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1.
Carbohydr Polym ; 255: 117475, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436239

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.


Assuntos
Engenharia Celular/métodos , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/biossíntese , Escherichia coli Extraintestinal Patogênica/imunologia , Glicoconjugados/biossíntese , Antígenos O/biossíntese , Sequência de Aminoácidos , Animais , Animais não Endogâmicos , Anticorpos Antibacterianos/biossíntese , Sequência de Carboidratos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/mortalidade , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Vacinas contra Escherichia coli/imunologia , Escherichia coli Extraintestinal Patogênica/patogenicidade , Feminino , Glicoconjugados/administração & dosagem , Glicoconjugados/genética , Glicoconjugados/imunologia , Glicosilação , Imunização , Imunogenicidade da Vacina , Imunoglobulina G/biossíntese , Redes e Vias Metabólicas/genética , Camundongos , Antígenos O/genética , Antígenos O/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Análise de Sobrevida , Vacinas Conjugadas
2.
Nat Commun ; 11(1): 4035, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788578

RESUMO

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.


Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Polifosfatos/metabolismo , Animais , Apresentação do Antígeno/imunologia , Polaridade Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon Tipo I/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Fenótipo , Sepse/imunologia , Análise de Sobrevida , Transcriptoma/genética
3.
PLoS One ; 15(6): e0234484, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511271

RESUMO

Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1ß, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Inflamação/tratamento farmacológico , Ativação de Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/imunologia , Estresse Oxidativo/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Fumarato de Dimetilo/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/imunologia , Flavanonas/farmacologia , Humanos , Inflamação/imunologia , Isotiocianatos/farmacologia , Leucócitos Mononucleares , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Estresse Oxidativo/imunologia , Pirazinas/farmacologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/efeitos dos fármacos , Células THP-1 , Tionas , Tiofenos
4.
J Anim Sci ; 98(5)2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32300795

RESUMO

The objective of this experiment was to investigate the impact of an F18 enterotoxigenic Escherichia coli (ETEC) challenge on growth performance, aspects of intestinal function, and selected immune responses of piglets, as well as to evaluate potential protective effects of direct-fed microbial (DFM) blends. Seventy-two weaned piglets (6.4 ± 0.2 kg body weight [BW]; ~21 d of age) were assigned to one of four treatments: 1) NC: Nonchallenged (n = 10), 2) positive challenged control (PC): F18 ETEC-challenged (n = 10), 3) PC + DFM1 (n = 8; three strains of Bacillus amyloliquefaciens; 7.5 × 105 colony-forming units [cfu]/g), or 4) PC + DFM2 (n=8; 2 strains of B. amyloliquefaciens and one strain of Bacillus subtilis; 1.5 × 105 cfu/g). Feed intake and BW were recorded on day 0, 7, and 17. Pigs were sham-infected either with 6 mL phosphate-buffered saline or inoculated with 6 mL F18 ETEC (~1.9 × 109 cfu/mL) on day 7 (0 d postinoculation [dpi]). All ETEC-challenged pigs were confirmed to be genetically susceptible to F18. Pigs had ad libitum access to feed and water throughout the 17-d trial. Fecal scores were visually ranked and rectal temperatures were recorded daily. To evaluate ETEC shedding, fecal swabs were collected on dpi 0, 1, 2, 3, 5, 7, and 10. Blood samples were collected on dpi 0, 1, 2, 4, 7, and 10. Ileal tissues were collected at necropsy on dpi 10. All challenged treatments had lower final BW, decreased average daily gain (ADG), and average daily feed intake (ADFI) during the 10-d postchallenge period (P < 0.01). The DFM2 treatment increased E. coli shedding on dpi 2 and decreased iton dpi 7 (P < 0.05) compared with the PC. Rectal temperature decreased across all challenged treatments (P < 0.01). Ileal mRNA abundance of occludin (OCLN) and zonula occludens-1 (ZO-1) decreased in PC and DFM1 compared with NC (P < 0.05). Pigs fed DFM2 had intermediate ileal mRNA abundance of OCLN and increased ZO-1 mRNA compared with pigs in PC (P < 0.05). Interleukin 8 (IL-8) increased in the plasma of PC and DFM2 on dpi 2 compared with NC (P < 0.05). Mucosal IL-8 increased in PC compared with NC (P < 0.05). All challenged treatments tended to have elevated tumor necrosis factor-α (TNF-α) mRNA abundance compared with NC (P < 0.10). Challenged pigs had reduced secretory immunoglobulin A and villus height compared with NC pigs (P < 0.05). The impact of an ETEC challenge on intestinal function and the immune system has been revealed, information critical to developing improved treatment regimes.


