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1.
Nature ; 609(7925): 144-150, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35850148

RESUMO

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.


Assuntos
Antitoxinas , Bacteriófagos , Retroelementos , Salmonella typhimurium , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Bacteriófagos/metabolismo , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Conformação de Ácido Nucleico , Prófagos/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos/genética , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/virologia , Sistemas Toxina-Antitoxina/genética
2.
PLoS Pathog ; 18(3): e1010308, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35231068

RESUMO

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Macrófagos , Camundongos , Testes de Sensibilidade Microbiana , Polimixinas/farmacologia , Proteômica
3.
Nature ; 559(7713): 259-263, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29973719

RESUMO

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine1,2. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.


Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Benzaldeídos/farmacologia , Colistina/farmacologia , Combinação de Medicamentos , Interações Medicamentosas , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Aditivos Alimentares/farmacologia , Larva/efeitos dos fármacos , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Filogenia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Especificidade da Espécie
4.
Mol Syst Biol ; 17(2): e10188, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33590968

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a system-wide scale so that potential host-directed therapies can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.


Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Estabilidade Proteica , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Antivirais/farmacologia , COVID-19/virologia , Humanos , Proteoma , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Temperatura , Replicação Viral/efeitos dos fármacos
5.
Bioinformatics ; 35(12): 2017-2028, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388198

RESUMO

MOTIVATION: Type III secreted effectors (T3SEs) can be injected into host cell cytoplasm via type III secretion systems (T3SSs) to modulate interactions between Gram-negative bacterial pathogens and their hosts. Due to their relevance in pathogen-host interactions, significant computational efforts have been put toward identification of T3SEs and these in turn have stimulated new T3SE discoveries. However, as T3SEs with new characteristics are discovered, these existing computational tools reveal important limitations: (i) most of the trained machine learning models are based on the N-terminus (or incorporating also the C-terminus) instead of the proteins' complete sequences, and (ii) the underlying models (trained with classic algorithms) employed only few features, most of which were extracted based on sequence-information alone. To achieve better T3SE prediction, we must identify more powerful, informative features and investigate how to effectively integrate these into a comprehensive model. RESULTS: In this work, we present Bastion3, a two-layer ensemble predictor developed to accurately identify type III secreted effectors from protein sequence data. In contrast with existing methods that employ single models with few features, Bastion3 explores a wide range of features, from various types, trains single models based on these features and finally integrates these models through ensemble learning. We trained the models using a new gradient boosting machine, LightGBM and further boosted the models' performances through a novel genetic algorithm (GA) based two-step parameter optimization strategy. Our benchmark test demonstrates that Bastion3 achieves a much better performance compared to commonly used methods, with an ACC value of 0.959, F-value of 0.958, MCC value of 0.917 and AUC value of 0.956, which comprehensively outperformed all other toolkits by more than 5.6% in ACC value, 5.7% in F-value, 12.4% in MCC value and 5.8% in AUC value. Based on our proposed two-layer ensemble model, we further developed a user-friendly online toolkit, maximizing convenience for experimental scientists toward T3SE prediction. With its design to ease future discoveries of novel T3SEs and improved performance, Bastion3 is poised to become a widely used, state-of-the-art toolkit for T3SE prediction. AVAILABILITY AND IMPLEMENTATION: http://bastion3.erc.monash.edu/. CONTACT: selkrig@embl.de or wyztli@163.com or or trevor.lithgow@monash.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Aprendizado de Máquina , Algoritmos , Sequência de Aminoácidos , Proteínas de Bactérias , Biologia Computacional , Bactérias Gram-Negativas , Software
6.
Mol Syst Biol ; 12(12): 888, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27909043

RESUMO

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making.


