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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.
FASEB J ; 33(8): 9087-9099, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075211

RESUMO

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-ß-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the ß-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

3.
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.

4.
EBioMedicine ; 23: 100-110, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28803120

RESUMO

The intracellular human bacterial pathogen Chlamydia trachomatis pursues effective strategies to protect infected cells against death-inducing stimuli. Here, we show that Chlamydia trachomatis infection evokes 3-phosphoinositide-dependent protein kinase-1 (PDPK1) signaling to ensure the completion of its developmental cycle, further leading to the phosphorylation and stabilization of MYC. Using biochemical approaches and imaging we demonstrate that Chlamydia-induced PDPK1-MYC signaling induces host hexokinase II (HKII), which becomes enriched and translocated to the mitochondria. Strikingly, preventing the HKII interaction with mitochondria using exogenous peptides triggers apoptosis of infected cells as does inhibiting either PDPK1 or MYC, which also disrupts intracellular development of Chlamydia trachomatis. These findings identify a previously unknown pathway activated by Chlamydia infection, which exhibits pro-carcinogenic features. Targeting the PDPK1-MYC-HKII-axis may provide a strategy to overcome therapeutic resistance of infection.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Apoptose , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/fisiologia , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Enzimática , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação
5.
Dermatol Pract Concept ; 5(3): 55-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26336627
6.
Dermatol Pract Concept ; 5(2): 55-6, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26114052
8.
Proc Natl Acad Sci U S A ; 111(38): E4024-32, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25201986

RESUMO

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.


Assuntos
Arginase/metabolismo , Granuloma/enzimologia , Hipóxia/enzimologia , Macrófagos/enzimologia , Mycobacterium tuberculosis , Tuberculose Pulmonar/enzimologia , Animais , Arginase/genética , Arginase/imunologia , Arginina/genética , Arginina/imunologia , Arginina/metabolismo , Proliferação de Células/genética , Modelos Animais de Doenças , Granuloma/genética , Granuloma/imunologia , Granuloma/patologia , Humanos , Hipóxia/genética , Hipóxia/imunologia , Hipóxia/patologia , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico/genética , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia
9.
Nature ; 512(7515): 387-92, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25119038

RESUMO

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mycobacterium tuberculosis/imunologia , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Antibacterianos/metabolismo , Células da Medula Óssea/citologia , Citocinas/imunologia , Citocinas/metabolismo , Retroalimentação Fisiológica , Humanos , Ligantes , Ativação de Macrófagos , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fenazinas/metabolismo , Pigmentos Biológicos/química , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
10.
J Infect Dis ; 210(12): 1928-37, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24943726

RESUMO

Bacillus Calmette-Guérin (BCG) has been used for vaccination against tuberculosis for nearly a century. Here, we analyze immunity induced by a live tuberculosis vaccine candidate, recombinant BCG ΔureC::hly vaccine (rBCG), with proven preclinical and clinical safety and immunogenicity. We pursue in-depth analysis of the endogenous mycobacteria-specific CD4(+) T-cell population, comparing the more efficacious rBCG with canonical BCG to determine which T-cell memory responses are prerequisites for superior protection against tuberculosis. rBCG induced higher numbers and proportions of antigen-specific memory CD4(+) T cells than BCG, with a CXCR5(+)CCR7(+) phenotype and low expression of the effector transcription factors T-bet and Bcl-6. We found that the superior protection of rBCG, compared with BCG, correlated with higher proportions and numbers of these central memory T cells and of T follicular helper cells associated with specific antibody responses. Adoptive transfer of mycobacteria-specific central memory T cells validated their critical role in protection against pulmonary tuberculosis.


Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Mycobacterium bovis/imunologia , Tuberculose/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Vacina BCG/genética , Feminino , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Mycobacterium bovis/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
14.
Proc Natl Acad Sci U S A ; 110(18): 7407-12, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23589855

RESUMO

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.


Assuntos
Seleção Clonal Mediada por Antígeno/imunologia , MicroRNAs/metabolismo , Células T Matadoras Naturais/imunologia , Animais , Proliferação de Células , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células T Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
15.
Eur J Immunol ; 43(7): 1828-38, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23575529

RESUMO

The CD8αß coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-ß on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-ß mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-ß. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-ß occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-ß can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .


Assuntos
Adenosina Difosfato Ribose/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adenosina Difosfato Ribose/imunologia , Animais , Separação Celular , Citometria de Fluxo , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NAD/imunologia , NAD/metabolismo
16.
Biochem J ; 449(3): 631-42, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23095086

RESUMO

The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short ß-bulge in ß2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria gonorrhoeae/metabolismo , Porinas/química , Porinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Cristalografia por Raios X , DNA Bacteriano/genética , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidade , Peptidoglicano/metabolismo , Fosfatos/metabolismo , Porinas/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Virulência/genética , Virulência/fisiologia
17.
FASEB J ; 26(4): 1717-26, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22247334

RESUMO

Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effector proteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Citosol/metabolismo , Via Secretória/fisiologia , Shigella flexneri/patogenicidade , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Cristalografia por Raios X , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Shigella flexneri/genética , Shigella flexneri/metabolismo
18.
PLoS Pathog ; 7(8): e1002163, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21829362

RESUMO

Many infectious gram-negative bacteria, including Salmonella typhimurium, require a Type Three Secretion System (T3SS) to translocate virulence factors into host cells. The T3SS consists of a membrane protein complex and an extracellular needle together that form a continuous channel. Regulated secretion of virulence factors requires the presence of SipD at the T3SS needle tip in S. typhimurium. Here we report three-dimensional structures of individual SipD, SipD in fusion with the needle subunit PrgI, and of SipD:PrgI in complex with the bile salt, deoxycholate. Assembly of the complex involves major conformational changes in both SipD and PrgI. This rearrangement is mediated via a π bulge in the central SipD helix and is stabilized by conserved amino acids that may allow for specificity in the assembly and composition of the tip proteins. Five copies each of the needle subunit PrgI and SipD form the T3SS needle tip complex. Using surface plasmon resonance spectroscopy and crystal structure analysis we found that the T3SS needle tip complex binds deoxycholate with micromolar affinity via a cleft formed at the SipD:PrgI interface. In the structure-based three-dimensional model of the T3SS needle tip, the bound deoxycholate faces the host membrane. Recently, binding of SipD with bile salts present in the gut was shown to impede bacterial infection. Binding of bile salts to the SipD:PrgI interface in this particular arrangement may thus inhibit the T3SS function. The structures presented in this study provide insight into the open state of the T3SS needle tip. Our findings present the atomic details of the T3SS arrangement occurring at the pathogen-host interface.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Membrana/química , Subunidades Proteicas/química , Salmonella typhimurium/fisiologia , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ácido Desoxicólico/química , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão , Ressonância de Plasmônio de Superfície
19.
Eur J Immunol ; 41(4): 981-91, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21337542

RESUMO

Even though Mycobacterium tuberculosis (Mtb) remains one of the top microbial killers, more than 90% of the 2 billion infected individuals never develop active tuberculosis (TB), indicating efficient immune control of infection in these individuals. Immune mechanisms promoting either control or reactivation of TB are incompletely understood. Kinetic analyses of T-cell responses against Mtb in C57BL/6 mice revealed surface expression of inducible co-stimulatory molecule (ICOS) on >30% of all CD4(+) T cells, suggesting a pivotal role of this costimulatory molecule of the CD28 family in TB control. Surprisingly, Mtb-infected ICOS(-/-) mice showed lower bacterial burden during the late chronic stage of infection as compared to WT controls. ICOS deficiency resulted in a reduced Mtb-specific CD8(+) T-cell response during late-stage infection. In contrast, the polyclonal CD4(+) Th1 response against Mtb was increased, most likely caused by diminished numbers and frequencies of Tregs. Thus, by altering effector T-cell populations differentially, ICOS signaling modulates TB control in the late stage of infection.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Tuberculose/metabolismo , Tuberculose/patologia
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