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1.
Am J Vet Res ; 80(8): 792-798, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339765

RESUMO

OBJECTIVE: To investigate effects of body condition on permeability of intestinal mucosa in horses. ANIMALS: 13 horses (7 obese and 6 lean) from 8 to 15 years of age. PROCEDURES: Body condition score was assessed, and an oral sugar test (OST) was performed to evaluate glucose and insulin dynamics. Horses were allowed a 2-week diet acclimation period and were then euthanized. Tissue samples were collected from the jejunum, ileum, cecum, pelvic flexure, right dorsal colon, and rectum. Mucosal permeability was assessed by measuring transepithelial resistance and lipopolysaccharide (LPS) flux across tissue samples mounted in Ussing chambers. RESULTS: 5 obese horses and 1 lean horse had evidence of insulin dysregulation, whereas 1 obese and 5 lean horses had no abnormalities in results of the OST. Results for the OST were not available for 1 obese horse. Mucosal transepithelial resistance did not differ in any intestinal segment between obese and lean horses. Obese horses had a significantly higher LPS flux across jejunal mucosa, compared with results for lean horses, but there were no significant differences between obese and lean horses for other intestinal segments. CONCLUSIONS AND CLINICAL RELEVANCE: Obese horses may have had greater paracellular mucosal permeability of jejunal mucosa to LPS, compared with that for lean horses. This finding was consistent with data for the gastrointestinal mucosa of humans and mice and supported the hypothesis that obese horses may be at higher risk from chronic exposure to increased amounts of LPS, compared with the risk for lean horses.


Assuntos
Doenças dos Cavalos/metabolismo , Mucosa Intestinal/metabolismo , Obesidade/veterinária , Animais , Constituição Corporal , Ceco/metabolismo , Colo/metabolismo , Absorção Gastrointestinal , Glucose/metabolismo , Cavalos , Insulina/metabolismo , Jejuno/metabolismo , Lipopolissacarídeos/metabolismo , Obesidade/metabolismo , Permeabilidade
2.
Biochemistry (Mosc) ; 84(4): 398-406, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228931

RESUMO

To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.


Assuntos
Elementos de DNA Transponíveis/genética , Yersinia pestis/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Carboidratos , Farmacorresistência Bacteriana/genética , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Mutagênese , Polimixina B/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/genética
3.
Subcell Biochem ; 92: 9-37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214983

RESUMO

Gram-negative bacteria have an outer membrane that is positioned at the frontline of the cell's interaction with the environment and that serves as a barrier against noxious molecules including many antibiotics. This protective function mainly relies on lipopolysaccharide, a complex glycolipid located in the outer leaflet of the outer membrane. In this chapter we will first summarize lipopolysaccharide structure, functions and biosynthetic pathway and then we will discuss how it is transported and assembled to the cell surface. This is a remarkably complex process, as amphipathic lipopolysaccharide molecules must traverse three different cellular compartments to reach their final destination.


Assuntos
Membrana Celular/metabolismo , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Transporte Biológico
4.
BMC Infect Dis ; 19(1): 548, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226940

