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1.
Sci Rep ; 9(1): 10878, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350436

RESUMO

As a first host barrier, the skin is constantly exposed to environmental insults that perturb its integrity. Tight regulation of skin homeostasis is largely controlled by the aryl hydrocarbon receptor (AhR). Here, we demonstrate that Henna and its major pigment, the naphthoquinone Lawsone activate AhR, both in vitro and in vivo. In human keratinocytes and epidermis equivalents, Lawsone exposure enhances the production of late epidermal proteins, impacts keratinocyte differentiation and proliferation, and regulates skin inflammation. To determine the potential use of Lawsone for therapeutic application, we harnessed human, murine and zebrafish models. In skin regeneration models, Lawsone interferes with physiological tissue regeneration and inhibits wound healing. Conversely, in a human acute dermatitis model, topical application of a Lawsone-containing cream ameliorates skin irritation. Altogether, our study reveals how a widely used natural plant pigment is sensed by the host receptor AhR, and how the physiopathological context determines beneficial and detrimental outcomes.

2.
Nat Cell Biol ; 21(7): 812-823, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31235935

RESUMO

Wnt signalling stimulated by binding of R-spondin (Rspo) to Lgr-family members is crucial for gastrointestinal stem cell renewal. Infection of the stomach with Helicobacter pylori stimulates increased secretion of Rspo by myofibroblasts, leading to an increase in proliferation of Wnt-responsive Axin2+Lgr5- stem cells in the isthmus of the gastric gland and finally gastric gland hyperplasia. Basal Lgr5+ cells are also exposed to Rspo3, but their response remains unclear. Here, we demonstrate that-in contrast to its known mitogenic activity-Rspo3 induces differentiation of basal Lgr5+ cells into secretory cells that express and secrete antimicrobial factors, such as intelectin-1, into the lumen. The depletion of Lgr5+ cells or the knockout of Rspo3 in myofibroblasts leads to hypercolonization of the gastric glands with H. pylori, including the stem cell compartment. By contrast, systemic administration or overexpression of Rspo3 in the stroma clears H. pylori from the gastric glands. Thus, the Rspo3-Lgr5 axis simultaneously regulates both antimicrobial defence and mucosal regeneration.

3.
MBio ; 10(3)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113891

RESUMO

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many Actinobacteria Mycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis We found that the growth deficit of mft deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of M. tuberculosis In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosis in vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

4.
EMBO Rep ; 20(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30872316

RESUMO

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

5.
Nat Commun ; 10(1): 1194, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886143

RESUMO

Chronic infections of the fallopian tubes with Chlamydia trachomatis (Ctr) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation-demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer.


Assuntos
Infecções por Chlamydia/genética , Chlamydia trachomatis/imunologia , Ilhas de CpG/genética , Metilação de DNA/imunologia , Interações entre Hospedeiro e Microrganismos/genética , Células-Tronco/metabolismo , Fatores Etários , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Doença Crônica , Ilhas de CpG/imunologia , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/microbiologia , Epigênese Genética/genética , Epigênese Genética/imunologia , Epitélio/imunologia , Epitélio/metabolismo , Epitélio/microbiologia , Tubas Uterinas/imunologia , Tubas Uterinas/metabolismo , Tubas Uterinas/microbiologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Microscopia Intravital , Microscopia Confocal , Organoides/imunologia , Organoides/metabolismo , Organoides/microbiologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/microbiologia , Sorogrupo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Análise de Célula Única , Células-Tronco/imunologia , Células-Tronco/microbiologia , Técnicas de Cultura de Tecidos
6.
EBioMedicine ; 33: 230-241, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29937069

RESUMO

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.


Assuntos
Células Epiteliais Alveolares/citologia , Brônquios/citologia , Técnicas de Cultura de Células/métodos , Meios de Cultura/farmacologia , Vírus da Influenza A/fisiologia , Células Epiteliais Alveolares/virologia , Amidas/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Galinhas , Meios de Cultura/química , Dibenzazepinas/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Alimentadoras/citologia , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Piridinas/farmacologia , Replicação Viral
7.
Gastroenterology ; 154(5): 1391-1404.e9, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29273450

RESUMO

BACKGROUND & AIMS: Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. METHODS: MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-ß-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. RESULTS: In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human ß-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. CONCLUSIONS: H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.