Assuntos
Bacillus amyloliquefaciens/fisiologia , Bacillus subtilis/fisiologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/veterinária , Imunidade Inata , Probióticos/análise , Doenças dos Suínos/prevenção & controle , Animais , Derrame de Bactérias , Dieta/veterinária , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fezes/microbiologia , Feminino , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/fisiologia , Masculino , Distribuição Aleatória , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Desmame
5.
Sci Rep ; 10(1): 4611, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165720

RESUMO

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.


Assuntos
Anticorpos/imunologia , Dissacarídeos/imunologia , Imunoglobulina G/imunologia , Anticorpos/isolamento & purificação , Anticorpos/farmacologia , Dissacarídeos/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática , Escherichia coli/imunologia , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/farmacologia , Sepse/sangue , Sepse/diagnóstico , Sepse/etiologia
6.
Proc Natl Acad Sci U S A ; 117(14): 7941-7949, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32179676

RESUMO

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients' intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.


Assuntos
Translocação Bacteriana/imunologia , Receptores ErbB/metabolismo , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Microbioma Gastrointestinal/imunologia , Leite Humano/imunologia , Sepse Neonatal/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Aleitamento Materno , Colo/metabolismo , Colo/microbiologia , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Leite Humano/metabolismo , Sepse Neonatal/metabolismo , Sepse Neonatal/microbiologia , Transdução de Sinais/imunologia , Fatores de Tempo
7.
Proc Natl Acad Sci U S A ; 117(14): 7971-7980, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205444

RESUMO

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA4 or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA4 and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Infecções por Escherichia coli/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Pneumonia/imunologia , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células Cultivadas , Ilhas de CpG/imunologia , DNA Bacteriano/imunologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Voluntários Saudáveis , Humanos , Lipoxinas/farmacologia , Lipoxinas/uso terapêutico , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Pneumonia/patologia , Cultura Primária de Células , Receptores de Formil Peptídeo/imunologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/imunologia , Receptores de Lipoxinas/metabolismo
8.
Sci Rep ; 10(1): 5109, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198370

RESUMO

Splenic immune function was enhanced in diet-induced-obese (DIO) mice caused by Escherichia coli. The changes in spleen function on apoptosis were still unknown. Two hundred mice in groups Lean-E. coli and DIO-E. coli were intranasal instillation of E. coli. And another two hundred mice in groups Lean-PBS and DIO-PBS were given phosphate-buffered saline (PBS). Subsequently, spleen histology was analyzed. Then the rates of spleen cell (SC) apoptosis, and expression of the genes and proteins of Bcl-2, Bax, caspase-3 and caspase-9 were quantified in each group at 0 h (uninfected), 12 h, 24 h, and 72 h postinfection. The SC apoptosis rates of the DIO-E. coli groups were lower than those of the DIO-PBS groups at 12, 24 and 72 h (p < 0.05). Anti-apoptotic Bcl-2 expression gene and protein of the DIO-E. coli groups were higher than those of the DIO-PBS groups (p < 0.05). Gene expressions of pro-apoptotic Bax, caspase-3 and caspase-9 of the DIO-E. coli groups were lower than those of DIO-PBS groups at 12, 24 and 72 h (p < 0.05). The SC apoptosis rates of the Lean-E. coli groups were higher than those of the Lean- PBS groups at 12 h and 24 h (p < 0.05). Interestingly, the SC apoptosis rates in the DIO-E. coli groups were lower than those of the Lean-E. coli groups at 12 h (p < 0.05). In conclusion, our results suggested that the DIO mice presented stronger anti-apoptotic abilities than Lean mice in non-fatal acute pneumonia induced by E. coli infection, which is more conducive to protecting the spleen and improving the immune defense ability of the body.


