RESUMO
Inflammatory bowel disease (IBD) is a chronic disease of the gastrointestinal tract affecting millions of people. Here, we investigated the expression and functions of poly(ADP-ribose) polymerase 14 (Parp14), an important regulatory protein in immune cells, with an IBD patient cohort as well as two mouse colitis models, that is, IBD-mimicking oral dextran sulfate sodium (DSS) exposure and oral Salmonella infection. Parp14 was expressed in the human colon by cells in the lamina propria, but, in particular, by the epithelial cells with a granular staining pattern in the cytosol. The same expression pattern was evidenced in both mouse models. Parp14-deficiency caused increased rectal bleeding as well as stronger epithelial erosion, Goblet cell loss, and immune cell infiltration in DSS-exposed mice. The absence of Parp14 did not affect the mouse colon bacterial microbiota. Also, the colon leukocyte populations of Parp14-deficient mice were normal. In contrast, bulk tissue RNA-Seq demonstrated that the colon transcriptomes of Parp14-deficient mice were dominated by abnormalities in inflammation and infection responses both prior and after the DSS exposure. Overall, the data indicate that Parp14 has an important role in the maintenance of colon epithelial barrier integrity. The prognostic and predictive biomarker potential of Parp14 in IBD merits further investigation.
Assuntos
Colite , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Poli(ADP-Ribose) Polimerases , Animais , Feminino , Humanos , Masculino , Camundongos , Colite/genética , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Colo/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/deficiênciaRESUMO
Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Toxina Pertussis , Toxinas Bacterianas/toxicidade , Citosol , Anticorpos Antibacterianos , Chaperonina com TCP-1RESUMO
The northern bat (Eptesicus nilssonii) is the most northern bat species in the world. Its distribution covers whole Eurasia, and the species is thus well adapted to different habitat types. However, recent population declines have been reported and rapid conservation efforts are needed. Here we present a high-quality de novo genome assembly of a female northern bat from Finland (BLF_Eptnil_asm_v1.0). The assembly was generated using a combination of Pacbio and Omni-C technologies. The primary assembly comprises 726 scaffolds spanning 2.0 Gb, represented by a scaffold N50 of 102 Mb, a contig N50 of 66.2 Mb, and a BUSCO completeness score of 93.73%. Annotation of the assembly identified 20,250 genes. This genome will be an important resource for the conservation and evolutionary genomic studies especially in understanding how rapid environmental changes affect northern species.
Assuntos
Quirópteros , Animais , Feminino , Quirópteros/genética , Genoma , Genômica , Evolução Biológica , CromossomosRESUMO
Bordetella pertussis toxin (PT) and Clostridium botulinum C2 toxin are ADP-ribosylating toxins causing severe diseases in humans and animals. They share a common translocation mechanism requiring the cellular chaperones Hsp90 and Hsp70, cyclophilins, and FK506-binding proteins to transport the toxins' enzyme subunits into the cytosol. Inhibitors of chaperone activities have been shown to reduce the amount of transported enzyme subunits into the cytosol of cells, thus protecting cells from intoxication by these toxins. Recently, domperidone, an approved dopamine receptor antagonist drug, was found to inhibit Hsp70 activity. Since Hsp70 is required for cellular toxin uptake, we hypothesized that domperidone also protects cells from intoxication with PT and C2. The inhibition of intoxication by domperidone was demonstrated by analyzing the ADP-ribosylation status of the toxins' specific substrates. Domperidone had no inhibitory effect on the receptor-binding or enzyme activity of the toxins, but it inhibited the pH-driven membrane translocation of the enzyme subunit of the C2 toxin and reduced the amount of PTS1 in cells. Taken together, our results indicate that domperidone is a potent inhibitor of PT and C2 toxins in cells and therefore might have therapeutic potential by repurposing domperidone to treat diseases caused by bacterial toxins that require Hsp70 for their cellular uptake.
Assuntos
Toxinas Bacterianas , Toxinas Botulínicas , Animais , Humanos , Bordetella pertussis/metabolismo , Domperidona/farmacologia , Toxinas Botulínicas/toxicidade , Toxinas Bacterianas/metabolismo , Toxina Pertussis , ADP Ribose Transferases/metabolismoRESUMO
Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough.
Assuntos
Coqueluche , Humanos , Criança , Toxina Pertussis/farmacologia , Coqueluche/microbiologia , Bordetella pertussis , Proteínas , Linhagem CelularRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-associated bacterial keratitis is highly intractable, with strong resistance to ß-lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin-binding protein PBP2a has been implicated in the sensitization of ß-lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye-drop system capable of penetrating biofilms and targeting bacteria for chemo-gene therapy in MRSA-caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA-specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, ß-lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down-regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1ß ï¼IL-1ßï¼ and tumor neocrosis factor-alpha ï¼TNF-αï¼, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA-induced keratitis.