RESUMO

BACKGROUND: This retrospective study evaluated the performance of a lipoarabinomannan (LAM)-based immunological method for diagnosing pleural tuberculosis (TB) from pleural effusion samples. Results were compared to those obtained using conventional culture and molecular testing methods. METHODS: Suspected pleural TB patients who visited Beijing Chest Hospital for medical care between January 2016 and June 2017 were retrospectively analysed in the study. Pleural effusion samples were tested for Mycobacterium tuberculosis (MTB) using the BACTEC MGIT 960 System, GeneXpert, and an anti-LAM antibody assay (LAM assay). RESULTS: Pleural effusion samples were collected from a total of 219 retrospectively recruited participants suspected of having pleural TB. Thirteen of 155 confirmed pleural TB cases tested positive for MTB via MGIT culture, for a sensitivity of 8.4% [95% confidence interval (CI): 4.0-12.8%]. In addition, GeneXpert and LAM testing identified 22 and 55 pleural TB cases, for sensitivities of 14.2% (95% CI: 8.7-19.7%) and 35.5% (95% CI: 28.1-43.6%), respectively. The specificities of these two assays were 100.0% (95% CI: 92.9-100.0%) and 96.9% (95% CI: 88.2-99.5%), respectively. Combined application of culture and LAM testing identified 60 positive cases, for a sensitivity of 38.7% (95% CI: 31.0-46.4%) that was significantly higher than that of MGIT culture alone (P < 0.01). Similarly, use of LAM testing in combination with GeneXpert led to correct diagnosis of 40.0% (95% CI: 32.3-47.7%) of pleural TB cases, a higher rate than obtained using GeneXpert alone (P < 0.01). In addition, the specificity of the combined assay of GeneXpert and LAM testing was 96.9% (95% CI: 88.2-99.5%). Patients aged 25 to 44 years were more likely to have positive LAM assay results than those ≥65 years of age (P = 0.02). Meanwhile, the proportion of diabetic patients with positive LAM assay results was significantly lower than that of the non-diabetes group (P = 0.03). CONCLUSIONS: An anti-LAM antibody detection assay showed potential for diagnosis of pleural TB from pleural effusion samples. Combined use of the LAM assay with MGIT culture or GeneXpert methods could improve sensitivity for improved pleural TB diagnosis compared to results of individual conventional tests alone.


Assuntos
Algoritmos , Testes Imunológicos/métodos , Lipopolissacarídeos/imunologia , Mycobacterium tuberculosis , Derrame Pleural/diagnóstico , Reação em Cadeia da Polimerase/métodos , Tuberculose Pleural/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Tipagem Bacteriana/métodos , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Derrame Pleural/microbiologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Fatores de Tempo , Tuberculose Pleural/microbiologia
5.
Cell Mol Life Sci ; 76(18): 3667-3678, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31062071

RESUMO

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1ß release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.


Assuntos
Cardiolipinas/farmacologia , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Ligação Competitiva , Cardiolipinas/química , Cardiolipinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL10/metabolismo , Células HEK293 , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
Br J Anaesth ; 123(1): 51-59, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084986

RESUMO

BACKGROUND: Macrophage phagocytosis constitutes an essential part of the host defence against microbes and the resolution of inflammation. Hyperglycaemia during sepsis is reported to reduce macrophage function, and thus, potentiate inflammatory deterioration. We investigated whether high-glucose concentrations augment lipopolysaccharide-induced reduction in macrophage phagocytosis via the endoplasmic stress-C/EBP homologous protein (CHOP) pathway using animal and laboratory investigations. METHODS: Peritoneal macrophages of artificially ventilated male Wistar rats, divided into four groups based on target blood glucose concentrations achieved by glucose administration with or without lipopolysaccharide, were obtained after 24 h. Human macrophages were also cultured in normal or high glucose with or without lipopolysaccharide exposure for 72 h. Changes in the phagocytic activity, intranuclear CHOP expression, and intracellular Akt phosphorylation status of macrophages were evaluated. These changes were also evaluated in human macrophages after genetic knock-down of CHOP by specific siRNA transfection or resolvin D2 treatment. RESULTS: Lipopolysaccharide impaired phagocytosis, increased intranuclear expression of CHOP, and inhibited Akt phosphorylation in both rat peritoneal and human macrophages. Hyperglycaemic glucose concentrations augmented these changes. Genetic knock-down of CHOP restored phagocytic ability and Akt phosphorylation in human macrophages. Furthermore, resolvin D2 co-incubation restored the inhibited phagocytosis and Akt phosphorylation along with the inhibition of intranuclear CHOP expression in human macrophages. CONCLUSIONS: These findings imply that controlling endoplasmic reticulum stress might provide new strategies for restoring reduced macrophage phagocytosis in sepsis-induced hyperglycaemia.


