ABSTRACT
Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. Here, we show that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, we identified L. plantarum mutants sensitive to AMPs. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, our work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.
Subject(s)
Antimicrobial Peptides , Drosophila , Animals , Antimicrobial Cationic Peptides/genetics , Aging , InflammationABSTRACT
The methyltransferase FliB posttranslationally modifies surface-exposed É-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.
Subject(s)
Bacterial Proteins/metabolism , Flagellin/metabolism , Iron-Sulfur Proteins/metabolism , Lysine/metabolism , Methyltransferases/metabolism , S-Adenosylmethionine/metabolism , Salmonella typhi/enzymology , Bacterial Proteins/genetics , Flagellin/chemistry , Iron-Sulfur Proteins/genetics , Lysine/chemistry , Methylation , Methyltransferases/geneticsABSTRACT
Mouse guanylate-binding proteins (mGBPs) are recruited to various invasive pathogens, thereby conferring cell-autonomous immunity against these pathogens. However, whether and how human GBPs (hGBPs) target M. tuberculosis (Mtb) and L. monocytogenes (Lm) remains unclear. Here, we describe hGBPs association with intracellular Mtb and Lm, which was dependent on the ability of bacteria to induce disruption of phagosomal membranes. hGBP1 formed puncta structures which were recruited to ruptured endolysosomes. Furthermore, both GTP-binding and isoprenylation of hGBP1 were required for its puncta formation. hGBP1 was required for the recovery of endolysosomal integrity. In vitro lipid-binding assays demonstrated direct binding of hGBP1 to PI4P. Upon endolysosomal damage, hGBP1 was targeted to PI4P and PI(3,4)P2-positive endolysosomes in cells. Finally, live-cell imaging demonstrated that hGBP1 was recruited to damaged endolysosomes, and consequently mediated endolysosomal repair. In summary, we uncover a novel interferon-inducible mechanism in which hGBP1 contributes to the repair of damaged phagosomes/endolysosomes.
Subject(s)
GTP-Binding Proteins , Phagosomes , Humans , Animals , Mice , GTP-Binding Proteins/metabolism , Phagosomes/metabolism , Interferons/metabolism , Endosomes/metabolismABSTRACT
Retraction: Emoto, M., Emoto, Y., Yoshizawa, I., Kita, E., Shimizu, T., Hurwitz, R., Brinkmann, V. and Kaufmann, S.H.E. (2010), α-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1+ cells into the liver. Eur. J. Immunol., 40: 1328-1341. DOI: https://doi.org/10.1002/eji.200939594 The above article, published online on 16 February 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the Chairman of the Executive Committee of the European Journal of Immunology and Wiley-VCH Verlag GmbH & Co. KGaA. The retraction has been agreed following an investigation carried out by Gunma University (http://www.gunma-u.ac.jp/wp-content/uploads/2017/10/chosakekka29.pdf). The investigation was unable to determine the validity of the images for which Professor Emoto, the article's corresponding author, was responsible. As a result, the journal has made the decision to retract the article.
ABSTRACT
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.
Subject(s)
Host-Pathogen Interactions , Immunity, Innate , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/metabolism , Animals , Cell Line , Endocytosis/genetics , Endocytosis/immunology , Extracellular Space , Host-Pathogen Interactions/immunology , Humans , Interferon Type I/metabolism , Macrophages/immunology , Macrophages/metabolism , Membrane Proteins/metabolism , Mice , Models, Molecular , Nucleotides, Cyclic/chemistry , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/genetics , Protein Binding , Protein Conformation , Protein Multimerization , Second Messenger Systems , Signal Transduction , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Adenosine Diphosphate Sugars/metabolism , Helicobacter pylori/metabolism , Heptoses/metabolism , Pathogen-Associated Molecular Pattern Molecules/metabolism , Adenosine Diphosphate Sugars/chemistry , Adenosine Diphosphate Sugars/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gene Deletion , Genes, Bacterial , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Helicobacter pylori/genetics , Helicobacter pylori/immunology , Heptoses/chemistry , Heptoses/immunology , Humans , Immunity, Innate , NF-kappa B/metabolism , Pathogen-Associated Molecular Pattern Molecules/chemistry , Pathogen-Associated Molecular Pattern Molecules/immunology , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Mycobacterium tuberculosis/immunology , Pigments, Biological/metabolism , Pseudomonas aeruginosa/immunology , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Pattern Recognition/metabolism , Animals , Anti-Bacterial Agents/metabolism , Bone Marrow Cells/cytology , Cytokines/immunology , Cytokines/metabolism , Feedback, Physiological , Humans , Ligands , Macrophage Activation , Mice , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Phenazines/metabolism , Pigments, Biological/chemistry , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/metabolism , Pyocyanine/metabolism , Virulence Factors/chemistry , Virulence Factors/metabolismABSTRACT
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.
