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1.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1431-1439, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748601

RESUMO

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 µg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.


Assuntos
Células da Medula Óssea , Macrófagos , Fagocitose , Animais , Células da Medula Óssea/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Lipopolissacarídeos/metabolismo , Macrófagos/classificação , Macrófagos/fisiologia , Camundongos
2.
Nat Commun ; 11(1): 3797, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732998

RESUMO

Receptor-mediated perception of surface-exposed carbohydrates like lipo- and exo-polysaccharides (EPS) is important for non-self recognition and responses to microbial associated molecular patterns in mammals and plants. In legumes, EPS are monitored and can either block or promote symbiosis with rhizobia depending on their molecular composition. To establish a deeper understanding of receptors involved in EPS recognition, we determined the structure of the Lotus japonicus (Lotus) exopolysaccharide receptor 3 (EPR3) ectodomain. EPR3 forms a compact structure built of three putative carbohydrate-binding modules (M1, M2 and LysM3). M1 and M2 have unique ßαßß and ßαß folds that have not previously been observed in carbohydrate binding proteins, while LysM3 has a canonical ßααß fold. We demonstrate that this configuration is a structural signature for a ubiquitous class of receptors in the plant kingdom. We show that EPR3 is promiscuous, suggesting that plants can monitor complex microbial communities though this class of receptors.


Assuntos
Lipopolissacarídeos/metabolismo , Lotus/microbiologia , Lotus/fisiologia , Mesorhizobium/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Mesorhizobium/genética , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas/genética , Dobramento de Proteína , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/fisiologia , Simbiose/fisiologia
3.
PLoS Pathog ; 16(8): e1008639, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790743

RESUMO

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Leptospira/imunologia , Leptospirose/imunologia , Lipopolissacarídeos/metabolismo , Lipoproteínas/metabolismo , Antígenos O/metabolismo , Receptor 4 Toll-Like/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Citocinas/metabolismo , Feminino , Leptospirose/metabolismo , Leptospirose/microbiologia , Leptospirose/patologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Antígenos O/genética , Transdução de Sinais , Receptor 2 Toll-Like/fisiologia
4.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
5.
Nat Commun ; 11(1): 3384, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636379

RESUMO

Targeting single mediators has failed to reduce the mortality of sepsis. We developed a telodendrimer (TD) nanotrap (NT) to capture various biomolecules via multivalent, hybrid and synergistic interactions. Here, we report that the immobilization of TD-NTs in size-exclusive hydrogel resins simultaneously adsorbs septic molecules, e.g. lipopolysaccharides (LPS), cytokines and damage- or pathogen-associated molecular patterns (DAMPs/PAMPs) from blood with high efficiency (92-99%). Distinct surface charges displayed on the majority of pro-inflammatory cytokines (negative) and anti-inflammatory cytokines (positive) allow for the selective capture via TD NTs with different charge moieties. The efficacy of NT therapies in murine sepsis is both time-dependent and charge-dependent. The combination of the optimized NT therapy with a moderate antibiotic treatment results in a 100% survival in severe septic mice by controlling both infection and hyperinflammation, whereas survival are only 50-60% with the individual therapies. Cytokine analysis, inflammatory gene activation and tissue histopathology strongly support the survival benefits of treatments.


Assuntos
Dendrímeros/química , Inflamação/terapia , Nanopartículas/química , Sepse/terapia , Adsorção , Animais , Antibacterianos/uso terapêutico , Citocinas/metabolismo , Feminino , Humanos , Hidrogéis , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanomedicina , Padrões Moleculares Associados a Patógenos , Células RAW 264.7
6.
Nat Commun ; 11(1): 3276, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581219

RESUMO

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.


Assuntos
Caspases Iniciadoras/metabolismo , Citosol/microbiologia , Proteínas de Ligação ao GTP/metabolismo , Lipopolissacarídeos/metabolismo , Salmonella/metabolismo , Linhagem Celular , Ativação Enzimática , Células Epiteliais/metabolismo , Células HeLa , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Ligação Proteica , Piroptose , Eletricidade Estática
7.
J Appl Oral Sci ; 28: e20190699, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401938

RESUMO

Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.


Assuntos
Cavidade Pulpar/microbiologia , Dinoprostona/metabolismo , Leucotrieno B4/metabolismo , Lipopolissacarídeos/metabolismo , Periodontite Periapical/microbiologia , Animais , Araquidonato 5-Lipoxigenase/análise , Araquidonato 5-Lipoxigenase/metabolismo , Reabsorção Óssea/metabolismo , Reabsorção Óssea/microbiologia , Ciclo-Oxigenase 2/análise , Ciclo-Oxigenase 2/metabolismo , Cavidade Pulpar/metabolismo , Cavidade Pulpar/patologia , Dinoprostona/análise , Expressão Gênica , Leucotrieno B4/análise , Masculino , Camundongos Endogâmicos C57BL , Periodontite Periapical/metabolismo , Periodontite Periapical/patologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
8.
Mol Cell ; 78(4): 683-699.e11, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32386575

RESUMO

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Glicosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Proteína de Transporte de Acila/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Parede Celular/metabolismo , Galactanos/metabolismo , Glicosiltransferases/genética , Lipopolissacarídeos/metabolismo , Mutação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Filogenia , Conformação Proteica , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 117(21): 11207-11216, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32424102

RESUMO

Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular, whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Here, we describe Escherichia coli phage U136B, showing it relies on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC and 2) the structural barrier molecule lipopolysaccharide (LPS). Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, we used fluctuation experiments and experimental evolution to obtain phage-resistant mutants. Using these mutants, we compared the accessibility of specific mutations (revealed in the fluctuation experiments) to their actual success during ecological competition and coevolution (revealed in the evolution experiments). Both tolC and LPS-related mutants arise readily during fluctuation assays, with tolC mutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance via tolC mutations occurs with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically confer increased antibiotic resistance. We discuss the molecular mechanisms underlying this surprising pleiotropic result, consideration for applied phage biology, and the importance of ecology in evolution of phage resistance. We envision that phages may be useful for the reversal of antibiotic resistance, but such applications will need to account for unexpected pleiotropy and evolutionary context.


Assuntos
Colífagos/fisiologia , Farmacorresistência Bacteriana/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Pleiotropia Genética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Biblioteca Gênica , Genes Bacterianos , Especificidade de Hospedeiro , Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Mutação
10.
Am J Physiol Gastrointest Liver Physiol ; 318(6): G1070-G1087, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390462

RESUMO

Lipopolysaccharides (LPS) are potent pro-inflammatory molecules that enter the systemic circulation from the intestinal lumen by uncertain mechanisms. We investigated these mechanisms and the effect of exogenous glucagon-like peptide-2 (GLP-2) on LPS transport in the rodent small intestine. Transmucosal LPS transport was measured in Ussing-chambered rat jejunal mucosa. In anesthetized rats, the appearance of fluorescein isothiocyanate (FITC)-LPS into the portal vein (PV) and the mesenteric lymph was simultaneously monitored after intraduodenal perfusion of FITC-LPS with oleic acid and taurocholate (OA/TCA). In vitro, luminally applied LPS rapidly appeared in the serosal solution only with luminal OA/TCA present, inhibited by the lipid raft inhibitor methyl-ß-cyclodextrin (MßCD) and the CD36 inhibitor sulfosuccinimidyl oleate (SSO), or by serosal GLP-2. In vivo, perfusion of FITC-LPS with OA/TCA rapidly increased FITC-LPS appearance into the PV, followed by a gradual increase of FITC-LPS into the lymph. Rapid PV transport was inhibited by the addition of MßCD or by SSO, whereas transport into the lymph was inhibited by chylomicron synthesis inhibition. Intraveous injection of the stable GLP-2 analog teduglutide acutely inhibited FITC-LPS transport into the PV, yet accelerated FITC-LPS transport into the lymph via Nω-nitro-l-arginine methyl ester (l-NAME)- and PG97-269-sensitive mechanisms. In vivo confocal microscopy in mouse jejunum confirmed intracellular FITC-LPS uptake with no evidence of paracellular localization. This is the first direct demonstration in vivo that luminal LPS may cross the small intestinal barrier physiologically during fat absorption via lipid raft- and CD36-mediated mechanisms, followed by predominant transport into the PV, and that teduglutide inhibits LPS uptake into the PV in vivo.NEW & NOTEWORTHY We report direct in vivo confirmation of transcellular lipopolysaccharides (LPS) uptake from the intestine into the portal vein (PV) involving CD36 and lipid rafts, with minor uptake via the canonical chylomicron pathway. The gut hormone glucagon-like peptide-2 (GLP-2) inhibited uptake into the PV. These data suggest that the bulk of LPS absorption is via the PV to the liver, helping clarify the mechanism of LPS transport into the PV as part of the "gut-liver" axis. These data do not support the paracellular transport of LPS, which has been implicated in the pathogenesis of the "leaky gut" syndrome.


Assuntos
Gorduras/metabolismo , Intestino Delgado/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Fármacos Gastrointestinais/farmacologia , Células HEK293 , Humanos , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
11.
Life Sci ; 253: 117606, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32320707

RESUMO

BACKGROUND/AIMS: In cirrhosis, the levels of proinflammatory cytokines are high in the liver and blood. Endotoxin decreases level of consciousness in cirrhotic rats. Phosphatidylserine exists in the cell membrane structure and is essential for the survival of neurons. Phosphatidylserine receptor is found in phagocytic cells and also activates the signaling of membrane proteins in apoptotic process. Therefore this study was aimed to explore the hypothesis that hepatic encephalopathy is prevented by phosphatidylserine treatment and if so, whether this is associated with altered level of proinflammatory cytokines in the brain. METHODS: Cirrhosis was induced by surgical ligation of the bile duct in male Wister rats. The groups were treated with phosphatidylserine and saline for 4 weeks. Brain IL6, TNFα and the expression of phosphatidylserine receptor were assessed. Intraperitoneal injections of either saline or lipopolysaccharide (0.1 mg/kg) were administered to each group. Finally, animal behavior, blood ammonia and the expression of toll like receptor 4 were examined in the brain. RESULTS: Cirrhosis in rats was associated with altered expression of toll-like receptor4 in brain cortex and phosphatidylserine treatment increases toll-like receptor4 receptor expression. Phosphatidylserine had anti-inflammatory effect in healthy rats but no effect in cirrhotic rats. Chronic phosphatidylserine treatment decreased blood ammonia in BDL cirrhotic rats treated with lipopolysaccharide. CONCLUSION: The brain of cirrhotic rat is more susceptible to acute endotoxemia and chronic phosphatidylserine treatment decreases blood ammonia and encephalopathy in cirrhotic rats by encountering endotoxin. Phosphatidylserine may boost immune system against endotoxin.


Assuntos
Anti-Inflamatórios/farmacologia , Endotoxemia/tratamento farmacológico , Encefalopatia Hepática/tratamento farmacológico , Encefalopatia Hepática/prevenção & controle , Cirrose Hepática/metabolismo , Fosfatidilserinas/farmacologia , Animais , Anti-Inflamatórios/metabolismo , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Ductos Biliares/metabolismo , Encéfalo , Citocinas/metabolismo , Endotoxinas/metabolismo , Encefalopatia Hepática/complicações , Lipopolissacarídeos/metabolismo , Fígado , Cirrose Hepática Experimental , Masculino , Fagócitos/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Receptor 4 Toll-Like/metabolismo , Resultado do Tratamento
12.
PLoS One ; 15(4): e0230482, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32310973

RESUMO

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype.


Assuntos
Lesão Pulmonar Aguda , Modelos Animais de Doenças , Transfusão de Eritrócitos , Lipopolissacarídeos/metabolismo , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Animais , Incompatibilidade de Grupos Sanguíneos/metabolismo , Complemento C5a/metabolismo , DNA/sangue , Eritrócitos/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Masculino , Infiltração de Neutrófilos , Ratos , Ratos Wistar , Reação Transfusional/patologia
13.
PLoS Pathog ; 16(4): e1008469, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324807

RESUMO

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Bombyx/microbiologia , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Modelos Moleculares , Ligação Proteica , Virulência/fisiologia
14.
Nat Commun ; 11(1): 1229, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144274

RESUMO

Liquid-liquid phase separation (LLPS) explains many intracellular activities, but its role in extracellular functions has not been studied to the same extent. Here we report how LLPS mediates the extracellular function of galectin-3, the only monomeric member of the galectin family. The mechanism through which galectin-3 agglutinates (acting as a "bridge" to aggregate glycosylated molecules) is largely unknown. Our data show that its N-terminal domain (NTD) undergoes LLPS driven by interactions between its aromatic residues (two tryptophans and 10 tyrosines). Our lipopolysaccharide (LPS) micelle model shows that the NTDs form multiple weak interactions to other galectin-3 and then aggregate LPS micelles. Aggregation is reversed when interactions between the LPS and the carbohydrate recognition domains are blocked by lactose. The proposed mechanism explains many of galectin-3's functions and suggests that the aromatic residues in the NTD are interesting drug design targets.


Assuntos
Aglutinação , Galectina 3/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Agregados Proteicos , Glicosilação , Lipopolissacarídeos/metabolismo , Micelas , Domínios Proteicos
15.
Life Sci ; 253: 117539, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32165213

RESUMO

AIMS: Lipopolysaccharide (LPS)-induced intestinal injury is a common clinical feature of sepsis. Aggravated inflammation and higher sensitivity to infection are associated with high-fat diet (HFD) in patients with type 2 diabetes and/or obesity. However, the mechanism by which HFD exacerbates LPS-induced intestinal injury has not been elucidated. This study aims to examine the effects of HFD on intestinal injury induced by LPS and the underlying mechanism. MAIN METHODS: Mice were fed with HFD or regular chow for 12weeks and were then challenged with LPS. Vas2870 was administered to mice that received HFD before the initiation of the diet. The levels of tight junction protein expression, oxidative stress, organ injury, and nicotinamide adenine dinucleotide phosphate (NADPH)-associated proteins were assessed periodically. KEY FINDINGS: LPS treatment resulted in severe intestinal pathological injury and increased oxidative stress, evidenced by significantly increased serum diamine oxidase, reactive oxygen species, malondialdehyde, and intestinal fatty acid binding protein contents. Additionally, a decrease in tight junction protein expression was observed, indicating a loss of tight junction integrity. LPS treatment induced the expression of Nox2 and Nox4. All the effects were more severe in HFD mice. Treatment with vas2870 conferred protection against LPS-induced intestinal injury in HFD-fed mice, partially reduced oxidative stress, and rescued the expression of tight junction proteins. CONCLUSION: HFD aggravated LPS-induced intestine injury through exacerbating intestinal Nox-related oxidative stress, which led to a loss of the integrity of tight junctions and consequently increased intestinal permeability.


Assuntos
Dieta Hiperlipídica , Intestinos/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Adsorção , Animais , Benzoxazóis/administração & dosagem , Benzoxazóis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Obesidade/metabolismo , Oxirredução , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Triazóis/administração & dosagem , Triazóis/metabolismo
16.
Nat Microbiol ; 5(5): 688-696, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32123346

RESUMO

The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1-6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7-11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15-17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interferon Tipo I/metabolismo , Infecções por Rickettsia/imunologia , Rickettsia/metabolismo , Animais , Proteínas de Transporte/metabolismo , Caspases Iniciadoras/genética , Linhagem Celular , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata , Fatores Reguladores de Interferon , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Rickettsia/genética , Rickettsia/patogenicidade
17.
Chemistry ; 26(28): 6247-6256, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32166806

RESUMO

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos/química , Lipídeos de Membrana/química , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Hemólise , Lipopolissacarídeos/metabolismo , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína
18.
Biochemistry (Mosc) ; 85(2): 234-240, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32093599

RESUMO

Helicobacter pylori is an important human pathogen that causes gastritis, gastric and duodenal ulcers, and gastric cancer. O-polysaccharides of H. pylori lipopolysaccharide (LPS) are composed of (ß1→3)-poly(N-acetyllactosamine) (polyLacNAc) decorated with multiple α-L-fucose residues. In many strains, their terminal LacNAc units are mono- or di-fucosylated to mimic Lewis X (Lex) and/or Lewis Y (Ley) oligosaccharides. The studies in rhesus macaques as a model of human infection by H. pylori showed that this bacterium adapts to the host during colonization by expressing host Lewis antigens. Here, we characterized LPS from H. pylori strains used in the previous study, including the parental J166 strain and the three derivatives (98-149, 98-169, and 98-181) isolated from rhesus macaques after long-term colonization. Chemical and NMR spectroscopic analyses of the LPS showed that the parent strain expressed Lex, Ley, and H type 1 terminal oligosaccharide units. The daughter strains were similar to the parental one in the presence of the same LPS core and fucosylated polyLacNAc chain of the same length but differed in the terminal oligosaccharide units. These were Lex in the isolates 98-149 and 98-169, which corresponded to the Lea phenotype of the host animals, and Ley was found in the 98-181 isolate from the macaque characterized by the Leb phenotype. As Lea and Leb are isomers of Lex and Ley, respectively, the observed correlation confirmed adaptation of the expression of terminal oligosaccharide units in H. pylori strains to the properties of the host gastric mucosa. The 98-181 strain also acquired glucosylation of the polyLacNAc chain and was distinguished by a lower expression of fucosylated internal LacNAc units (internal Lex) as a result of decoration of polyLacNAc with ß-glucopyranose, which may also play a role in the bacterial adaptation.


Assuntos
Helicobacter pylori/química , Lipopolissacarídeos/química , Macaca mulatta/microbiologia , Oligossacarídeos/genética , Polissacarídeos/metabolismo , Animais , Glicosilação , Helicobacter pylori/metabolismo , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Fenótipo , Polissacarídeos/química
19.
Animal ; 14(S1): s65-s77, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32024569

RESUMO

Improvements in feed intake of dairy cows entering the early lactation period potentially decrease the risk of metabolic disorders, but before developing approaches targeting the intake level, mechanisms controlling and dysregulating energy balance and feed intake need to be understood. This review focuses on different inflammatory pathways interfering with the neuroendocrine system regulating feed intake of periparturient dairy cows. Subacute inflammation in various peripheral organs often occurs shortly before or after calving and is associated with increased pro-inflammatory cytokine levels. These cytokines are released into the circulation and sensed by neurons located in the hypothalamus, the key brain region regulating energy balance, to signal reduction in feed intake. Besides these peripheral humoral signals, glia cells in the brain may produce pro-inflammatory cytokines independent of peripheral inflammation. Preliminary results show intensive microglia activation in early lactation, suggesting their involvement in hypothalamic inflammation and the control of feed intake of dairy cows. On the other hand, pro-inflammatory cytokine-induced activation of the vagus nerve transmits signalling to the brain, but this pathway seems not exclusively necessary to signal feed intake reduction. Yet, less studied in dairy cows so far, the endocannabinoid system links inflammation and the hypothalamic control of feed intake. Distinct endocannabinoids exert anti-inflammatory action but also stimulate the posttranslational cleavage of neuronal proopiomelanocortin towards ß-endorphin, an orexigen promoting feed intake. Plasma endocannabinoid concentrations and hypothalamic ß-endorphin levels increase from late pregnancy to early lactation, but less is known about the regulation of the hypothalamic endocannabinoid system during the periparturient period of dairy cows. Dietary fatty acids may modulate the formation of endocannabinoids, which opens new avenues to improve metabolic health and immune status of dairy cows.


Assuntos
Bovinos/fisiologia , Citocinas/metabolismo , Metabolismo Energético , Inflamação/veterinária , Leite/metabolismo , Transdução de Sinais , Animais , Dieta/veterinária , Feminino , Hipotálamo/metabolismo , Lactação , Lipopolissacarídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Gravidez
20.
Oxid Med Cell Longev ; 2020: 8630275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32089785

RESUMO

Background: Neurodegenerative diseases (ND) as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis represent a growing cause of disability in the developed countries. The underlying physiopathology is still unclear. Several lines of evidence suggest a role for oxidative stress and NADPH oxidase 2 (NOX2) in the neuropathological pathways that lead to ND. Furthermore, recent studies hypothesized a role for gut microbiota in the neuroinflammation; in particular, lipopolysaccharide (LPS) derived from Gram-negative bacteria in the gut is believed to play a role in causing ND by increase of oxidative stress and inflammation. The aim of this study was to assess NOX2 activity as well as serum 8-iso-prostaglandin F2α (8-iso-PGF2α (8-iso-PGF2. Methods: One hundred and twenty-eight consecutive subjects, including 64 ND patients and 64 controls (CT) matched for age and gender, were recruited. A cross-sectional study was performed to compare serum activity of soluble NOX2-dp (sNOX2-dp), blood levels of isoprostanes, serum H2O2, and LPS in these two groups. Serum zonulin was used to assess gut permeability. Results: Compared with CT, ND patients had higher values of sNOX2-dp, 8-iso-PGF2α (8-iso-PGF2p < 0.001), zonulin (Rs = 0.411; p < 0.001), zonulin (Rs = 0.411; p < 0.001), zonulin (Rs = 0.411; α (8-iso-PGF2p < 0.001), zonulin (Rs = 0.411; p < 0.001), zonulin (Rs = 0.411; α (8-iso-PGF2p < 0.001), zonulin (Rs = 0.411; ß, 0.459; p < 0.001), zonulin (Rs = 0.411; α (8-iso-PGF2ß, 0.459; p < 0.001), zonulin (Rs = 0.411; R 2 = 57%). Conclusion: This study provides the first report attesting that patients with ND have high NOX2 activation that could be potentially implicated in the process of neuroinflammation.


Assuntos
Lipopolissacarídeos/metabolismo , NADPH Oxidase 2/metabolismo , Doenças Neurodegenerativas/genética , Idoso , Feminino , Humanos , Masculino , Doenças Neurodegenerativas/fisiopatologia , Estresse Oxidativo
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