Assuntos
Hiperglicemia/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fator de Transcrição CHOP/metabolismo , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Humanos , Masculino , Ratos , Ratos Wistar , Transdução de Sinais , Fator de Transcrição CHOP/genética
7.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 31(4): 468-473, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31109423

RESUMO

OBJECTIVE: To evaluate the effect and mechanism of rivaroxaban, an inhibitor of coagulation factor Xa (FXa), on endotoxin-induced injury to human umbilical vein endothelial cells (HUVEC). METHODS: When cultured HUVEC grow to 80% fusion, they were divided into four groups according to the random number method: blank control group (DMEM medium), lipopolysaccharide (LPS) group (cells were challenged by 100 µg/L LPS for 16 hours), FXa+LPS group (cells were challenged by LPS for 16 hours after they were cultured with 100 nmol/L FXa for 24 hours), and FXa +RIV+LPS group (cells were challenged by LPS for 16 hours after they were cultured with 100 nmol/L FXa and 1 µmol/L rivaroxaban for 24 hours). After each group of cells were challenged with LPS, the cell activity was detected by the cell proliferation and toxicity kit (CCK-8); the cell migration ability was detected by cell scratch experiments; the abilities of cells migration were measured by scratch-wound-healing assay; the apoptosis of cells were evaluated using flow cytometry; the endothelial barrier of cells was assessed by Transwell and Evans blue; the levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1ß, IL-6) were detected by the enzyme linked immunosorbent assay (ELISA); the expressions of nuclear factor-ΚB (NF-ΚB) and mitogen activated protein kinase (MAPK) signaling pathway were detected by Western Blot. RESULTS: Compared with blank control group, the cell viability in LPS group was significantly decreased, and the migration ability, number of apoptotic cells, and barrier permeability of endothelial cells was significantly increased, the levels of TNF-α, IL-1ß and IL-6 were significantly increased, and the expressions of phosphorylation of c-Jun N-terminal kinase (p-JNK), phosphorylation of p38MAPK (p-p38MAPK), phosphorylation of transforming growth factor kinase 1 (p-TAK1) and phosphorylation of NF-ΚBp65 (p-NF-ΚBp65) were significantly increased. It indicated that LPS could stimulate the inflammatory response of vascular endothelial cells, and had a significant impact on cell activity, apoptosis and function. There was no significant difference in above indexes between FXa+LPS group and LPS group, except for the level of IL-6 being higher in FXa+LPS group. Compared with FXa+LPS group, in FXa+RIV+LPS group, the cell activity was significantly increased (A value: 0.42±0.02 vs. 0.33±0.02), and migration ability was significantly decreased (folds: 1.78±0.17 vs. 2.24±0.20), the number of apoptotic cells was significantly decreased [(11.30±0.70)% vs. (21.03±0.19)%], and permeability of monolayers endothelial cells was significantly decreased [(149±12)% vs. (253±15)%], the levels of inflammatory cytokines were significantly decreased [IL-1ß (ng/L): 163.2±20.7 vs. 477.8±20.2, IL-6 (ng/L): 69.3±0.5 vs. 238.0±24.1, TNF-α (ng/L): 117.0±13.1 vs. 196.2±4.5], the expressions of p-TAK1 and p-NF-ΚBp65 were significantly decreased (p-TAK1/TAK1: 0.74±0.09 vs. 1.85±0.15, p-NF-ΚBp65/NF-ΚBp65: 1.15±0.17 vs. 2.36±0.20), with statistically significant differences (all P < 0.05). There was no significant difference in the p-JNK, p-p38MAPK expressions between FXa+RIV+LPS group and FXa+LPS group (p-JNK/JNK: 1.64±0.12 vs. 1.65±0.15, p-p38MAPK/p38MAPK: 2.31±0.32 vs. 2.35±0.20, both P > 0.05). CONCLUSIONS: Rivaroxaban can effectively relieve the inflammatory response of HUVEC stimulated by LPS, which may be related to the inhibition of NF-ΚB signaling pathway activation rather than MAPK signaling pathway.


Assuntos
Endotoxinas/efeitos adversos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Rivaroxabana/farmacologia , Humanos , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
8.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052177

RESUMO

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by deficits in social interaction and communication, and repetitive behaviors. In addition, co-morbidities such as gastro-intestinal problems have frequently been reported. Mutations and deletion of proteins of the SH3 and multiple ankyrin repeat domains (SHANK) gene-family were identified in patients with ASD, and Shank knock-out mouse models display autism-like phenotypes. SHANK3 proteins are not only expressed in the central nervous system (CNS). Here, we show expression in gastrointestinal (GI) epithelium and report a significantly different GI morphology in Shank3 knock-out (KO) mice. Further, we detected a significantly altered microbiota composition measured in feces of Shank3 KO mice that may contribute to inflammatory responses affecting brain development. In line with this, we found higher E. coli lipopolysaccharide levels in liver samples of Shank3 KO mice, and detected an increase in Interleukin-6 and activated astrocytes in Shank3 KO mice. We conclude that apart from its well-known role in the CNS, SHANK3 plays a specific role in the GI tract that may contribute to the ASD phenotype by extracerebral mechanisms.


Assuntos
Transtorno do Espectro Autista/microbiologia , Microbioma Gastrointestinal , Proteínas do Tecido Nervoso/genética , Animais , Astrócitos/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
9.
J Dairy Sci ; 102(6): 5706-5712, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954263

RESUMO

Antimicrobial peptides are a common defense against bacterial infections in many species and a significant part of the innate immune response of the bovine mammary gland. The objective of this study was to investigate the influence of epigenetic factors on vitamin D and toll-like receptor-mediated induction of ß-defensins in mammary epithelial cells. Primary bovine mammary epithelial cells were treated with lipopolysaccharide (LPS, 0 or 100 ng/mL), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0 or 10 nM], and 5-aza-2'-deoxycytidine (5-Aza, inhibitor of DNA methyltransferase, 0 or 5 µM) or trichostatin A (TSA, inhibitor of histone deacetylase, 0 or 80 nM) in a factorial arrangement. Effects of treatments on ß-defensin gene expression along with genes for cytokines and enzymes known to be induced by LPS or 1,25(OH)2D3 were evaluated by quantitative PCR. The LPS treatment induced expression of ß-defensin (DEFB)3, DEFB5, DEFB7, DEFB10, enteric ß-defensin (EBD), lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide (TAP); whereas, the 1,25(OH)2D3 treatment increased DEFB5 and DEFB7 expression and decreased LAP. The 5-Aza treatment increased expression of DEFB3, DEFB5, DEFB10, EBD, LAP, and TAP in the presence and absence of LPS. The TSA treatment increased expression of DEFB3, DEFB4, DEFB5, DEFB7, and DEFB10 in the absence of LPS but decreased LPS-induced expression of and LAP and TAP. Together these results indicate that ß-defensin expression in bovine mammary epithelial cells is likely influenced by DNA methylation and histone acetylation. Investigation of environmental and nutritional factors that influence epigenetic control of ß-defensins in the mammary gland may be beneficial for improving resistance to intramammary infections.


Assuntos
Bovinos/metabolismo , Células Epiteliais/metabolismo , Histona Desacetilases/metabolismo , Lipopolissacarídeos/metabolismo , Glândulas Mamárias Animais/metabolismo , Metiltransferases/metabolismo , Vitamina D/análogos & derivados , beta-Defensinas/genética , Animais , Bovinos/genética , Metilação de DNA , Feminino , Histona Desacetilases/genética , Glândulas Mamárias Animais/citologia , Metiltransferases/genética , Vitamina D/metabolismo , beta-Defensinas/metabolismo
10.
Oxid Med Cell Longev ; 2019: 6043245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944694

RESUMO

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS from Escherichia coli for fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.


Assuntos
Escherichia coli/genética , Leucócitos/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Escherichia coli/metabolismo , Humanos , Estresse Oxidativo , Espécies Reativas de Oxigênio
11.
Clin Hemorheol Microcirc ; 71(2): 175-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958337

RESUMO

 Polymers are often contaminated with lipopolysaccharides also known as endotoxins. Even small amounts of endotoxins can have strong effects on endothelial cell function so that the endothelialisation of cardiovascular implants might be hampered. An open question is how endothelial cells seeded on a body foreign substrate respond to shear load after adding Lipid A (LPA), the domain, which is responsible for much of the toxicity of gram-negative bacteria, and whether morphological changes of endothelial cells occur.LPA supplementation to the culture medium in increasing concentrations (5, 25 and 50µg/ml) resulted in progressive reductions of the density of adherent HUVEC after shear load (p < 0.001). 48% of the HUVEC in control cultures (0µg/ml LPA) were still adherent after 2 hours of shearing at 6 dyne/cm2, while 80 minutes after addition of 50µg/ml LPA, 88% of the HUVEC had already detached from the substrate and after 100 minutes no more HUVEC were attached.The results demonstrate that endotoxins are of extreme importance for the behavior of HUVEC and that in vivo pathologies can be increasingly simulated in vitro.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Lipopolissacarídeos/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos
12.
Genes Genet Syst ; 94(2): 71-80, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-30971625

RESUMO

In Bacillus subtilis, extracytoplasmic function (ECF) sigma factors are activated by reduction of phosphatidylglycerol (PG) content, absence of glucolipids, or absence of lipoteichoic acid (LTA). LTA is synthesized by polymerization of the glycerophosphate moiety of PG onto diglucosyldiacylglycerol (DGlcDG), a major glucolipid in B. subtilis, in the plasma membrane. Thus, reduction of PG content or absence of glucolipids might cause some changes in LTA, and hence we investigated whether reduction of PG content or absence of glucolipids induces the activation of ECF sigma factors independently from an ensuing change in LTA. Disruption of ugtP, responsible for glucolipid synthesis, in cells lacking LTA caused an additive increase of activation levels of σM, σX, σV and σY (3.1-, 2.2-, 2.1- and 1.4-fold, respectively), relative to their activation levels in cells lacking LTA alone. Reduction of PG content (by repressing Pspac-pgsA) in the cells lacking LTA caused an additive increase of activation levels of σM, σW and σV (2.3-, 1.9- and 2.2-fold, respectively). These results suggested that absence of glucolipids or reduction of PG alone, not the possible secondary alteration in LTA, leads to changes that affect the regulation systems of some ECF sigma factors in the plasma membrane.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Fator sigma/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Glicolipídeos/metabolismo , Lipopolissacarídeos/metabolismo , Fosfatidilgliceróis/metabolismo , Fator sigma/genética , Ácidos Teicoicos/metabolismo
13.
Nat Immunol ; 20(5): 527-533, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962589

RESUMO

Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.


Assuntos
Caspases/imunologia , Citosol/imunologia , Imunidade Inata/imunologia , Lipopolissacarídeos/imunologia , Animais , Caspases/metabolismo , Citosol/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Modelos Imunológicos , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Piroptose/imunologia
14.
MBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015326

RESUMO

Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections. C. trachomatis sexually transmitted infections are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we studied the ability of chlamydial LPS to activate the proinflammatory canonical and noncanonical inflammasome pathways in mouse bone marrow-derived macrophages (BMDM). We show, in comparison to Escherichia coli LPS, that C. trachomatis LPS-treated BMDM produce significantly less IL-6, TNF, and type I interferon mRNA, indicating that downstream signaling through the canonical TLR4 myddosome and triffosome pathways was blocked. We confirmed this in C. trachomatis LPS-treated BMDM by showing the lack of NF-κB and IRF3 phosphorylation, respectively. Interestingly, C. trachomatis LPS bound CD14 and promoted its endocytosis; however; it did not promote efficient TLR4/MD-2 dimerization or endocytosis, known requirements for myddosome and triffosome signaling pathways. We further found that transfection of BMDM with C. trachomatis LPS did not cause pyroptotic cell ballooning, cytotoxicity, or IL-1ß secretion, all characteristic features of noncanonical inflammasome activation. Western blotting confirmed that cytosolic C. trachomatis LPS failed to signal through caspase-11, as shown by the lack of gasdermin D, caspase-1, or IL-1ß proteolytic cleavage. We propose that chlamydiae evolved a unique LPS structure as a pathogenic strategy to avoid canonical and noncanonical innate immune signaling and conclude that this strategy might explain the high incidence of asymptomatic infections.IMPORTANCE Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections (STI). C. trachomatis STI are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we found that C. trachomatis LPS is not capable of engaging the canonical TLR4/MD-2 or noncanonical caspase-11 inflammatory pathways. The inability of C. trachomatis LPS to trigger innate immunity inflammatory pathways is related to its unique fatty acid structure. Evolutionary modification of the LPS structure likely evolved as a pathogenic strategy to silence innate host defense mechanisms. The findings might explain the high incidence of asymptomatic chlamydial genital infection.


Assuntos
Chlamydia trachomatis/imunologia , Chlamydia trachomatis/patogenicidade , Evasão da Resposta Imune , Imunidade Inata , Lipopolissacarídeos/metabolismo , Fatores de Virulência/metabolismo , Animais , Citocinas/biossíntese , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Perfilação da Expressão Gênica , Macrófagos/imunologia , Camundongos Endogâmicos C57BL
15.
Int J Med Microbiol ; 309(3-4): 213-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31010630

RESUMO

Clinical isolates of Klebsiella pneumoniae are often resistant to beta-lactam antibiotics via the acquisition of extended spectrum beta lactamase (ESBL) enzymes paired with loss of one or both major outer membrane porins. It has been well established that loss of OmpK35 and/or OmpK36 correlates with increased minimum inhibitory concentrations of antibiotics that target the peptidoglycan. However, little is known concerning the downstream effects porin loss might have on other major virulence factors such as the polysaccharide capsule or LPS. Furthermore, it is unknown whether these cumulative changes impact pathogenesis. Therefore, the focus of this study was to identify alterations in production of the major virulence factors due to porin loss; and to investigate the effect these changes have on host pathogen interactions. Our data demonstrates that loss of a single porin is paired with reductions in capsule, increased LPS content, and up-regulated transcription of compensatory porin genes. In contrast, loss of both porins resulted in a significant increase in capsule production. Loss of OmpK35 alone or dual porin loss was further associated with reduced oxidative burst by macrophages and increased ability of the bacteria to survive phagocytic killing. These data indicate that porin loss is accompanied by a suite of changes in other virulence-associated factors. These cumulative changes act to nullify any negative fitness effect due to lack of the nonspecific porin proteins, allowing the bacteria to grow and survive phagocytic immune responses.


Assuntos
Klebsiella pneumoniae/fisiologia , Klebsiella pneumoniae/patogenicidade , Macrófagos/microbiologia , Porinas/deficiência , Fatores de Virulência/metabolismo , Animais , Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Viabilidade Microbiana , Porinas/genética , Células RAW 264.7 , Transcrição Genética , Fatores de Virulência/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
16.
Prep Biochem Biotechnol ; 49(6): 616-622, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929584

RESUMO

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110 kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1 mg/200 ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.


Assuntos
Clonagem Molecular/métodos , Proteínas de Escherichia coli/biossíntese , Proteínas Hemolisinas/biossíntese , Proteínas Recombinantes/biossíntese , Escherichia coli Uropatogênica/enzimologia , Acilação , Aciltransferases/genética , Cromatografia de Afinidade/métodos , Ensaios Enzimáticos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/isolamento & purificação , Lipopolissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Escherichia coli Uropatogênica/genética
17.
J Toxicol Sci ; 44(4): 283-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944281

RESUMO

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Polimixina B/farmacologia , Proteínas/efeitos adversos , Proteínas/imunologia , Testes Cutâneos/métodos , Pele/imunologia , Clorometilcetonas de Aminoácidos/administração & dosagem , Clorometilcetonas de Aminoácidos/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Meios de Cultura , Expressão Gênica/efeitos dos fármacos , Humanos , Imunização , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/metabolismo , Muramidase , Ovalbumina , Ovomucina , Polimixina B/administração & dosagem , Polimixina B/metabolismo , Ligação Proteica , Células THP-1 , Temperatura Ambiente , Fatores de Tempo
18.
Res Vet Sci ; 124: 321-327, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31035220

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a worrying cause of diarrhoea in calves and the drug multiresistance phenotype concerning various antibiotic families are of concern. Resistance mechanisms associated with envelope changes (porin expression, efflux pump overexpression, lipolysaccahride (LPS) modification) were studied in 14 ETEC isolates selected for their resistance. We performed determinations of (i) antimicrobials Minimal Inhibitory Concentrations with or without the efflux pump inhibitor phenylalanine arginine ß-naphthylamide; (ii) colistin and polymyxin MICs with and without EDTA, (iii) intracellular accumulation of chloramphenicol in presence of an energy uncoupler of pump energy, (iv) and immunodetection of porins and evaluation of porin trimers thermostability. Results indicated that 9 strains presented significant efflux mechanisms overexpression, among them 8 were resistant to colistin and polymyxin B due to a modification of LPS structure as evidenced by EDTA effect and silver staining electrophoresis. The high resistant strains to colistin and polymyxin exhibited identical LPS patterns. Studies of E. coli porins indicated that the majority of strains didn't show modification in their amount, however analysis of porin thermostability showed that porin trimers of some resistant strains were relatively heat-labile, suggesting a misassembly of the functional trimer. The multidrug resistance (MDR) phenotypes detected in these selected ETEC corresponded to association of LPS modifications, abordive assembly of porin trimers and active efflux which drastically alter the antibiotic activity currently used to combat enteric infections caused by this pathogen.


Assuntos
Antibacterianos/farmacologia , Doenças dos Bovinos/microbiologia , Diarreia/veterinária , Farmacorresistência Bacteriana Múltipla , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Infecções por Escherichia coli/veterinária , Lipopolissacarídeos/metabolismo , Animais , Bovinos , Cloranfenicol/farmacologia , Indústria de Laticínios , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/fisiologia , Infecções por Escherichia coli/microbiologia , Fluoroquinolonas/farmacologia , Membranas/efeitos dos fármacos , Membranas/fisiologia , Testes de Sensibilidade Microbiana , Permeabilidade , Polimixinas/farmacologia
19.
Mar Drugs ; 17(5)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027390

RESUMO

PT-peptide is derived from the anti-lipopolysaccharide factor of the swimming crab Portunus trituberculatus. The peptide, consisting of 34 amino acids, contains a lipopolysaccharide binding domain. In this study, we investigated the effect of PT-peptide encapsulated in raw milk-derived extracellular vesicles (EVs), designated as EVs-PT peptide, on immune regulation. The results showed that raw milk-derived EVs efficaciously delivered the PT-peptide into monocytes and elevated immune activity, including reactive oxygen species level, superoxide anion production, and phagocytosis. PT-peptide and EVs-PT peptide also elevated the secretion of cytokines, such as interferon-γ, interleukin-6, and tumor necrosis factor-α in human monocytic THP-1 cells. These results suggest that the PT-peptide could be developed as an immune stimulator.


Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Proteínas de Artrópodes/administração & dosagem , Braquiúros , Sistemas de Liberação de Medicamentos/métodos , Monócitos/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Composição de Medicamentos/métodos , Vesículas Extracelulares/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/metabolismo , Leite/química , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo
20.
ACS Appl Mater Interfaces ; 11(17): 15389-15400, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951282

RESUMO

The antimicrobial effects of Laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ potential measurements, and confocal microscopy. As a result of the net negative charge of Laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the Laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite their net positive charge, peptide-loaded Laponite nanoparticles did not kill Gram-negative Escherichia coli bacteria or disrupt anionic model liposomes. They did however cause bacteria flocculation, originating from the interaction of Laponite and bacterial lipopolysaccharide (LPS). Free LL-37, in contrast, is potently antimicrobial through membrane disruption but does not induce bacterial aggregation in the concentration range investigated. Through LL-37 loading of Laponite nanoparticles, the combined effects of bacterial flocculation and membrane lysis are observed. However, bacteria aggregation seems to be limited to Gram-negative bacteria as Laponite did not cause flocculation of Gram-positive Bacillus subtilis bacteria nor did it bind to lipoteichoic acid from bacterial envelopes. Taken together, the present investigation reports several novel phenomena by demonstrating that nanoparticle charge does not invariably control membrane destabilization and by identifying the ability of anionic Laponite nanoparticles to effectively flocculate Gram-negative bacteria through LPS binding. As demonstrated in cell experiments, such aggregation results in diminished LPS-induced cell activation, thus outlining a promising approach for confinement of infection and inflammation caused by such pathogens.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Nanopartículas/química , Silicatos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Nanopartículas/metabolismo , Tamanho da Partícula , Silicatos/metabolismo
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