Assuntos
Ceratite , Staphylococcus aureus Resistente à Meticilina , Óxido de Zinco , Humanos , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , DNA/metabolismo , Ampicilina/metabolismo , Ampicilina/farmacologia , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia , Ceratite/tratamento farmacológico , Ceratite/genética , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/metabolismoRESUMO
Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.
Assuntos
NAD , Toxina Pertussis/química , Fatores de Virulência de Bordetella/química , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Bordetella pertussis , Citosol/metabolismo , NAD/metabolismoRESUMO
The paradigm of antivirulence therapy dictates that bacterial pathogens are specifically disarmed but not killed by neutralizing their virulence factors. Clearance of the invading pathogen by the immune system is promoted. As compared to antibiotics, the pathogen-selective antivirulence drugs hold promise to minimize collateral damage to the beneficial microbiome. Also, selective pressure for resistance is expected to be lower because bacterial viability is not directly affected. Antivirulence drugs are being developed for stand-alone prophylactic and therapeutic treatments but also for combinatorial use with antibiotics. This Review focuses on drug modalities that target bacterial exotoxins after the secretion or release-upon-lysis. Exotoxins have a significant and sometimes the primary role as the disease-causing virulence factor, and thereby they are attractive targets for drug development. We describe the key pre-clinical and clinical trial data that have led to the approval of currently used exotoxin-targeted drugs, namely the monoclonal antibodies bezlotoxumab (toxin B/TcdB, Clostridioides difficile), raxibacumab (anthrax toxin, Bacillus anthracis), and obiltoxaximab (anthrax toxin, Bacillus anthracis), but also to challenges with some of the promising leads. We also highlight the recent developments in pre-clinical research sector to develop exotoxin-targeted drug modalities, i.e., monoclonal antibodies, antibody fragments, antibody mimetics, receptor analogs, neutralizing scaffolds, dominant-negative mutants, and small molecules. We describe how these exotoxin-targeted drug modalities work with high-resolution structural knowledge and highlight their advantages and disadvantages as antibiotic alternatives.
Assuntos
Bacillus anthracis , Toxinas Bacterianas , Clostridioides difficile , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , ExotoxinasRESUMO
Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not ß-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.
Assuntos
Anti-Infecciosos/farmacologia , Toxina Pertussis/antagonistas & inibidores , alfa-Defensinas/farmacologia , Animais , Peptídeos Antimicrobianos , Bordetella pertussis/metabolismo , Criança , Humanos , Toxina Pertussis/metabolismo , Fatores de Virulência de Bordetella , CoquelucheRESUMO
Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.
Assuntos
Bordetella pertussis/enzimologia , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Leucocitose , Chaperonas Moleculares , Toxina Pertussis/toxicidade , Animais , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Células CHO , Cricetulus , Células Epiteliais/microbiologia , Células HEK293 , Humanos , Leucocitose/induzido quimicamente , Leucocitose/tratamento farmacológico , Leucocitose/metabolismo , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require thousands of mice annually. Here, we describe Interference in Gαi-mediated Signal Transduction (iGIST), an animal-free kinetic bioassay for detection of PTX, by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells coexpressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high-affinity agonist octreotide; and a luminescent 3'5'-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in the pg/mL range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e., a commercial PTX-toxoid-containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.
Assuntos
Bioensaio , Toxina Pertussis , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , CamundongosRESUMO
Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Förster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory α-subunit of the heterotrimeric G-protein Gαi showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD+ hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose-response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format.
Assuntos
Cisteína/análise , Transferência Ressonante de Energia de Fluorescência , Peptídeos/química , Cisteína/metabolismo , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor ErbB-4/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3 , Mapas de Interação de ProteínasRESUMO
We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/química , Guanosina Trifosfato/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/metabolismo , Descoberta de Drogas , Toxina Pertussis/antagonistas & inibidores , Toxina Pertussis/metabolismo , Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/patogenicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NAD/metabolismoRESUMO
Protein ADP-ribosylation is a reversible post-translational modification, which alters protein activity, localization, interactome or stability, leading to perturbation of cell signaling. This review summarizes the emerging data indicating that host cell ADP-ribosylating enzymes, poly(ADP-ribose) polymerases (PARPs), influence the course of a bacterial infection, in parallel to ADP-ribosylating bacterial toxins. Host cell PARP targeting could be an efficient therapeutic approach to treat certain bacterial infections, possibly by repurposing the approved or clinical trial PARP inhibitors developed for cancer therapy.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Poli(ADP-Ribose) Polimerases/imunologia , ADP-Ribosilação/efeitos dos fármacos , Animais , Bactérias/classificação , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/imunologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Arthropod-borne diseases remain a major health-threat for humans and animals worldwide. To estimate the distribution of pathogenic agents and especially Bartonella spp., we conducted tick microbiome analysis and determination of the infection status of wild animals, pets and pet owners in the state of Hesse, Germany. RESULTS: In total, 189 engorged ticks collected from 163 animals were tested. Selected ticks were analyzed by next generation sequencing (NGS) and confirmatory PCRs, blood specimens of 48 wild animals were analyzed by PCR to confirm pathogen presence and sera of 54 dogs, one cat and 11 dog owners were analyzed by serology. Bartonella spp. were detected in 9.5% of all ticks and in the blood of 17 roe deer. Further data reveal the presence of the human and animal pathogenic species of genera in the family Spirochaetaceae (including Borrelia miyamotoi and Borrelia garinii), Bartonella spp. (mainly Bartonella schoenbuchensis), Rickettsia helvetica, Francisella tularensis and Anaplasma phagocytophilum in ticks. Co-infections with species of several genera were detected in nine ticks. One dog and five dog owners were seropositive for anti-Bartonella henselae-antibodies and one dog had antibodies against Rickettsia conorii. CONCLUSIONS: This study provides a snapshot of pathogens circulating in ticks in central Germany. A broad range of tick-borne pathogens are present in ticks, and especially in wild animals, with possible implications for animal and human health. However, a low incidence of Bartonella spp., especially Bartonella henselae, was detected. The high number of various detected pathogens suggests that ticks might serve as an excellent sentinel to detect and monitor zoonotic human pathogens.
