumoniae (cHsp60, GroEL-1)-enhanced GM-CSF release, suggesting a role of these proteins in epithelial cell activation. Our data demonstrate that C. pneumoniae triggers an early proinflammatory signaling cascade involving p38 MAPK-dependent NF-kappaB activation, resulting in subsequent GM-CSF release. C. pneumoniae-induced epithelial cytokine liberation may contribute significantly to inflammatory airway diseases like chronic obstructive pulmonary disease (COPD) or bronchial asthma.

Critical investigation of the CD14 promoter polymorphism: lack of a role for in vitro cytokine response and membrane CD14 expression.

Blood of volunteers, genotyped for the CD14 C(-159)-->T polymorphism, showed no difference in cytokine release when stimulated with nine CD14-dependent immune stimuli. An analysis of the published data on the proposed association of CD14 genotype with membrane CD14 density revealed no significant correlation, questioning a functional impact of the CD14 polymorphism.