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We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis , C. muridarum , C. caviae , C. pecorum , and C. abortus However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis , indicating the potential of horizontal pathogenic gene transfer via a plasmid. IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species., (Copyright © 2018 Shima et al.)

Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O 2 ≈ 20%) and hypoxic conditions (O 2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae .

Community-acquired pneumonia is caused by intra- and extracellular bacteria, with some of these bacteria also being linked to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease. Chlamydia pneumoniae is an obligate intracellular pathogen that is highly sensitive to micro-environmental conditions controlling both pathogen growth and host immune responses. The availability of nutrients, as well as changes in oxygen, pH and interferon-γ levels, have been shown to directly influence the chlamydial life cycle and clearance. Although the lung has been traditionally regarded as a sterile environment, sequencing approaches have enabled the identification of a large number of bacteria in healthy and diseased lungs. The influence of the lung microbiota on respiratory infections has not been extensively studied so far and data on chlamydial infections are currently unavailable. In the present study, we speculate on how lung microbiota might interfere with acute and chronic infections by focusing exemplarily on the obligate intracellular C. pneumoniae. Furthermore, we consider changes in the gut microbiota as an additional player in the control of lung infections, especially in view the increasing evidence suggesting the involvement of the gut microbiota in various immunological processes throughout the human body., (© 2016 Federation of European Biochemical Societies.)

The obligatory intracellular bacterium Chlamydia pneumoniae (C. pneumoniae) can survive and multiply in neutrophil granulocytes. Since neutrophils are short living cells, inhibition of neutrophil apoptosis appears to play a major role in the productive infection of neutrophils by C. pneumoniae. In the present study, we have investigated which survival pathways and which events of the apoptotic process are modulated in C. pneumoniae-infected neutrophils. All infection experiments were carried out using primary human neutrophils in vitro. We show that infection with C. pneumoniae activates PI3K/Akt as well as the ERK1/2 and p38 MAP kinases and present evidence that activation of the PI3K/Akt and ERK1/2 pathways are essential to initiate the apoptosis delay in C. pneumoniae-infected neutrophils. Both the PI3K/Akt and ERK1/2 pathways are involved in the maintained expression of the anti-apoptotic protein Mcl-1. In addition, we also showed that the PI3K/Akt pathway leads to the activation of NF-κB-dependent release of IL-8 by infected neutrophils. Infection with C. pneumoniae activates the PI3K/Akt and ERK1/2 MAPK survival pathways in neutrophils, induces the NF-κB dependent release of IL-8 and leads to the maintenance of Mcl-1 expression in neutrophils., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN-γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN-γ-induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN-γ-induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN-γ-induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor-2α (eIF2α) and down-regulation of the vesicle-associated membrane protein-associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN-γ-induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER-related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN-γ-induced persistent infection., (© 2015 John Wiley & Sons Ltd.)

Chlamydia pneumoniae is a Gram-negative bacterium that causes acute or chronic respiratory infections. As obligate intracellular pathogens, chlamydiae efficiently manipulate host cell processes to ensure their intracellular development. Here we focused on the interaction of chlamydiae with the host cell transcription factor activator protein 1 (AP-1) and its consequence on chlamydial development. During Chlamydia pneumoniae infection, the expression and activity of AP-1 family proteins c-Jun, c-Fos, and ATF-2 were regulated in a time- and dose-dependent manner. We observed that the c-Jun protein and its phosphorylation level significantly increased during C. pneumoniae development. Small interfering RNA knockdown of the c-Jun protein in HEp-2 cells reduced the chlamydial load, resulting in smaller inclusions and significantly lower chlamydial recovery. Furthermore, inhibition of the c-Jun-containing AP-1 complexes using tanshinone IIA changed the replicative infection phenotype into a persistent one. Tanshinone IIA-dependent persistence was characterized by smaller, aberrant inclusions, a strong decrease in the chlamydial load, and significantly reduced chlamydial recovery, as well as by the reversibility of the reduced recovery after the removal of tanshinone IIA. Interestingly, not only was tanshinone IIA treatment accompanied by a significant decrease of ATP levels, but fluorescence live cell imaging analysis by two-photon microscopy revealed that tanshinone IIA treatment also resulted in a decreased fluorescence lifetime of protein-bound NAD(P)H inside the chlamydial inclusion, indicating that chlamydial reticulate bodies have decreased metabolic activity. In all, these data demonstrate that the AP-1 transcription factor is involved in C. pneumoniae development, with tanshinone IIA treatment resulting in persistence., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Chlamydia pneumoniae (Cpn) are obligate intracellular bacteria that cause acute infections of the upper and lower respiratory tract and have been implicated in chronic inflammatory diseases. Although of significant clinical relevance, complete genome sequences of only four clinical Cpn strains have been obtained. All of them were isolated from the respiratory tract and shared more than 99% sequence identity. Here we investigate genetic differences on the whole-genome level that are related to Cpn tissue tropism and pathogenicity., Results: We have sequenced the genomes of 18 clinical isolates from different anatomical sites (e.g. lung, blood, coronary arteries) of diseased patients, and one animal isolate. In total 1,363 SNP loci and 184 InDels have been identified in the genomes of all clinical Cpn isolates. These are distributed throughout the whole chlamydial genome and enriched in highly variable regions. The genomes show clear evidence of recombination in at least one potential region but no phage insertions. The tyrP gene was always encoded as single copy in all vascular isolates. Phylogenetic reconstruction revealed distinct evolutionary lineages containing primarily non-respiratory Cpn isolates. In one of these, clinical isolates from coronary arteries and blood monocytes were closely grouped together. They could be distinguished from all other isolates by characteristic nsSNPs in genes involved in RB to EB transition, inclusion membrane formation, bacterial stress response and metabolism., Conclusions: This study substantially expands the genomic data of Cpn and elucidates its evolutionary history. The translation of the observed Cpn genetic differences into biological functions and the prediction of novel pathogen-oriented diagnostic strategies have to be further explored.

Chlamydial infections cause a wide range of acute and chronic diseases. Chlamydia trachomatis is the most common sexually transmitted bacterium while Chlamydia pneumoniae causes infections of the upper and lower respiratory tract. Chlamydia are obligate, intracellular bacteria with a biphasic developmental cycle that involves unique metabolic changes. Aside from entering an actively replicating state, Chlamydia may also implement persistent infections depending on different microenvironmental factors. In addition, changes in local oxygen availability and the composition of surrounding host microbiota are suggested to affect chlamydial growth and metabolism. Both bacteria and host cells endure characteristic metabolic changes during infection. Technical developments in recent years enable us to separately characterize chlamydial and host cell metabolism in living cells. This article focuses on novel approaches to analyze chlamydial metabolism such as NAD(P)H fluorescence lifetime imaging by two-photon microscopy. In addition, we provide an overview regarding promising future possibilities to further elucidate host-pathogen metabolic interactions.

Chlamydia pneumoniae infections of the respiratory tract are common and are associated with acute and chronic diseases such as community-acquired pneumonia (CAP) and chronic obstructive pulmonary disease (COPD). Recent studies have shown that reduced environmental oxygen availability promotes chlamydial growth in infected host cells. The underlying mechanisms remain unclear. We performed a targeted siRNA screen coupled with an automated high-throughput microscopic analysis to identify key host cell genes that play a role in promoting the hypoxic growth of C. pneumoniae. A total of 294 siRNAs - targeting 98 selected genes including central mediators of metabolic, trafficking and signaling pathways - were tested on chlamydial inclusion formation in C. pneumoniae infected A549 cells under normoxic (20% O2) and hypoxic (2% O2) conditions 48 h post infection. Evaluation of the different functional clusters of genes revealed that under hypoxic conditions, enhanced growth of C. pneumoniae was centrally mediated by the host cell glycolytic pathway. Inhibition of the phosphofructokinase (PFK), lactate dehydrogenase (LDH), glycerol-3-phosphate dehydrogenase (GPD2) and the forkheadbox O3 (FOXO3) gene-expression by siRNAs abrogated chlamydial progeny. The pivotal role of host cell glycolysis in chlamydial development under hypoxia was further confirmed by pharmacological inhibition of the pathway by 2-fluoro-deoxy-glucose. The results indicate that the microenvironment of the host cell determines the fate of C. pneumoniae by controlling pathogen-induced metabolic pathways., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Infections with Chlamydia pneumoniae cause several respiratory diseases, such as community-acquired pneumonia, bronchitis or sinusitis. Here, we present an integrated non-targeted metabolomics analysis applying ultra-high-resolution mass spectrometry and ultra-performance liquid chromatography mass spectrometry to determine metabolite alterations in C. pneumoniae-infected HEp-2 cells. Most important permutations are elaborated using uni- and multivariate statistical analysis, logD retention time regression and mass defect-based network analysis. Classes of metabolites showing high variations upon infection are lipids, carbohydrates and amino acids. Moreover, we observed several non-annotated compounds as predominantly abundant after infection, which are promising biomarker candidates for drug-target and diagnostic research.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, whereby it is customary to distinguish between early familial FAD and late-onset AD (LOAD). The development of LOAD, the most prevalent form of AD, is believed to be a multifactorial process that may also involve infections with bacterial or viral pathogens. After the first report on the presence of Chlamydia pneumoniae (Cpn) in brains of patients with AD appeared in 1998, this bacterium has most often been implicated in AD pathogenesis. However, while some studies demonstrate a clear association between Cpn infection and AD, others have failed to confirm these findings. This might be due to heterogeneity of the specimens analyzed and lack of standardized detection methods. Additionally, non-availability of suitable chlamydial infection models severely hampers research in the field. In this review, we will critically discuss the possible role of Cpn in the pathogenesis of LOAD in light of the available data. We will also present three mutually non-exclusive hypotheses how Cpn might contribute to the pathogenesis of AD.

Chlamydia pneumoniae is an obligatory intracellular bacterium causing chronic inflammatory diseases in humans. We studied the role of the nutritive factors, iron and tryptophan, towards the course of infection and immune response pathways in C. pneumoniae infected endothelial cells and monocytes. Human endothelial (EA.hy923) and monocytic cells (THP-1) were infected with C. pneumoniae, supplemented with iron or 1-methyltryptophan (1-MT), an inhibitor of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO), and subsequently stimulated with IFN-gamma or left untreated. The number of infected cells, the morphology and quantity of C. pneumoniae inclusion bodies, IDO activity and innate immune effector pathways were analysed. While neither iron challenge, IDO inhibition or IFN-gamma treatment had a significant effect on C. pneumoniae morphology or numbers within THP-1 monocytic cells, iron supplementation to EA.hy926 cells resulted in promotion of C. pneumoniae proliferation and differentiation while IFN-gamma had an inhibitory effect. Furthermore, the number of infected endothelial cells was significantly decreased upon 1-MT treatment. C. pneumoniae infection induced a pro-inflammatory immune response as evidenced by increased IDO activity, neopterin formation or TNF-alpha production in THP-1 but not in endothelial cells. These pathways were superinduced upon IFN-gamma treatment and partly modulated by iron supplementation. Our results demonstrate that the infectious cycle of C. pneumoniae behaves differently between monocytic and endothelial cells. While the intracellular pathogen remains in a persistent form within monocytes, it can differentiate and proliferate within endothelial cells indicating that endothelial cells are a preferred environment for Chlamydia. Nutritive factors such as iron have subtle effects on C. pneumoniae biology in endothelial, but not monocytic cells. Our results contribute to a better understanding of C. pneumoniae infection and its role in chronic inflammatory diseases such as atherosclerosis., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Chlamydia pneumoniae is a frequent pathogen of the respiratory tract, and persistent infections with this obligate intracellular bacterium have been associated with different severe sequelae. Although T-cell activation during acute C. pneumoniae infections has been described, little is known about the frequency or the role of the C. pneumoniae-specific memory T cells that reside in the human body after the resolution of the infection. In the present study, the C. pneumoniae-induced T-cell responses in peripheral blood mononuclear cells of 56 healthy volunteers were analyzed and compared to the donor's serum antibody reactivity toward whole C. pneumoniae as well as recombinant C. pneumoniae antigens. Following short-term stimulation with C. pneumoniae, both gamma interferon (IFN-gamma)- and interleukin-2 (IL-2)-producing CD4(+) T-cell responses could be detected in 16 of 56 healthy individuals. C. pneumoniae-activated CD4(+) T cells expressed CD154, a marker for T-cell receptor-dependent activation, and displayed a phenotype of central memory T cells showing dominant IL-2 production but also IFN-gamma production. Interestingly, individuals with both IFN-gamma- and IL-2-producing responses showed significantly decreased immunoglobulin G reactivity toward C. pneumoniae RpoA and DnaK, antigens known to be strongly upregulated during chlamydial persistence, compared to IgG reactivity of seropositive individuals with no T-cell response or CD4(+) T-cell responses involving the production of a single cytokine (IFN-gamma or IL-2). Our results demonstrate that memory CD4(+) T cells responding to C. pneumoniae stimulation can be detected in the circulation of healthy donors. Furthermore, among seropositive individuals, the presence or the absence of dual IFN-gamma- and IL-2-producing T-cell responses was associated with distinct patterns of antibody responses toward persistence-associated C. pneumoniae antigens.