Assuntos
Doenças do Gato/transmissão , Cervos/microbiologia , Doenças do Cão/transmissão , Infecções por Bactérias Gram-Negativas/transmissão , Microbiota , Doenças Transmitidas por Carrapatos/transmissão , Carrapatos/microbiologia , Animais , Doenças do Gato/epidemiologia , Doenças do Gato/microbiologia , Gatos , Cervos/parasitologia , Doenças do Cão/epidemiologia , Doenças do Cão/microbiologia , Cães , Feminino , Alemanha/epidemiologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Masculino , Animais de Estimação , Risco , Alinhamento de Sequência/veterinária , Estudos Soroepidemiológicos , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/microbiologiaRESUMO
Galectin-3-binding protein (Gal-3BP) is a ubiquitous and multifunctional secreted glycoprotein originally identified and mainly studied in the context of neoplastic transformation and cancer progression. However, Gal-3BP expression is induced in viral infection and by a multitude of molecules that either mimic or are characteristic for an ongoing inflammation and microbial infection, such as IFN-α, IFN-ß, IFN-γ, TNF-α, poly(I:C), dsRNA, and dsDNA. Furthermore, Gal-3BP belongs to the scavenger receptor cysteine-rich (SRCR) domain-containing protein family, by virtue of its N-terminal SRCR domain. The SRCR domain is found in soluble or membrane-associated innate immunity-related proteins and is implicated in self-nonself discrimination. This review summarizes the current knowledge of structural features of Gal-3BP and its proposed intracellular and extracellular innate immunity functions with special emphasis on viral and bacterial infections.
Assuntos
Antígenos de Neoplasias/fisiologia , Infecções Bacterianas/imunologia , Biomarcadores Tumorais/fisiologia , Proteínas de Transporte/fisiologia , Glicoproteínas/fisiologia , Imunidade Inata , Viroses/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Líquidos Corporais/química , Química Encefálica , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Família Multigênica , Especificidade de Órgãos , Domínios Proteicos , Especificidade da Espécie , Relação Estrutura-Atividade , Vísceras/químicaRESUMO
The deer ked (Lipoptena cervi) is distributed in Europe, North America, and Siberia and mainly infests cervids as roe deer, fallow deer, and moose. From a one health perspective, deer keds occasionally bite other animals or humans and are a potential vector for Bartonella schoenbuchensis. This bacterium belongs to a lineage of ruminant-associated Bartonella spp. and is suspected to cause dermatitis and febrile diseases in humans. In this study, we analyzed the microbiome from 130 deer keds collected from roe deer, fallow deer and humans in the federal states of Hesse, Baden-Wuerttemberg, and Brandenburg, Germany. Endosymbiontic Arsenophonus spp. and Bartonella spp. represented the biggest portion (~90%) of the microbiome. Most Bartonella spp. (n = 93) were confirmed to represent B. schoenbuchensis. In deer keds collected from humans, no Bartonella spp. were detected. Furthermore, Acinetobacter spp. were present in four samples, one of those was confirmed to represent A. baumannii. These data suggest that deer keds harbor only a very narrow spectrum of bacteria which are potentially pathogenic for animals of humans.
RESUMO
Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to gamma-secretase cleavage among human RTKs. The screen covering 45 of the 55 human RTKs identified 12 new as well as all nine previously published gamma-secretase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the gamma-secretase component presenilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on gamma-secretase cleavage. Taken together, these data indicate that gamma-secretase-mediated cleavage provides an additional signaling mechanism for numerous human RTKs.