Inhibition of tRNA Synthetases Induces Persistence in Chlamydia.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.

Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes.

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via ß-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.

Amphipathic ß2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae.

We demonstrate in the current work that small cationic antimicrobial ß2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 µM, i.e. < 3.4 µg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the ß2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these ß2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic ß2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

Transforming activities of Chlamydia pneumoniae in human mesothelial cells

Knowledge in viral oncology has made considerable progress in the field of cancer fight. However, the role of bacteria as mediators of oncogenesis has not yet been elucidated. As cancer still is the leading cause of death in developed countries, understanding the long-term effects of bacteria has become of great importance as a possible means of cancer prevention. This study reports that Chlamydia pneumoniae infection induce transformation of human mesothelial cells. Mes1 cells infected with C. pneumoniae at a multiplicity of infection of 4 inclusion-forming units/cell showed many intracellular inclusion bodies. After a 7-day infection an increased proliferative activity was also observed. Real-time PCR analysis revealed a strong induction of calretinin, Wilms’ tumour gene 1, osteopontin, matrix metalloproteinases-2, and membrane-type 1 metal- matrix metalloproteinases-2, and membrane-type 1 metal- membrane-type 1 metalloproteinases gene expression in Mes1 cell, infected for a longer period (14 days). The results were confirmed by western blot analysis. Zymography analysis showed that C. pneumoniae modulated the in-vitro secretion of MMP-2 in Mes1 cells both at 7 and 14 days. Cell invasion, as measured by matrigel-coated filter, increased after 7 and 14 days infection with C. pneumoniae, compared with uninfected Mes1 cells. The results of this study suggest that C. pneumoniae infection might support cellular transformation, thus increasing lung cancer risk (AU)

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AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae.

Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation.

Roquefort cheese proteins inhibit Chlamydia pneumoniae propagation and LPS-induced leukocyte migration.

Inflammation in atherosclerosis, which could be associated with some subclinical infections such as C. pneumoniae, is one of the key factors responsible for the development of clinical complications of this disease. We report that a proprietary protein extract isolated from Roquefort cheese inhibits the propagation of C. pneumoniae in a human HL cell line in a dose-dependent manner, as revealed by the immunofluorescence analysis. These changes were accompanied by a significant reduction in the infective progeny formation over the protein extract range of 0.12-0.5 µg/mL. Moreover, short term feeding of mice with Roquefort cheese (twice, 10 mg per mouse with an interval of 24 hours) led to the inhibition of the migration of peritoneal leukocytes caused by intraperitoneal injection of E. coli lipopolysaccharide. These changes were complemented by a reduction in neutrophil count and a relative increase in peritoneal macrophages, suggesting that ingestion of Roquefort could promote regenerative processes at the site of inflammation. The ability of this protein to inhibit propagation of Chlamydia infection, as well as the anti-inflammatory and proregenerative effects of Roquefort itself, may contribute to the low prevalence of cardiovascular mortality in France where consumption of fungal fermented cheeses is the highest in the world.

Characterization and intracellular localization of putative Chlamydia pneumoniae effector proteins.

We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation.

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10.

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.

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.

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle.

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.

Effective macrophage redox defense against Chlamydia pneumoniae depends on L-type Ca2+ channel activation.

Macrophage immune capability depends on their efficient redox potential expressed in the effective release of reactive oxygen species (ROS) and nitric oxide. In this study the effect of the activation of a specialized Ca(2+) channel on macrophage redox function during Chlamydia pneumoniae infection was explored. C. pneumoniae exhibited a profound and sustained Ca(2+) influx capacity, with evidence of activity attributable to their lipopolysaccharide (cLPS) content. Also the organism showed an additional Ca(2+) influx signal in macrophages exposed to thapsigargin, and there was evidence for the operation of a single ion channel of the L type as demonstrated by the effect of L-type channel antagonists (methoxyverapamil and nimodipine) despite exposure to Ca(2+)-rich medium. C. pneumoniae or cLPS induced intracellular ROS and NO generation in a manner consistent with dependence on intracellular calcium. L-type Ca(2+) channel blocking significantly prompted C. pneumoniae inclusion formation. These findings suggest that Ca(2+) influx signal and redox function in C. pneumoniae-infected macrophages depend on L-type Ca(2+) channel activation.

Chlamydia pneumoniae survival in macrophages is regulated by free Ca2+ dependent reactive nitrogen and oxygen species.

OBJECTIVES: Despite an efficient macrophage immune capability, Chlamydia pneumoniae infects host cells and causes chronic diseases. To gain better insights into C. pneumoniae survival mechanisms in macrophages, its growth in regular RAW-264.7 cells (nitric oxide sufficient NO (+)) and RAW-264.7 cells (nitric oxide insufficient NO (-)) were studied. METHODS: Role of Ca(2+), NO and reactive oxygen species (ROS) during C. pneumoniae infection in macrophages were determined. RESULTS: RAW-264.7 NO (-) cells supported significantly Chlamydia growth, showing an upregulation of ROS, superoxide dismutase (SOD) and catalase activities as compared with RAW-264.7 NO (+) cell. Ascorbic acid, inducible nitric oxide synthase inhibitor and glutathione significantly prompted Chlamydia inclusion formation. Cytosolic Ca(2+) had regulatory effect on organism growth, NO generation, SOD and catalase activities in both cell types. CONCLUSIONS: These findings suggest that minimal Ca(2+) signaling in macrophages at early stages of infection, NO and ROS release have modulatory effects onC. pneumoniae survival, onset of persistence and chronicity, processes which are needed for the initiation of diseases in which C. pneumoniae has been implicated as a possible etiologic agent.

Inflammation, infection and antimicrobial therapy in coronary heart disease--where do we currently stand?

Traditional atherosclerotic risk factors such as hypertension, smoking, hyperlipidaemia and diabetes mellitus, account for only about 50% of the clinical occurrence of coronary heart disease (CHD). The infectious hypothesis proposes that various microorganisms, in particular, Chlamydia pneumoniae, may serve as potential etiological factors, linking inflammation and atherosclerosis (or its clinical manifestations). Evidence from seroepidemiology, pathology, animal models, molecular biology and immunology, and human antibiotic intervention studies, collectively have suggested a largely positive association between C. pneumoniae infection and CHD. As CHD is a multifactorial disease, it is possible that C. pneumoniae may interact with conventional cardiovascular risk factors and predispose certain genetically susceptible people to atherosclerotic disease. However, the precise nature of a causal or coincidental link between C. pneumoniae and CHD remains to be determined. The results of ongoing antibiotic intervention studies may help to further clarify the role of infection and inflammation in CHD, but until such a role is proven beyond reasonable doubt, antimicrobial therapy cannot yet be justified in the treatment or prevention of CHD. A current perspective is presented in this review.

Quantitation of bacterial adherence by image analysis.

In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved. We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells. We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained. We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae. To demonstrate an application of this method, we studied the attachment of H. influenzae and S. pneumoniae to target epithelial cell lines derived from the respiratory tract.