Subject(s)
Arginase/metabolism , Granuloma/enzymology , Hypoxia/enzymology , Macrophages/enzymology , Mycobacterium tuberculosis , Tuberculosis, Pulmonary/enzymology , Animals , Arginase/genetics , Arginase/immunology , Arginine/genetics , Arginine/immunology , Arginine/metabolism , Cell Proliferation/genetics , Disease Models, Animal , Granuloma/genetics , Granuloma/immunology , Granuloma/pathology , Humans , Hypoxia/genetics , Hypoxia/immunology , Hypoxia/pathology , Lung/enzymology , Lung/immunology , Lung/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Knockout , Nitric Oxide/genetics , Nitric Oxide/immunology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/immunology , Nitric Oxide Synthase Type II/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/pathologyABSTRACT
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.
Subject(s)
Clonal Selection, Antigen-Mediated/immunology , MicroRNAs/metabolism , Natural Killer T-Cells/immunology , Animals , Cell Proliferation , Ligands , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Natural Killer T-Cells/cytology , Receptors, Antigen, T-Cell, alpha-beta/metabolismABSTRACT
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.
Subject(s)
BCG Vaccine/immunology , CD4-Positive T-Lymphocytes/immunology , Immunologic Memory , Mycobacterium bovis/immunology , Tuberculosis/prevention & control , Animals , BCG Vaccine/administration & dosage , BCG Vaccine/genetics , Female , Gene Deletion , Male , Mice, Inbred C57BL , Mutagenesis, Insertional , Mycobacterium bovis/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
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(+) .
Subject(s)
Adenosine Diphosphate Ribose/metabolism , CD8 Antigens/immunology , CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Adenosine Diphosphate Ribose/immunology , Animals , Cell Separation , Flow Cytometry , Lymphocyte Activation/physiology , Mice , Mice, Inbred C57BL , NAD/immunology , NAD/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Neisseria gonorrhoeae/metabolism , Porins/chemistry , Porins/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/genetics , Base Sequence , Binding Sites/genetics , Cell Line , Crystallography, X-Ray , DNA, Bacterial/genetics , Host-Pathogen Interactions , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/pathogenicity , Peptidoglycan/metabolism , Phosphates/metabolism , Porins/genetics , Protein Conformation , Sequence Homology, Amino Acid , Static Electricity , Virulence/genetics , Virulence/physiologyABSTRACT
In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.
Subject(s)
Aplysia , Arginine , Lysine , Polyethylene Glycols , Animals , Arginine/pharmacology , Arginine/chemistry , Lysine/pharmacology , Lysine/chemistry , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Humans , Mice , Xenograft Model Antitumor Assays , Marine Toxins/pharmacology , Marine Toxins/therapeutic use , Marine Toxins/chemistry , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , L-Amino Acid Oxidase/pharmacology , L-Amino Acid Oxidase/metabolism , L-Amino Acid Oxidase/chemistry , Female , Cell Line, TumorABSTRACT
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.
Subject(s)
Antigens, Bacterial/chemistry , Bacterial Proteins/chemistry , Bacterial Secretion Systems/physiology , Host-Pathogen Interactions/physiology , Membrane Proteins/chemistry , Protein Subunits/chemistry , Salmonella typhimurium/physiology , Animals , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Crystallography, X-Ray , Deoxycholic Acid/chemistry , Membrane Proteins/metabolism , Membranes/chemistry , Membranes/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits/metabolism , Recombinant Fusion Proteins , Surface Plasmon ResonanceABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Cytosol/metabolism , Secretory Pathway/physiology , Shigella flexneri/pathogenicity , Amino Acid Sequence , Bacterial Outer Membrane Proteins/genetics , Crystallography, X-Ray , Gene Knockdown Techniques , HeLa Cells , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Sequence Alignment , Shigella flexneri/genetics , Shigella flexneri/metabolismABSTRACT
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.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , Immunologic Memory , Inducible T-Cell Co-Stimulator Protein , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Tuberculosis/metabolism , Tuberculosis/pathologyABSTRACT
Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.