Assuntos
Colesterol/metabolismo , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Gastrite/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Interferon gama/metabolismo , Transdução de Sinais , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Microambiente Celular , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/imunologia , Gastrite/microbiologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/imunologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/imunologia , Interleucina-6/metabolismo , Interleucinas/metabolismo , Janus Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Mutação , Cultura Primária de Células , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Fator de Transcrição STAT1/metabolismo , Fatores de Tempo
8.
PLoS Pathog ; 13(10): e1006676, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29040326

RESUMO

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue.


Assuntos
Tecido Adiposo/imunologia , Tecido Adiposo/microbiologia , Tuberculose/imunologia , Tuberculose/microbiologia , Latência Viral/imunologia , Adipócitos/microbiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Mycobacterium tuberculosis/imunologia
9.
mSystems ; 2(4)2017 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28845460

RESUMO

Nutrient acquisition from the host environment is crucial for the survival of intracellular pathogens, but conceptual and technical challenges limit our knowledge of pathogen diets. To overcome some of these technical roadblocks, we exploited an experimentally accessible model for early infection of human macrophages by Mycobacterium tuberculosis, the etiological agent of tuberculosis, to study host-pathogen interactions with a multi-omics approach. We collected metabolomics and complete transcriptome RNA sequencing (dual RNA-seq) data of the infected macrophages, integrated them in a genome-wide reaction pair network, and identified metabolic subnetworks in host cells and M. tuberculosis that are modularly regulated during infection. Up- and downregulation of these metabolic subnetworks suggested that the pathogen utilizes a wide range of host-derived compounds, concomitant with the measured metabolic and transcriptional changes in both bacteria and host. To quantify metabolic interactions between the host and intracellular pathogen, we used a combined genome-scale model of macrophage and M. tuberculosis metabolism constrained by the dual RNA-seq data. Metabolic flux balance analysis predicted coutilization of a total of 33 different carbon sources and enabled us to distinguish between the pathogen's substrates directly used as biomass precursors and the ones further metabolized to gain energy or to synthesize building blocks. This multiple-substrate fueling confers high robustness to interventions with the pathogen's metabolism. The presented approach combining multi-omics data as a starting point to simulate system-wide host-pathogen metabolic interactions is a useful tool to better understand the intracellular lifestyle of pathogens and their metabolic robustness and resistance to metabolic interventions. IMPORTANCE The nutrients consumed by intracellular pathogens are mostly unknown. This is mainly due to the challenge of disentangling host and pathogen metabolism sharing the majority of metabolic pathways and hence metabolites. Here, we investigated the metabolic changes of Mycobacterium tuberculosis, the causative agent of tuberculosis, and its human host cell during early infection. To this aim, we combined gene expression data of both organisms and metabolite changes during the course of infection through integration into a genome-wide metabolic network. This led to the identification of infection-specific metabolic alterations, which we further exploited to model host-pathogen interactions quantitatively by flux balance analysis. These in silico data suggested that tubercle bacilli consume up to 33 different nutrients during early macrophage infection, which the bacteria utilize to generate energy and biomass to establish intracellular growth. Such multisubstrate fueling strategy renders the pathogen's metabolism robust toward perturbations, such as innate immune responses or antibiotic treatments.

10.
Nature ; 548(7668): 451-455, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28813421

RESUMO

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.


Assuntos
Infecções por Helicobacter/metabolismo , Homeostase , Células-Tronco/citologia , Células-Tronco/metabolismo , Estômago/citologia , Células Estromais/metabolismo , Trombospondinas/metabolismo , Animais , Proteína Axina/metabolismo , Proliferação de Células , Células Epiteliais/citologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Antro Pilórico/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Nicho de Células-Tronco , Células Estromais/citologia , Via de Sinalização Wnt
11.
Front Immunol ; 8: 1792, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29375545

RESUMO

Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.