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.

Background: Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae., Methodology/principal Findings: We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ss production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-alpha response., Conclusions/significance: Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.

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.

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.

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.

The Chlamydia pneumoniae bacteriophage was first identified in isolate AR-39. Its relevance for chlamydial biology and pathogenicity remains unknown. In this study, a collection of 36 C. pneumoniae isolates was screened and the phage was detected in eight. As the positive isolates differed by several polymorphisms, they presumably belonged to different genetic lineages. It was investigated whether different genotypes of the phage also existed and whether they could be assigned to chlamydial genotypes as evidence of coevolution. Sequencing of >3000 bp of the 4524 bp phage genome revealed complete identity to the published sequences. Thus, it was hypothesized that the genetic conservation was related to easy transmissibility of the phage between C. pneumoniae isolates. Cocultivation of phage positive and negative isolates followed by cloning and identification of different C. pneumoniae genotypes demonstrated for the first time transmissibility of the bacteriophage from one isolate to the other. These observations indicate that the phage is capable of infecting C. pneumoniae isolates of different genetic backgrounds and suggest that all C. pneumoniae strains might be susceptible. The successful in vitro infection of C. pneumoniae with the phage provides the basis for studying its pathogenetic relevance in isolates of identical genetic background and provides a potential tool for genetic manipulation of C. pneumoniae.

Rationale: Chlamydia pneumoniae (Cpn) infection may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available comparing persistent and acute infection of this pathogen in the human respiratory tract., Objectives: To study Cpn-induced innate immune responses in lung tissue from patients with COPD and control subjects ex vivo and in vitro., Methods: Cpn detection was done by nested polymerase chain reaction, in situ hybridization, and immunohistochemistry ex vivo in unstimulated tissue and in vitro using an acute Cpn infection model. As main endpoints for the assessment of early cellular responses, nuclear factor (NF)-kappaB activation and CXC chemokine ligand (CXCL)-8 expression were evaluated. The role of Toll-like receptors (TLRs) as recognition molecules in Cpn-induced innate responses was tested by blocking experiments., Measurements and Main Results: Fifteen percent of patients with COPD were chronically infected with Cpn in contrast to 0% of control subjects (p < 0.05). There were no differences in CXCL-8 and NF-kappaB expression between infected and noninfected COPD tissue ex vivo. In contrast, acute in vitro infection induced an intense innate immune response including up-regulation of TLR2. Blocking experiments demonstrated the predominant role of TLR2 in induction of the early immune response, whereas no influence on chlamydial infection rates was observed., Conclusions: Acute in vitro infection of human lung tissue with Cpn elicited a marked innate response via TLR2, whereas chronic chlamydial infection in patients with COPD was not associated with enhanced cellular activation. These findings suggest different roles of Cpn during acute and chronic stages of pulmonary infection.