Assuntos
Células-Tronco Embrionárias Humanas/citologia , Proteínas Quinases/metabolismo , Biologia de Sistemas/métodos , Diferenciação Celular , Bases de Dados de Proteínas , Regulação Enzimológica da Expressão Gênica , Células HeLa , Células-Tronco Embrionárias Humanas/enzimologia , Humanos , Fosforilação , Proteômica/métodos , Transdução de Sinais
7.
Biochim Biophys Acta ; 1843(8): 1542-50, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24135059

RESUMO

Membrane proteins with a ß-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of ß-barrel protein assembly into membranes, and the components of the ß-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bactérias Gram-Negativas/química , Mitocôndrias/metabolismo , Transporte Proteico/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Mitocôndrias/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
8.
Eur J Cell Biol ; 103(4): 151448, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39128247

RESUMO

Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

9.
Nat Microbiol ; 8(11): 2196-2212, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37770760

RESUMO

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface ( https://apps.embl.de/combact/ ).


Assuntos
Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus , Antibacterianos/farmacologia , Bactérias Gram-Positivas , Combinação de Medicamentos
10.
Mol Biol Evol ; 28(5): 1581-91, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21081480

RESUMO

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.


Assuntos
Eucariotos/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Transporte Proteico/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Eucariotos/ultraestrutura , Evolução Molecular , Transferência Genética Horizontal , Interações Hidrofóbicas e Hidrofílicas , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA
11.
Cell Rep ; 34(2): 108602, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33440154

RESUMO

Protein kinases lie at the heart of cell-signaling processes and are often mutated in disease. Kinase target recognition at the active site is in part determined by a few amino acids around the phosphoacceptor residue. However, relatively little is known about how most preferences are encoded in the kinase sequence or how these preferences evolved. Here, we used alignment-based approaches to predict 30 specificity-determining residues (SDRs) for 16 preferences. These were studied with structural models and were validated by activity assays of mutant kinases. Cancer mutation data revealed that kinase SDRs are mutated more frequently than catalytic residues. We have observed that, throughout evolution, kinase specificity has been strongly conserved across orthologs but can diverge after gene duplication, as illustrated by the G protein-coupled receptor kinase family. The identified SDRs can be used to predict kinase specificity from sequence and aid in the interpretation of evolutionary or disease-related genomic variants.


Assuntos
Eucariotos/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/genética , Animais , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Transdução de Sinais
12.
Cell Host Microbe ; 29(8): 1316-1332.e12, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34237247

RESUMO

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Domínios e Motivos de Interação entre Proteínas , Salmonella enterica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Bactérias , Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Feminino , Células HeLa , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Células RAW 264.7 , Salmonella enterica/genética , Salmonella typhimurium/metabolismo
13.
mSystems ; 5(2)2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127419

RESUMO

The molecular architecture and function of the Gram-negative bacterial cell envelope are dictated by protein composition and localization. Proteins that localize to the inner membranes (IM) and outer membranes (OM) of Gram-negative bacteria play critical and distinct roles in cellular physiology; however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. Here, we employed multiplexed quantitative mass spectrometry using tandem mass tag (TMT) labeling to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing Escherichia coli K-12 cells on a sucrose density gradient. The migration patterns for >1,600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli For 559 proteins that are currently annotated as peripherally associated with the IM (G. Orfanoudaki and A. Economou, Mol Cell Proteomics 13:3674-3687, 2014, https://doi.org/10.1074/mcp.O114.041137) and that display potential for dual localization to either the IM or cytoplasm, we could allocate 110 proteins to the IM and 206 proteins to the soluble cell fraction based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of these cases, we were able to find supportive evidence for our localization findings in the literature. We anticipate that our systems-level analysis of the E. coli membrane proteome will serve as a useful reference data set to query membrane protein localization, as well as to provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species.IMPORTANCE Current knowledge of protein localization, particularly outer membrane proteins, is highly dependent on bioinformatic predictions. To date, no systematic experimental studies have directly compared protein localization spanning the inner and outer membranes of E. coli By combining sucrose density gradient fractionation of inner membrane (IM) and outer membrane (OM) proteins with multiplex quantitative proteomics, we systematically quantified localization patterns for >1,600 proteins, providing high-confidence localization annotations for 1,368 proteins. Of these proteins, we resolve the predominant localization of 316 proteins that currently have dual annotation (cytoplasmic and IM) in protein databases and identify new annotations for 42 additional proteins. Overall, we present a novel quantitative methodology to systematically map membrane proteins in Gram-negative bacteria and use it to unravel the biological complexity of the membrane proteome architecture in E. coli.