12.
BMC Genomics ; 17(1): 837, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27784279

RESUMO

BACKGROUND: Mycobacteria, along with exospore forming Streptomyces, belong to the phylum actinobacteria. Mycobacteria are generally believed to be non-differentiating. Recently however, we showed that the mycobacterial model organism M. smegmatis is capable of forming different types of morphologically distinct resting cells. When subjected to starvation conditions, cells of M. smegmatis exit from the canonical cell division cycle, segregate and compact their chromosomes, and become septated and multi-nucleoided. Under zero nutrient conditions the differentiation process terminates at this stage with the formation of Large Resting Cells (LARCs). In the presence of traces of carbon sources this multi-nucleoided cell stage completes cell division and separates into Small Resting Cells (SMRCs). Here, we carried out RNA-seq profiling of SMRC and LARC development to characterize the transcriptional program underlying these starvation-induced differentiation processes. RESULTS: Changes among the top modulated genes demonstrated that SMRCs and LARCs undergo similar transcriptional changes. The formation of multi-nucleoided cells (i.e. LARCs and the LARC-like intermediates observed during SMRC formation) was accompanied by upregulation of septum formation functions FtsZ, FtsW, and PbpB, as well as the DNA translocase FtsK. The observed compaction of chromosomes was accompanied by an increase of the transcript level of the DNA binding protein Hlp, an orthologue of the Streptomyces spore-specific chromosome condensation protein HupS. Both SMRC and LARC development were accompanied by similar temporal expression patterns of candidate regulators, including the transcription factors WhiB2, WhiB3, and WhiB4, which are orthologues of the Streptomyces sporulation regulators WhiB, WhiD and WblA, respectively. CONCLUSIONS: Transcriptional analyses of the development of mycobacterial resting cell types suggest that these bacteria harbor a novel differentiation program and identify a series of potential regulators. This provides the basis for the genetic dissection of this actinobacterial differentiation process.


Assuntos
Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/genética , Transcriptoma , Análise por Conglomerados , Perfilação da Expressão Gênica , Genes Bacterianos , Sequenciamento de Nucleotídeos em Larga Escala
13.
Int J Med Microbiol ; 306(7): 517-528, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27424770

RESUMO

Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.


Assuntos
Ciclo Celular , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Proteína Forkhead Box M1/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Propionibacterium acnes/patogenicidade , Próstata/microbiologia , Proteína Forkhead Box M1/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Análise em Microsséries , Peptídeos/análise , Peptídeos/genética , Reação em Cadeia da Polimerase em Tempo Real
14.
MBio ; 7(3)2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27222470

RESUMO

UNLABELLED: The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG ΔureC::hly In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG ΔureC::hly ΔnuoG vaccination was safer than BCG and improved protection over that of parental BCG ΔureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG ΔureC::hly ΔnuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety. IMPORTANCE: Autophagy and apoptosis are fundamental processes allowing cells to degrade their components or kill themselves, respectively. The immune system has adopted these mechanisms to eliminate intracellular pathogens. Residing in host cells, the causative agent of tuberculosis, Mycobacterium tuberculosis, has evolved strategies to set cellular programs of autophagy and apoptosis "on hold." The mycobacterial gene nuoG was found to prevent host cell apoptosis. We have deleted nuoG in the live vaccine candidate BCG ΔureC::hly, which is in phase II clinical development, to leave cellular apoptosis "on go" upon immunization. In preclinical models, this strategy boosted immunity and improved protection from M. tuberculosis infection. Unexpectedly, we obtained compelling evidence that mycobacterial nuoG facilitates inhibition of autophagic pathways, suggesting a new role for this gene in the host-pathogen interplay in tuberculosis.


Assuntos
Vacina BCG/genética , Vacina BCG/imunologia , Complexo I de Transporte de Elétrons/genética , Deleção de Genes , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Vacina BCG/efeitos adversos , Carga Bacteriana , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Inflamassomos/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/microbiologia , Camundongos , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Vacinação
15.
PLoS Pathog ; 12(2): e1005408, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26829557

RESUMO

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.


Assuntos
Hidroliases/imunologia , Interferons/imunologia , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Macrófagos Alveolares/imunologia , Proteoma , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Hidroliases/genética , Hidroliases/metabolismo , Imunidade Inata , Interferons/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Modelos Imunológicos , Espécies Reativas de Oxigênio/metabolismo , Succinatos/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia
16.
Cell Microbiol ; 18(1): 137-47, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26243717

RESUMO

Helicobacter pylori strains carrying the cag pathogenicity island (cagPAI) provoke an increased inflammatory response, conferring an increased risk of ulcer formation and carcinogenesis. How the immune system recognizes the presence of cagPAI positive strains is yet unclear. By comparing the transcriptional response of wild type and MyD88/Trif(-/-) bone marrow macrophages to infection with H. pylori, we found that the majority of regulated genes were dependent on toll-like receptor (TLR) signalling. To determine the role of TLR-independent responses, we analysed the transcriptome of MyD88/Trif(-/-) bone marrow macrophages at different time points after infection with cagPAI positive versus negative strains. We identified a group of genes that exhibited different kinetic behaviour depending on whether cagPAI was present. Analysis of their gene expression kinetics demonstrated that this responsiveness to cagPAI was observed only in MyD88/Trif(-/-) macrophages. This group of cagPAI-sensing genes was enriched for AU-rich element containing early response genes involved in immune regulation, including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Recognition of cagPAI positive strains was found to be mediated by the type IV secretion system (cagT4SS), rather than its effector protein CagA. We hypothesize that anergic macrophages of the gastric mucosa initiate an innate immune response following detection of the T4SS of H. pylori.