Chlamydophila pneumoniae alter the expression of Toll-like receptor (TLR) 4 in alveolar type II (ATII)-cells. Subsequently nuclear factor kappaB (NF-kappaB) is activated and tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) are produced. Perfluorocarbons (PFC) are beneficial in animals with bacterial pneumonia and reduce production of TNF-alpha. Using isolated ATII-cells, it was studied whether PFC prevent C. pneumoniae-induced TNF-alpha and MIP-2 release and what the underlying pathway is. PF5080 preincubation prevented C. pneumoniae-induced secretion of TNF-alpha (43 +/- 10 versus 661 +/- 41 pg/mL) and MIP-2 (573 +/- 41 versus 4786 +/- 502 pg/mL). The C. pneumoniae-induced 2.2-fold increase of TNF-alpha Receptor 1 expression was reduced by PF5080. C. pneumoniae reduced cytoplasmatic IkappaBalpha (3.7 +/- 0.3 versus 14 +/- 1) and increased NF-kappaB p65 (31 +/- 7.5 versus 3.6 +/- 1.1) compared with control. PF5080 prevented NF-kappaB activation. TLR4 expression was 1.5-fold higher after C. pneumoniae incubation, but remained at control levels after PF5080 pretreatment. After 24 h of C. pneumoniae incubation, in 88 +/- 6% of cells bacteria were found in the perinuclear region and in 50% of these cells bacteria adhered to cellular surface. After PF5080 preincubation, C. pneumoniae were in 32 +/- 4% attached to and in 5 +/- 1% internalized in ATII-cells. Since PF5080 was found in ATII-cell membranes, PF5080 effect could be explained by an alteration of the cellular membrane, preventing activation of the inflammatory cascade.

Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

Chlamydophila (Chlamydia) pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen. Chronic C. pneumoniae infection has been suggested as a trigger/promoter of inflammation that may result in vascular lesions. Although the genome of C. pneumoniae has been sequenced completely this information has not yet led to an understanding of the mechanisms of acute infection and target cell activation nor to the identification of potential chlamydial virulence factors. Intriguingly, current antibiotic treatment options for acute chlamydial infection were proven to be ineffective with respect to clinical outcome in different groups of atherosclerotic patients. The reason might be that primary infection of vascular smooth muscle cells and blood monocytes with C. pneumoniae resembles rather a persistent, antibiotic-resistant, than an active infection. In this review we will focus on the importance of putative host cell receptors for C. pneumoniae and subsequently activated signal transduction pathways.

Atherosclerosis is characterized by inflammation and proliferation of vascular cells. The intracellular bacterium Chlamydia (Chlamydophila) pneumoniae uses blood monocytes [peripheral blood mononuclear cells (PBMCs)] for dissemination, has been found to persist in atherosclerotic lesions, and has been implicated in atherogenesis by small GTPase activation and T lymphocyte recruitment. Infection of human coronary artery smooth muscle cells with C. pneumoniae significantly induced mRNA and protein for the angiogenic transcription factor Egr-1, resulting in enhanced coronary artery smooth muscle cell proliferation, which was reduced by transfection with small interfering RNA duplexes targeted at Egr-1 mRNA. These effects required viable chlamydiae and depended on p44/42 mitogen-activated protein kinase activity but not on the p38 mitogen-activated protein kinase pathway. Postinfectious Egr-1 mRNA up-regulation in arterial vessels was confirmed ex vivo in a rat aortic ring model of focal vascular chlamydial infection. An in vivo model based on the injection of C. pneumoniae-infected PBMCs into mice confirmed Egr-1 mRNA up-regulation within 24 h of endovascular infection. Arterial injury from repeated direct chlamydial infections and cell-to-cell contact with C. pneumoniae-infected PBMCs might represent a chronic focus of proliferative activity linked to the media proliferation seen in advanced atherosclerosis. Overall, chlamydial infection induces a proliferative phenotype in vascular cells via transcription factor Egr-1 activation in vitro, ex vivo, and in vivo.

Objective: Chlamydia pneumoniae infection has been associated with atherosclerosis, but serodiagnosis is unreliable in predicting vascular infection. Direct detection of circulating chlamydial DNA in peripheral blood mononuclear cells (PBMCs) was thus evaluated as a marker for cardiovascular risk in a general population survey using the common carotid intima-media thickness (IMT) as surrogate marker of asymptomatic atherosclerosis., Methods and Results: C pneumoniae DNA in PBMCs was determined by nested polymerase chain reaction and associated with IMT for 1032 healthy participants of a general population survey who were within the highest or lowest IMT distribution quartile. C pneumoniae DNA was more prevalent in those with increased IMT (13.4% versus 10.7%), but this was not significant in univariate and of borderline significance in multivariate analysis. Testing for potential effect modifications by known strong determinants of an increased IMT in group interaction analysis revealed an independent association between C pneumoniae DNA and IMT in normotensive subjects (odds ratio [OR], 2.06; 95% CI, 1.05 to 4.03; P=0.04) and in those <70 years old (OR, 1.84; 95% CI, 1.06 to 3.19; P=0.03)., Conclusions: Asymptomatic atherosclerosis is associated with circulating C pneumoniae DNA independently of classical cardiovascular risk factors in normotensive subjects and those <70 years old. C pneumoniae has been implicated in atherogenesis. We determined the association of chlamydial DNA in peripheral blood mononuclear cells with the carotid intima-media thickness from 1032 healthy subjects from a general population survey. A stratified group interaction analysis revealed an independent association in normotensive subjects and those <70 years old.

Chlamydia pneumoniae uses blood monocytes (PBMC) for systemic dissemination, persists in atherosclerotic lesions, and has been implicated in the pathogenesis of atherosclerosis. During transmigration in a newly developed transendothelial migration model (TEM) C. pneumoniae-infected PBMC spread their infection to endothelial cells. Transmigrated PBMC retained their infectivity and transmitted the pathogen to smooth muscle cells in the lower chamber of the TEM. Detection of chlamydial HSP60 mRNA proved pathogen viability and virulence. We conclude that PBMC can spread chlamydial infection to vascular wall cells and we suggest the TEM as a novel tool to analyze host-pathogen interactions in vascular chlamydial infections.

Quinolone resistance of Chlamydia pneumoniae has not been described previously. Serial subcultures of C. pneumoniae under increasing moxifloxacin concentrations (0.0125 to 6.4 mg/liter) resulted in a 256-fold MIC increase compared to moxifloxacin-naive strains. GyrA gene sequencing revealed a novel point mutation with a Ser-->Asn substitution. Subcultures under rifalazil and macrolides did not alter the respective MICs.

Chlamydia pneumoniae uses peripheral blood monocytes (PBMC) for systemic dissemination and has been linked to atherogenesis by inflammation mediated via TLR2/4 and CD14. We found 12.8% of 610 coronary artery disease (CAD) patients of Central European background to be chronically infected with C. pneumoniae based on the repeated detection of chlamydial DNA in PBMC. Among those the -159C>T CD14 promoter polymorphism was more frequent (OR 1.7, 95% CI 1.08-2.65, P=0.0224) than among C. pneumoniae-negative subjects matched for age and gender. The Arg753Gln TLR2 and Asp299Gly TLR4 polymorphisms were not related to chlamydial infection. Susceptibility for chronic chlamydial infection of PBMC in CAD patients appears associated with the CD14-159C>T promoter polymorphism encoding for enhanced CD14 expression.

Peripheral blood monocytes (PBMC) promote vascular inflammation and atherosclerosis. Chlamydia pneumoniae (Cp) infection of PBMC is found in atherosclerotic patients, appears refractory to antibiotics, and may predispose to vascular damage. In Cp-infected human PBMC we analyzed the role of cyclooxygenase-2 (Cox-2) for the proatherosclerotic key mediators prostaglandin E2 (PGE2) and interstitial collagenase (MMP-1). Cp infection resulted in rapid and sustained Cox-2 mRNA and protein stimulation depending on p38 and p44/42 MAPkinases. Subsequent upregulation of PGE synthase and MMP-1 was completely abrogated by the selective Cox-2 inhibitor NS398. Enhanced synthesis of PGE2 and MMP-1 in Cp infected PBMC is mediated through initiation of the p38 and p44/42 MAPK pathways and requires sustained Cox-2 activation. Selective Cox-2 inhibitors, currently under investigation for cardiovascular risk reduction, may represent a novel therapeutic option for patients with endovascular Cp infection as they target the actuated pathological signal transduction cascade in persistently infected PBMC.