14.
Nat Microbiol ; 5(9): 1119-1133, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514074

RESUMO

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.


Assuntos
Catepsinas/metabolismo , Morte Celular/fisiologia , Macrófagos/metabolismo , Proteômica , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Animais , Catepsinas/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cistatina B/antagonistas & inibidores , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Lisossomos/metabolismo , Macrófagos/microbiologia , Camundongos , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteoma , Células RAW 264.7 , Infecções por Salmonella/microbiologia
15.
Sci Transl Med ; 11(502)2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341063

RESUMO

The functional interactions between the gut microbiota and the host are important for host physiology, homeostasis, and sustained health. We compared the skeletal muscle of germ-free mice that lacked a gut microbiota to the skeletal muscle of pathogen-free mice that had a gut microbiota. Compared to pathogen-free mouse skeletal muscle, germ-free mouse skeletal muscle showed atrophy, decreased expression of insulin-like growth factor 1, and reduced transcription of genes associated with skeletal muscle growth and mitochondrial function. Nuclear magnetic resonance spectrometry analysis of skeletal muscle, liver, and serum from germ-free mice revealed multiple changes in the amounts of amino acids, including glycine and alanine, compared to pathogen-free mice. Germ-free mice also showed reduced serum choline, the precursor of acetylcholine, the key neurotransmitter that signals between muscle and nerve at neuromuscular junctions. Reduced expression of genes encoding Rapsyn and Lrp4, two proteins important for neuromuscular junction assembly and function, was also observed in skeletal muscle from germ-free mice compared to pathogen-free mice. Transplanting the gut microbiota from pathogen-free mice into germ-free mice resulted in an increase in skeletal muscle mass, a reduction in muscle atrophy markers, improved oxidative metabolic capacity of the muscle, and elevated expression of the neuromuscular junction assembly genes Rapsyn and Lrp4 Treating germ-free mice with short-chain fatty acids (microbial metabolites) partly reversed skeletal muscle impairments. Our results suggest a role for the gut microbiota in regulating skeletal muscle mass and function in mice.


Assuntos
Microbioma Gastrointestinal/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Animais , Linhagem Celular , Microbioma Gastrointestinal/genética , Vida Livre de Germes , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL
16.
Sci Rep ; 8(1): 10646, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006625

RESUMO

In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal's survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved.


Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Vida Livre de Germes/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Microbiota/fisiologia , Animais , Corte , Drosophila melanogaster/microbiologia , Feminino , Locomoção/fisiologia , Masculino , Sono/fisiologia , Simbiose/fisiologia
17.
Genome Biol Evol ; 7(6): 1628-43, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-25994932

RESUMO

Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Evolução Molecular , Motivos de Aminoácidos , Proteínas da Membrana Bacteriana Externa/classificação , Bacteroidetes/genética , Variação Genética , Família Multigênica , Filogenia , Proteobactérias/genética
18.
Sci Rep ; 5: 12905, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26243377

RESUMO

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/fisiologia , Sequência Conservada , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
19.
Gut Microbes ; 5(3): 369-80, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24685620

RESUMO

Brain development is an energy demanding process that relies heavily upon diet derived nutrients. Gut microbiota enhance the host's ability to extract otherwise inaccessible energy from the diet via fermentation of complex oligosaccharides in the colon. This nutrient yield is estimated to contribute up to 10% of the host's daily caloric requirement in humans and fluctuates in response to environmental variations. Research over the past decade has demonstrated a surprising role for the gut microbiome in normal brain development and function. In this review we postulate that perturbations in the gut microbial-derived nutrient supply, driven by environmental variation, profoundly impacts upon normal brain development and function.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Alimentos , Trato Gastrointestinal/microbiologia , Metabolismo , Microbiota , Humanos
20.
Nat Commun ; 5: 5078, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25341963

RESUMO

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Nanopartículas/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Mutagênese Insercional/genética , Estrutura Secundária de Proteína , Técnicas de Microbalança de Cristal de Quartzo , Especificidade por Substrato
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