Assuntos
Helicobacter pylori/imunologia , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/microbiologia , Receptores Toll-Like/metabolismo , Sistemas de Secreção Tipo IV/imunologia , Animais , Perfilação da Expressão Gênica , Imunidade Inata , Camundongos
17.
Nat Commun ; 6: 8989, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26643275

RESUMO

The epithelial lining of the fallopian tube is of critical importance for human reproduction and has been implicated as a site of origin of high-grade serous ovarian cancer. Here we report on the establishment of long-term, stable 3D organoid cultures from human fallopian tubes, indicative of the presence of adult stem cells. We show that single epithelial stem cells in vitro can give rise to differentiated organoids containing ciliated and secretory cells. Continuous growth and differentiation of organoids depend on both Wnt and Notch paracrine signalling. Microarray analysis reveals that inhibition of Notch signalling causes downregulation of stem cell-associated genes in parallel with decreased proliferation and increased numbers of ciliated cells and that organoids also respond to oestradiol and progesterone treatment in a physiological manner. Thus, our organoid model provides a much-needed basis for future investigations of signalling routes involved in health and disease of the fallopian tube.


Assuntos
Células-Tronco Adultas/metabolismo , Células Epiteliais/metabolismo , Tubas Uterinas/metabolismo , Organoides/metabolismo , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Células-Tronco Adultas/citologia , Diferenciação Celular , Proliferação de Células , Cílios , Regulação para Baixo , Células Epiteliais/citologia , Tubas Uterinas/citologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Organoides/citologia , Comunicação Parácrina , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo
18.
MBio ; 6(5): e01187-15, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26374119

RESUMO

UNLABELLED: An estimated one-third of the world's population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type. IMPORTANCE: DNA methylation modifies the transcriptional program of cells. We have focused on two major populations of leukocytes involved in immune response to infectious diseases, granulocytes and monocytes, both of which are professional phagocytes that engulf and kill bacteria. We have interrogated how DNA methylation, gene expression, and protein translation differ in these two cell populations between healthy individuals and patients suffering from TB. To better understand the underlying biologic mechanisms, we harnessed a statistical enrichment analysis, taking advantage of predefined and well-characterized gene sets. Not only were there clear differences on various levels between the two populations, but there were also differences between TB patients and healthy controls in the transcriptome, proteome, and, for the first time, DNA methylome in these cells. Our pilot study emphasizes the value of a large-scale study of the DNA methylome taking into account our findings.


Assuntos
Biomarcadores/análise , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteômica/métodos , Tuberculose/patologia , Humanos , Dados de Sequência Molecular , Projetos Piloto , Análise de Sequência de DNA
19.
Mol Biochem Parasitol ; 201(2): 100-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26222913

RESUMO

Gametocytogenesis and gametogenesis in malaria parasites are complex processes of cell differentiation and development likely involving many gene products. Gametocytes develop in the blood of the vertebrate host but mature gametocytes are not activated until taken up by the mosquito vector. Several distinct mutants have been described that block gametogenesis but the detailed molecular causes for the mutant phenotypes are not understood. To investigate whether a block in gametogenesis also results in a changed transcriptional profile we studied two gene deletions mutants; act2(-) lacking stage-specific actin II and CDPK4(-) lacking calcium-dependent protein kinase 4. Whole genome microarray analysis was performed from RNA of mature gametocytes to compare the transcriptomes of the mutants with wild-type Plasmodium berghei. The microarray analysis identified ∼12% of all genes being differentially expressed in either or both mutants compared to normal gametocytes, as defined by at least two-fold change in transcript abundance. A large proportion of the differentially expressed genes overlapped in the two mutants, consistent with a related outcome of gametocyte arrest. Distinct profiles in each mutant were also observed. Among the down-regulated genes were thioredoxin 2 and members of the merozoite surface protein 7 family. Generation and characterization of a msp7(-)/mspr1(-)/mspr2(-) triple mutant and re-analysis of trx2(-) parasites revealed no impairment of life cycle progression. Together, our analysis provides a resource for molecular signatures of Plasmodium berghei gametogenesis and exemplifies the potential of expression profiling of distinct genetically arrested parasites.


Assuntos
Actinas/deficiência , Perfilação da Expressão Gênica , Plasmodium berghei/crescimento & desenvolvimento , Proteínas Quinases/deficiência , Proteínas de Protozoários/genética , Análise em Microsséries , Plasmodium berghei/genética
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