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay. Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05). Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation. Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis.

Chlamydia pneumoniae, a major cause of community-acquired pneumonia, primarily infects the respiratory tract. Chronic infection of nonrespiratory sites, such as the vascular wall, the brain or blood monocytes, requires evasion from the lungs and spreading via the bloodstream. The cell types involved in dissemination are insufficiently characterised. In this study, New Zealand White rabbits were infected intratracheally with C. pneumoniae, and lung manifestation and systemic dissemination were monitored by polymerase chain reaction and immunohistochemistry. Infection of the lungs was characterised by an early phase dominated by granulocytes and a late phase dominated by alveolar macrophages (AM). Granulocytes, AM and alveolar epithelial cells acted as host cells for chlamydiae, which remained detectable for up to 8 weeks. AM transported the pathogen to the peribronchiolar lymphatic tissue, and subsequently C. pneumoniae entered the spleen and the aorta via dissemination by peripheral blood monocytes. In conclusion, Chlamydia pneumoniae-infected alveolar macrophages transmigrate through the mucosal barrier, and give the pathogen access to the lymphatic system and the systemic circulation. Infected peripheral blood monocytes are the vector system within the bloodstream and transmit the infection to the vascular wall. This is the first description of granulocytes acting as a reservoir for Chlamydia pneumoniae early in infection.

Persistent growth forms of Chlamydia pneumoniae have been associated with chronic infections in vivo. We investigated the effects of first-line therapeutics on the induction of persistence by monitoring recoverable organisms, gene expression of membrane proteins, and morphology. We found that all of the antibiotics tested have distinct and subinhibitory concentrations at which they induce persistence.

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes respiratory infections and has been associated with cardiovascular disease. We compared respiratory and cardiovascular isolates to find genetic differences associated with pathogenicity. A polymorphic region encoding a tyrosine/tryptophan permease was found to differ between disease isolates. Respiratory strains contained multiple copies of the tyrP gene, and vascular strains contained a single copy. Single-nucleotide polymorphism analysis revealed the duplication to be a phylogenetically old event. Gene amplification was associated with higher mRNA levels and higher uptake of the substrate tyrosine, indicating an amino-acid transport-related phenotype associated with the tyrP genotype. Vascular strains, despite their reduced ability to transport tyrosine, do not appear to have a reduced growth rate in vitro. We hypothesize that the important difference between strains of vascular and respiratory origin may lie in the increased tendency of vascular strains to elicit persistent infection that is triggered by amino-acid starvation.

Balanced secretion of pro- and anti-inflammatory cytokines is essential in limiting pulmonary inflammation in respiratory infections. It was hypothesised that, in acute infection with Chlamydia pneumoniae, mononuclear cells from chronic obstructive pulmonary disease (COPD) patients lack the opportunity to compensate for the inflammatory immune response by secreting adequate amounts of anti-inflammatory cytokines. Alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs) from eight COPD patients and eight healthy controls were infected with C. pneumoniae in order to determine interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1RA) and IL-8 expression and messenger ribonucleic acid levels. Secretion of IL-1beta was significantly enhanced in AMs (six-fold) and PBMCs (four-fold) from COPD patients after infection with C. pneumoniae. Compared to the control group, release of its anti-inflammatory counterpart IL-1RA was diminished in COPD patients, resulting in a significantly higher IL-1beta/IL-1RA ratio in C. pneumoniae-infected AMs and PBMCs from COPD patients. Mononuclear cells from chronic obstructive pulmonary disease patients have less capacity for balancing the pro-inflammatory immune response caused by Chlamydia pneumoniae infection than those from healthy controls. These findings suggest that, during acute exacerbation with intracellular pathogens, chronic obstructive pulmonary disease patients are predisposed to inflammatory changes in the lungs.