Subject(s)
Apoptosis/physiology , Immunity, Innate/physiology , Neutrophil Activation/physiology , Neutrophils/physiology , Antibodies/pharmacology , Apoptosis/drug effects , Apoptosis/immunology , Catalase/antagonists & inhibitors , Catalase/pharmacology , Cell Death/drug effects , Cell Death/immunology , Cell Death/physiology , Cell Survival/drug effects , Cell Survival/immunology , Cell Survival/physiology , Chromatin/metabolism , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/ultrastructure , Enzyme Inhibitors/pharmacology , Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/pathology , Humans , Hydrogen Peroxide/pharmacology , Leukocyte Elastase/metabolism , Microscopy, Electron , NADPH Oxidases/antagonists & inhibitors , Neutrophil Activation/drug effects , Neutrophils/cytology , Neutrophils/microbiology , Nuclear Envelope/metabolism , Nuclear Envelope/ultrastructure , Onium Compounds/pharmacology , Phagocytosis/immunology , Reactive Oxygen Species/metabolism , Staphylococcus aureus/physiology , Tetradecanoylphorbol Acetate/pharmacology , Vacuoles/metabolism , Vacuoles/ultrastructure , fas Receptor/immunologyABSTRACT
Alpha-galactosylceramide (alpha-GalCer) activates invariant (i)NKT cells, which in turn stimulate immunocompetent cells. Although activation of iNKT cells appears critical for regulation of immune responses, it remains elusive whether protection against intracellular bacteria can be induced by alpha-GalCer. Here, we show that alpha-GalCer treatment ameliorates murine listeriosis, and inhibits inflammation following Listeria monocytogenes infection. Liver infiltration of Gr-1+ cells and gamma/delta T cells was accelerated by alpha-GalCer treatment. Gr-1+ cell and gamma/delta T-cell depletion exacerbated listeriosis in alpha-GalCer-treated mice, and this effect was more pronounced after depletion of Gr-1+ cells than that of gamma/delta T cells. Although GM-CSF and IL-17 were secreted by NKT cells after alpha-GalCer treatment, liver infiltration of Gr-1+ cells was not prevented by neutralizing mAb. In parallel to the numerical increase of CD11b+Gr-1+ cells in the liver following alpha-GalCer treatment, CD11b-Gr-1+ cells were numerically reduced in the bone marrow. In addition, respiratory burst in Gr-1+ cells was enhanced by alpha-GalCer treatment. Our results indicate that alpha-GalCer-induced antibacterial immunity is caused, in part, by accelerated infiltration of Gr-1+ cells and to a lesser degree of gamma/delta T cells into the liver. We also suggest that the infiltration of Gr-1+ cells is caused by an accelerated supply from the bone marrow.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Galactosylceramides/therapeutic use , Listeriosis/drug therapy , Liver/immunology , Monocytes/immunology , Receptors, Chemokine/analysis , Adjuvants, Immunologic/pharmacology , Animals , Chemotaxis, Leukocyte/drug effects , Drug Evaluation, Preclinical , Female , Galactosylceramides/pharmacology , Immunity, Innate , Listeriosis/immunology , Liver/microbiology , Liver/pathology , Lymphocyte Depletion , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Organ Specificity , Receptors, Antigen, T-Cell, gamma-delta/analysis , Respiratory Burst/drug effects , Specific Pathogen-Free Organisms , Spleen/immunology , Spleen/microbiology , Spleen/pathologyABSTRACT
Tuberculosis causes 2 million deaths per year, yet in most cases the immune response successfully contains the infection and prevents disease outbreak. Induced lymphoid structures associated with pulmonary granuloma are observed during tuberculosis in both humans and mice and could orchestrate host defense. To investigate whether granuloma perform lymphoid functions, mice lacking secondary lymphoid organs (SLO) were infected with Mycobacterium tuberculosis (MTB). As in WT mice, granuloma developed, exponential growth of MTB was controlled, and antigen-specific T-cell responses including memory T cells were generated in the absence of SLO. Moreover, adoptively transferred T cells were primed locally in lungs in a granuloma-dependent manner. T-cell activation was delayed in the absence of SLO, but resulted in a normal development program including protective subsets and functional recall responses that protected mice against secondary MTB infection. Our data demonstrate that protective immune responses can be generated independently of SLO during MTB infection and implicate local pulmonary T-cell priming as a mechanism contributing to host defense.
Subject(s)
Granuloma/immunology , Lymphoid Tissue/immunology , T-Lymphocytes/immunology , Tuberculosis, Pulmonary/immunology , Adoptive Transfer , Adult , Animals , Cell Separation , Chemokines/biosynthesis , Flow Cytometry , Gene Expression , Gene Expression Profiling , Granuloma/microbiology , Humans , Lasers , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Microdissection , Middle Aged , Mycobacterium tuberculosis/immunology , Oligonucleotide Array Sequence Analysis , Receptors, Chemokine/biosynthesisABSTRACT
The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection.