RESUMO
To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.
Assuntos
Proteínas de Bactérias , Chlamydophila pneumoniae , Endocitose , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteína cdc42 de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Chlamydophila pneumoniae/metabolismo , Cristalografia por Raios X , Células HeLa , Interações Hospedeiro-Patógeno , Mimetismo Molecular , Ligação Proteica , Nexinas de Classificação/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , RatosRESUMO
Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to C. pneumoniae infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating C. pneumoniae proteins, a screen in the model yeast Schizosaccharomyces pombe was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by C. pneumoniae EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced C. pneumoniae genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection.
Assuntos
Chlamydophila pneumoniae , Schizosaccharomyces , Humanos , Chlamydophila pneumoniae/metabolismo , Saccharomyces cerevisiae/metabolismo , Chlamydia trachomatis/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Chlamydia pneumoniae is a respiratory tract pathogen but can also infect the central nervous system (CNS). Recently, the link between C. pneumoniae CNS infection and late-onset dementia has become increasingly evident. In mice, CNS infection has been shown to occur weeks to months after intranasal inoculation. By isolating live C. pneumoniae from tissues and using immunohistochemistry, we show that C. pneumoniae can infect the olfactory and trigeminal nerves, olfactory bulb and brain within 72 h in mice. C. pneumoniae infection also resulted in dysregulation of key pathways involved in Alzheimer's disease pathogenesis at 7 and 28 days after inoculation. Interestingly, amyloid beta accumulations were also detected adjacent to the C. pneumoniae inclusions in the olfactory system. Furthermore, injury to the nasal epithelium resulted in increased peripheral nerve and olfactory bulb infection, but did not alter general CNS infection. In vitro, C. pneumoniae was able to infect peripheral nerve and CNS glia. In summary, the nerves extending between the nasal cavity and the brain constitute invasion paths by which C. pneumoniae can rapidly invade the CNS likely by surviving in glia and leading to Aß deposition.
Assuntos
Doença de Alzheimer , Infecções por Chlamydophila , Chlamydophila pneumoniae/metabolismo , Nervo Olfatório , Nervo Trigêmeo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/microbiologia , Animais , Infecções por Chlamydophila/complicações , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/microbiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Nervo Olfatório/metabolismo , Nervo Olfatório/microbiologia , Nervo Trigêmeo/metabolismo , Nervo Trigêmeo/microbiologiaRESUMO
Chlamydia pneumoniae (Cpn) has been reported to be involved in the pathogenesis of early atherosclerosis by inducing macrophage-derived foam cell formation in the presence of low-density lipoprotein (LDL). However, the biochemical mechanisms underlying Cpn-induced foam cell formation are still not fully elucidated. The present study showed that in LDL-treated THP-1-derived macrophages, Cpn not only upregulated the expression of scavenger receptor A1 (SR-A1) and acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1), but it also downregulated the expression of ATP binding cassette transporters (ABCA1 and ABCG1) at both the mRNA and protein levels. These processes facilitated cholesterol accumulation and promoted macrophage-derived foam cell formation. Treatment with the peroxisome proliferator-activated receptor (PPAR)-γ agonist rosiglitazone or the PPARα agonist fenofibrate decreased the number of foam cells induced by Cpn, while the PPARγ antagonist GW9662, the PPARα antagonist MK886, or PPARα/γ siRNAs enhanced the effect of Cpn on foam cell formation and gene expression of SR-A1, ACAT1, and ABCA1/G1. Moreover, the PPARγ agonist rosiglitazone reversed the downregulation of CD36 by Cpn, while PPARγ siRNA and the PPARγ inhibitor GW9662 further suppressed CD36 expression. However, the PPARα agonist, inhibitor, and siRNA all showed no effect on CD36 expression. In conclusion, the PPARα and PPARγ pathways are both involved in Cpn-induced macrophage-derived foam cell formation by upregulating SR-A1 and ACAT1 and downregulating ABCA1/G1 expression.
Assuntos
Chlamydophila pneumoniae , Células Espumosas , Transdução de Sinais , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aciltransferases , Antígenos CD36/genética , Antígenos CD36/metabolismo , Chlamydophila pneumoniae/metabolismo , Regulação para Baixo , Células Espumosas/metabolismo , Células Espumosas/microbiologia , Humanos , PPAR alfa/genética , PPAR gama , Células THP-1 , Regulação para CimaRESUMO
Chlamydia pneumoniae (Cpn) is a gram-negative intracellular pathogen that causes a variety of pulmonary diseases, and there is growing evidence that it may play a role in Alzheimer's disease (AD) pathogenesis. Cpn can interact functionally with host histones, altering the host's epigenetic regulatory system by introducing bacterial products into the host tissue and inducing a persistent inflammatory response. Because Cpn is difficult to propagate, isolate, and detect, a modified LPS-like neuroinflammation model was established using lyophilized cell free supernatant (CFS) obtained from infected cell cultures, and the effects of CFS were compared to LPS. The neuroprotective effects of Trichostatin A (TSA), givinostat, and RG108, which are effective on epigenetic mechanisms, and the antibiotic rifampin, were studied in this newly introduced model and in the presence of amyloid beta (Aß) 1-42. The neuroprotective effects of the drugs, as well as the effects of CFS and LPS, were evaluated in Aß-induced neurotoxicity using a real-time cell analysis system, total ROS, and apoptotic impact. TSA, RG108, givinostat, and rifampin all demonstrated neuroprotective effects in both this novel model and Aß-induced neurotoxicity. The findings are expected to provide early evidence on neuroprotective actions against Cpn-induced neuroinflammation and Aß-induced neurotoxicity, which could represent a new treatment option for AD, for which there are currently few treatment options.
Assuntos
Carbamatos/farmacologia , Infecções por Chlamydophila , Chlamydophila pneumoniae/metabolismo , Epigênese Genética/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Ftalimidas/farmacologia , Triptofano/análogos & derivados , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/prevenção & controle , Humanos , Inflamação/metabolismo , Fragmentos de Peptídeos/metabolismo , Células THP-1 , Triptofano/farmacologiaRESUMO
BACKGROUND: Chlamydia pneumoniae and Mycoplasma pneumoniae have been implicated in the pathogenesis of asthma and are responsible for chronic inflammation when host immune system fails to eradicate the bacteria. METHOD: We performed a prospective study on 410 patients who underwent a visit at the asthma clinic of CHU of Liege between June 2016 and June 2018 with serology testing for C. pneumoniae and M. pneumoniae. RESULTS: 65% of our asthmatic population had serum IgA and/or IgG towards C. pneumoniae, while only 12.6% had IgM and/or IgG against M. pneumoniae. Compared to seronegative asthmatics, asthmatics with IgA+ and IgG+ against C. pneumoniae were more often male and older with a higher proportion of patients with smoking history. They received higher doses of inhaled corticosteroids (ICS) and displayed lower FEV1/FVC ratio, higher RV/TLC ratio and lower conductance. They had higher levels of fibrinogen, though in the normal range and had lower sputum eosinophil counts. Patients with IgA- and IgG+ against C. pneumoniae were older and had higher blood monocyte counts and alpha-1-antitrypsin levels as compared to seronegative patients. Patients with IgM and/or IgG towards M. pneumoniae were more often males than seronegative asthmatics. In a subpopulation of 14 neutrophilic asthmatics with Chlamydia pneumoniae IgA + /IgG + treated with macrolides, we found a significant decrease in blood neutrophils and normalization of sputum neutrophil count but no effect on asthma quality of life and exacerbations. CONCLUSION: Positive Chlamydia serologic test is more common than positive Mycoplasma serology. Asthmatics with IgA and IgG against C. pneumoniae have more severe disease with increased airway obstruction, higher doses of ICS, more signs of air trapping and less type-2 inflammation.
Assuntos
Asma/epidemiologia , Infecções por Chlamydophila/epidemiologia , Chlamydophila pneumoniae , Mycoplasma pneumoniae , Pneumonia Bacteriana/epidemiologia , Pneumonia por Mycoplasma/epidemiologia , Adulto , Idoso , Asma/sangue , Asma/diagnóstico , Infecções por Chlamydophila/sangue , Infecções por Chlamydophila/diagnóstico , Chlamydophila pneumoniae/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycoplasma pneumoniae/metabolismo , Fenótipo , Pneumonia Bacteriana/sangue , Pneumonia Bacteriana/diagnóstico , Pneumonia por Mycoplasma/sangue , Pneumonia por Mycoplasma/diagnóstico , Estudos ProspectivosRESUMO
Chlamydia pneumoniae is an obligate intracellular pathogen that causes diseases of the upper and lower respiratory tract and is linked to a number of severe and chronic conditions. Here, we describe a large, C. pneumoniae-specific cluster of 13 genes (termed mbp1-13) that encode highly homologous chlamydial proteins sharing the capacity to bind to membranes. The gene cluster is localized on the chromosome between the highly diverse adhesin-encoding pmp genes pmp15 and pmp14. Comparison of human clinical isolates to the predicted ancestral koala isolate indicates that the cluster was acquired in the ancestor and was adapted / modified during evolution. SNPs and IN/DELs within the cluster are specific to isolates taken from different human tissues and show an ongoing adaptation. Most of the cluster proteins harbor one or two domains of unknown function (DUF575 and DUF562). During ectopic expression in human cells these DUF domains are crucial for the association of cluster proteins to the endo-membrane system. Especially DUF575 which harbors a predicted transmembrane domain is important for binding to the membrane, while presence of the DUF562 seems to be of regulatory function. For Mbp1, founding member of the cluster that exhibits a very limited sequence identity to the human Rab36 protein, we found a specific binding to vesicles carrying the early endosomal marker PtdIns(3)P and the endosomal Rab GTPases Rab11 and Rab14. This binding is dependent on a predicted transmembrane domain with an α-helical / ß-strand secondary structure, as the mutant version Mbp1mut, which lacks the ß-strand secondary structure, shows a reduced association to PtdIns(3)P-positive membranes carrying Rab11 and Rab14. Furthermore, we could not only show that Mbp1 associates with Rab36, but found this specific Rab protein to be recruited to the early C. pneumoniae inclusion. Detection of endogenous Mbp1 and Mbp4 reveal a colocalization to the chlamydial outer membrane protein Momp on EBs. The same colocalization pattern with Momp was observed when we ectopically expressed Mbp4 in C. trachomatis. Thus, we identified a C. pneumoniae-specific cluster of 13 membrane binding proteins (Mbps) localizing to the bacterial outer membrane system.
Assuntos
Chlamydophila pneumoniae , Proteínas da Membrana Bacteriana Externa/genética , Chlamydia trachomatis/genética , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Família Multigênica , Proteínas rab de Ligação ao GTPRESUMO
Protective immune response to chlamydial infection is largely dependent on cell-mediated immune responses with IFN-γ production. Recent studies have shown the critical role of NK cells in bridging innate and adaptive immune responses. In this study, we investigated the effect of NK cells on T cell responses during Chlamydophila pneumoniae (Cpn) lung infection. The results showed that NK cells play a protective role in Cpn infection and influence T cell immunity largely though modulating dendritic cells (DCs) function. Specifically, we found that NK depletion significantly impaired type 1 T cell responses, but enhanced FOXP3+Treg cells and IL-10-producing CD4+T cells. The alteration of T cell responses was associated with more disease severity and higher chlamydial growth in the lung. Further analysis of DC phenotype and cytokine profile found that DCs from NK cell-depleted mice expressed lower levels of co-stimulatory molecules and produced higher levels of IL-10 than those from control IgG-treated mice. More importantly, the adoptive transfer of DCs from NK cell-depleted mice induced a much lower degree of type 1 T cell responses but a higher amount of FOXP3+ Treg cells and IL-10-producing CD4+T cells in the recipient mice than DCs from IgG-treated mice. In contrast to the strong protective effect observed in recipients of DCs from IgG-treated mice, the recipients of DCs from NK cell-depleted mice failed to be protected against Cpn infection. The data suggest that NK cells play a critical role in coordinating innate and adaptive immunity in Cpn lung infection by modulating the DC function to influence T cell responses.
Assuntos
Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Células Matadoras Naturais/imunologia , Transferência Adotiva , Animais , Chlamydophila pneumoniae/metabolismo , Chlamydophila pneumoniae/patogenicidade , Citocinas/metabolismo , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Pneumonia Bacteriana/imunologiaRESUMO
Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.
Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia muridarum/efeitos dos fármacos , Chlamydia muridarum/metabolismo , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Pulmão/metabolismo , Animais , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Infecções por Chlamydia/genética , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina , Lipocalina-2/genética , Lipocalina-2/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Transcriptoma , Triptofano/análogos & derivados , Triptofano/antagonistas & inibidores , Triptofano/metabolismoRESUMO
Chlamydia pneumoniae is one of the two major species of the Chlamydiaceae family that have a profound effect on human health. C. pneumoniae is linked to a number of severe acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and infection by the pathogen might play a role in lung cancer. Following adhesion, Chlamydiae secrete effector proteins into the host cytoplasm that modulate the actin cytoskeleton facilitating internalization and infection. Members of the conserved TarP protein family comprise such effector proteins that polymerize actin, and in the case of the C. trachomatis TarP protein, has been shown to play a critical role in pathogenesis. In a previous study, we demonstrated that, upon bacterial invasion, the C. pneumoniae TarP family member CPn0572 is secreted into the host cytoplasm and recruits and associates with actin via an actin-binding domain conserved in TarP proteins. We have now extended our analysis of CPn0572 and found that the CPn0572 actin binding and modulating capability is more complex. With the help of the fission yeast system, a second actin modulating domain was identified independent of the actin binding domain. Microscopic analysis of HEp-2 cells expressing different CPn0572 deletion variants mapped this domain to the C-terminal part of the protein as CPn0572536-755 binds F-actin in vitro and colocalizes with aberrantly thickened actin cables in vivo. Finally, microscopic and bioinformatic analysis revealed the existence of a vinculin binding sequence in CPn0572. Our findings contribute to the understanding of the function of the TarP family and underscore the existence of several actin binding domains and a vinculin binding site for host actin modulation.
Assuntos
Proteínas de Bactérias/fisiologia , Chlamydophila pneumoniae/patogenicidade , Vinculina/metabolismo , Actinas/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/metabolismo , Biologia Computacional , Citoesqueleto/metabolismo , Humanos , Domínios Proteicos , Análise de Sequência de ProteínaRESUMO
Cardiovascular disease (CVD) is an important contributor to morbidity and mortality in American Indian communities. The Strong Heart Study (SHS) was initiated in response to the need for population based estimates of cardiovascular disease in American Indians. Previous studies within SHS have identified correlations between heart disease and deficiencies in mannose binding lectin (MBL), a motif recognition molecule of the innate immune system. MBL mediates the immune response to invading pathogens including Chlamydia pneumoniae (Cp), which has also been associated with the development and progression of CVD. However, a link between MBL2 genotype and Cp in contributing to heart disease has not been established. To address this, SHS collected baseline Cp antibody titers (IgA and IgG) and MBL2 genotypes for common functional variants from 553 individuals among twelve participating tribes. A single nucleotide polymorphism (SNP) in the promoter, designated X/Y, correlated significantly with increased Cp IgG titer levels, whereas another promoter SNP (H/L) did not significantly influence antibody levels to Cp. Two variants within exon 1 of MBL2, the A and B alleles, also displayed significant association with Cp antibody titers. Some MBL2 genotypes were absent from the population, suggesting linkage disequilibrium may be operating within the SHS cohort. Additional factors, such as increasing age and socioeconomic status, were also associated with increased Cp IgG antibody titers. This study demonstrates that MBL2 genotype associates with immune reactivity to C. pneumoniae in the SHS cohort. Thus, MBL2 may contribute to the progression of cardiovascular disease (CVD) among American Indians indirectly through pathogen interactions in addition to its previously defined roles.
Assuntos
Chlamydophila pneumoniae/metabolismo , Chlamydophila pneumoniae/patogenicidade , Lectina de Ligação a Manose/metabolismo , Idoso , Alelos , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Estudos de Casos e Controles , Chlamydophila pneumoniae/genética , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Indígenas Norte-Americanos , Desequilíbrio de Ligação/genética , Masculino , Lectina de Ligação a Manose/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genéticaAssuntos
Infecções por Chlamydophila , Chlamydophila pneumoniae/metabolismo , Pescoço/irrigação sanguínea , Doenças Vasculares , Infecções por Chlamydophila/diagnóstico , Infecções por Chlamydophila/terapia , Doença Crônica , Humanos , Pirimidinas , Pirróis , Doenças Vasculares/diagnóstico , Doenças Vasculares/terapiaRESUMO
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 1â% (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 0.1â% NCT within 1 min. Moreover, 0.025-0.1â% 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.
Assuntos
Anti-Infecciosos Locais/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/efeitos dos fármacos , Taurina/análogos & derivados , Infecções por Chlamydia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/metabolismo , Humanos , Taurina/farmacologiaRESUMO
Chlamydia pneumoniae is an obligate intracellular pathogen responsible for respiratory diseases, including pneumonia and bronchitis, and is highly involved in chronic diseases, including atherosclerosis, asthma, and Alzheimer's disease. We previously showed that the host apoptotic factor caspase-9 played a crucial role for chlamydial multiplication and host apoptosis inhibition by chlamydial infection. To identify chlamydial genes interacting with human caspase-9, yeast two-hybrid screening was performed and 5 chlamydial genes, including Cpj0838 and pmpG were isolated from the C. pneumoniae genomic library. Pull-down experiments showed that caspase-9 physically bound to the Cpj0838 product and chlamydial cells, which contain PmpG proteins. This study could provide a clue to understanding host-Chlamydia interactions, especially the apoptosis repression by Chlamydia infection.
Assuntos
Apoptose , Proteínas da Membrana Bacteriana Externa/metabolismo , Caspase 9/metabolismo , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Proteínas da Membrana Bacteriana Externa/genética , Chlamydophila pneumoniae/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Técnicas do Sistema de Duplo-HíbridoRESUMO
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 (O2 ≈ 20%) and hypoxic conditions (O2 < 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.
Assuntos
Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/patogenicidade , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Mitocôndrias/microbiologia , Mitocôndrias/fisiologia , Linhagem Celular , Chlamydophila pneumoniae/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Humanos , Hipóxia , Potencial da Membrana Mitocondrial/fisiologia , Oxigênio/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismoRESUMO
The initial steps in chlamydial infection involve adhesion and internalization into host cells and, most importantly, modification of the nascent inclusion to establish the intracellular niche. Here, we show that Chlamydia pneumoniae enters host cells via EGFR-dependent endocytosis into an early endosome with a phosphatidylinositol 3-phosphate (PI3P) membrane identity. Immediately after entry, the early chlamydial inclusion acquires early endosomal Rab GTPases including Rab4, Rab5, Rab7, as well as the two recycling-specific Rabs Rab11 and Rab14. While Rab5, Rab11 and Rab14 are retained in the vesicular membrane, Rab4 and Rab7 soon disappear. Loss of Rab7 enables the C. pneumoniae inclusion to escape delivery to, and degradation in lysosomes. Loss of Rab4 and retention of Rab11/ Rab14 designates the inclusion as a slowly recycling endosome-that is protected from degradation. Furthermore, we show that the Rab11/ Rab14 adaptor protein Rab11-Fip2 (Fip2) is recruited to the nascent inclusion upon internalization and retained in the membrane throughout infection. siRNA knockdown of Fip2 demonstrated that the protein is essential for internalization and infection, and expression of various deletion variants revealed that Fip2 regulates the intracellular positioning of the inclusion. Additionally, we show that binding to Rab11 and Fip2 recruits the unconventional actin motor protein myosin Vb to the early inclusion and that together they regulate the relocation of the nascent inclusion from the cell periphery to the perinuclear region, its final destination. Here, we characterize for the first time inclusion identity and inclusion-associated proteins to delineate how C. pneumoniae establishes the intracellular niche essential for its survival.
Assuntos
Proteínas de Transporte/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydophila pneumoniae/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Endocitose/fisiologia , Imunofluorescência , Humanos , ImunoprecipitaçãoRESUMO
Chlamydophila pneumoniae, a Gram-negative bacterium belongs to the family Chlamydiaceae, is known to cause community-acquired pneumonia and bronchitis. There is a need for genomic analyses of C. pneumoniae as its chronic infections result in reactive airway disease, lung cancer and asthma. Recent advancement in the sequencing techniques led to the generation of large genomic data. In order to utilize these data, sequence-based function predictions were used for annotating the uncharacterized genes. The genome of C. pneumoniae encodes 1052 proteins, which include a group of 366 functionally uncharacterized proteins, known as "hypothetical proteins" (HPs). Functions of these HPs were predicted by utilizing an integrated approach that combines varieties of bioinformatics tools. The functions of 142 proteins were successfully predicted and categorized into different classes of enzymes, transport proteins, binding proteins and virulence factors. Among these functionally annotated HPs, we were able to identify 12 virulent HPs. Furthermore, the HP with the highest virulence score was subjected to molecular dynamics (MD) simulations to better understand their dynamical behavior in explicit water conditions. These analyses could be utilized for an in-depth understanding of virulence mechanism. The functional knowledge of these proteins could be useful in drug design and discovery process of infections caused by C. pneumoniae.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydophila pneumoniae/metabolismo , Proteínas de Bactérias/genética , Chlamydophila pneumoniae/genética , Biologia Computacional , Genoma Bacteriano/genética , Simulação de Dinâmica MolecularRESUMO
Pathogenic Chlamydia species force entry into human cells via specific adhesin-receptor interactions and subsequently secrete effector proteins into the host cytoplasm, which in turn modulate host-cell processes to promote infection. One such effector, the C. trachomatis Tarp factor, nucleates actin polymerization in vitro. Here we show that its C. pneumoniae ortholog, CPn0572, associates with actin patches upon bacterial invasion. GFP-CPn0572 ectopically expressed in yeast and human cells co-localizes with actin patches and distinctly aberrantly thickened and extended actin cables. A 59-aa DUF 1547 (DUF) domain, which overlaps with the minimal actin-binding and protein oligomerization fragment required for actin nucleation in other Tarp orthologs, is responsible for the aberrant actin phenotype in yeast. Interestingly, GFP-CPn0572 in human cells associated with and led to the formation of non-actin microfilaments. This phenotype is strongly enhanced in human cells expressing the GFP-tagged DUF deletion variant (GFP-ΔDUF). Finally ectopic CPn0572 expression in yeast and in-vitro actin filament binding assays, demonstrated that CPn0572 stabilizes pre-assembled F-actin by displacing and/or inhibiting binding of the actin-severing protein cofilin. Remarkably, the DUF domain suffices to displace cofilin from F actin. Thus, in addition to its actin-nucleating activities, the C. pneumoniae CPn0572 also stabilizes preformed host actin filaments.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydophila pneumoniae/metabolismo , Proteínas de Bactérias/genética , Chlamydophila pneumoniae/fisiologia , Endocitose , Expressão Gênica , Células HEK293 , Humanos , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. The adhesion of Chlamydiae to the eukaryotic host cell is a pivotal step in pathogenesis. The adhesin family of polymorphic membrane proteins (Pmp) in Chlamydia pneumoniae consists of 21 members. Pmp21 binds to the epidermal growth factor receptor (EGFR). Pmps contain large numbers of FXXN (where X is any amino acid) and GGA(I/L/V) motifs. At least two of these motifs are crucial for adhesion by certain Pmp21 fragments. Here we describe how the two FXXN motifs in Pmp21-D (D-Wt), a domain of Pmp21, influence its self-interaction, folding, and adhesive capacities. Refolded D-Wt molecules form oligomers with high sedimentation values (8-85 S). These oligomers take the form of elongated protofibrils, which exhibit Thioflavin T fluorescence, like the amyloid protein fragment ß42. A mutant version of Pmp21-D (D-Mt), with FXXN motifs replaced by SXXV, shows a markedly reduced capacity to form oligomers. Secondary-structure assays revealed that monomers of both variants exist predominantly as random coils, whereas the oligomers form predominantly ß-sheets. Adhesion studies revealed that oligomers of D-Wt (D-Wt-O) mediate significantly enhanced binding to human epithelial cells relative to D-Mt-O and monomeric protein species. Moreover, D-Wt-O binds EGFR more efficiently than D-Wt monomers. Importantly, pretreatment of human cells with D-Wt-O reduces infectivity upon subsequent challenge with C. pneumoniae more effectively than all other protein species. Hence, the FXXN motif in D-Wt induces the formation of ß-sheet-rich oligomeric protofibrils, which are important for adhesion to, and subsequent infection of human cells.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydophila pneumoniae/metabolismo , Multimerização Proteica/fisiologia , Adesinas Bacterianas/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Chlamydophila pneumoniae/genética , Receptores ErbB/genética , Humanos , Mutação de Sentido Incorreto , Ligação ProteicaRESUMO
In evolving to an obligate intracellular niche, Chlamydia has streamlined its genome by eliminating superfluous genes as it relies on the host cell for a variety of nutritional needs like amino acids. However, Chlamydia can experience amino acid starvation when the human host cell in which the bacteria reside is exposed to interferon gamma (IFN-γ), which leads to a tryptophan (Trp)-limiting environment via induction of the enzyme indoleamine-2,3-dioxygenase (IDO). The stringent response is used to respond to amino acid starvation in most bacteria but is missing from Chlamydia Thus, how Chlamydia, a Trp auxotroph, responds to Trp starvation in the absence of a stringent response is an intriguing question. We previously observed that C. pneumoniae responds to this stress by globally increasing transcription while globally decreasing translation, an unusual response. Here, we sought to understand this and hypothesized that the Trp codon content of a given gene would determine its transcription level. We quantified transcripts from C. pneumoniae genes that were either rich or poor in Trp codons and found that Trp codon-rich transcripts were increased, whereas those that lacked Trp codons were unchanged or even decreased. There were exceptions, and these involved operons or large genes with multiple Trp codons: downstream transcripts were less abundant after Trp codon-rich sequences. These data suggest that ribosome stalling on Trp codons causes a negative polar effect on downstream sequences. Finally, reassessing previous C. pneumoniae microarray data based on codon content, we found that upregulated transcripts were enriched in Trp codons, thus supporting our hypothesis.