Assuntos
Apoptose/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Obesidade/imunologia , Pneumonia/imunologia , Baço/imunologia , Administração Intranasal , Animais , Caspase 3/biossíntese , Caspase 9/biossíntese , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/mortalidade , Macrófagos/imunologia , Masculino , Megacariócitos/imunologia , Camundongos , Camundongos Obesos , Neutrófilos/imunologia , Pneumonia/microbiologia , Pneumonia/mortalidade , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Baço/citologia , Proteína X Associada a bcl-2/biossíntese
9.
Arch Anim Nutr ; 74(4): 271-295, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32108496

RESUMO

Short and medium-chain fatty acids (SCFA and MCFA, respectively) are commonly used as feed additives in piglets to promote health and prevent post-weaning diarrhoea. Considering that the mechanism and site of action of these fatty acids can differ, a combined supplementation could result in a synergistic action. Considering this, it was aimed to assess the potential of two new in-feed additives based on butyrate or heptanoate, protected with sodium salts of MCFA from coconut distillates, against enterotoxigenic Escherichia coli (ETEC) F4+ using an experimental disease model. Two independent trials were performed in 48 early-weaned piglets fed a control diet (CTR) or a diet supplemented with MCFA-protected sodium butyrate (BUT+; Trial 1) or sodium heptanoate (HPT+; Trial 2). After 1 week of adaptation, piglets were challenged with a single oral inoculum of ETEC F4+ (minimum 1.4 · 109 cfu). One animal per pen was euthanised on days 4 and 8 post-inoculation (PI) and the following variables assessed: growth performance, clinical signs, gut fermentation, intestinal morphology, inflammatory mediators, pathogen excretion and colon microbiota. None of the additives recovered growth performance or reduced diarrhoea when compared to the respective negative controls. However, both elicited different responses against ETEC F4+. The BUT+ additive did not lead to reduce E. coli F4 colonisation but enterobacterial counts and goblet cell numbers in the ileum were increased on day 8 PI and this followed higher serum TNF-α concentrations on day 4 PI. The Firmicutes:Bacteroidetes ratio was nevertheless increased. Findings in the HPT+ treatment trial included fewer animals featuring E. coli F4 in the colon and reduced Enterobacteriaceae (determined by 16S RNA sequencing) on day 4 PI. In addition, while goblet cell numbers were lower on day 8 PI, total SCFA levels were reduced in the colon. Results indicate the efficacy of MCFA-protected heptanoate against ETEC F4+ and emphasise the potential trophic effect of MCFA-protected butyrate on the intestinal epithelium likely reinforcing the gut barrier.


Assuntos
Ácido Butírico/metabolismo , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/efeitos dos fármacos , Heptanoatos/metabolismo , Sus scrofa/fisiologia , Ração Animal/análise , Animais , Ácido Butírico/administração & dosagem , Cocos/química , Colo/efeitos dos fármacos , Colo/microbiologia , Dieta/veterinária , Suplementos Nutricionais/análise , Escherichia coli Enterotoxigênica/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Ácidos Graxos/administração & dosagem , Fermentação/efeitos dos fármacos , Trato Gastrointestinal/anatomia & histologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Heptanoatos/administração & dosagem , Masculino , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/microbiologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia
10.
FASEB J ; 34(1): 1018-1037, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914603

RESUMO

Recombinant antimicrobial peptide microcin J25 (MccJ25) causes potent antimicrobial activity against enterotoxigenic Escherichia coli (ETEC) in vitro; however, independently of this activity, its role in suppressing intestinal inflammation and epithelial barrier injury in vivo remains unclear. We investigated the therapeutic effects of MccJ25 on intestinal inflammation and epithelial barrier dysfunction and the underlying mechanism, using gentamicin for comparison. In a mouse model of intestinal inflammation, therapeutic administration of either MccJ25 or gentamicin after ETEC K88 infection attenuated clinical symptoms, reduced intestinal pathogen colonization, improved intestinal morphology, and decreased inflammatory pathologies and intestinal permeability, ultimately improving the hosts' health. MccJ25 also attenuated ETEC-induced mouse intestinal barrier dysfunction by enhancing tight junction proteins (TJPs). Using the human epithelial cell line Caco-2, we verified the epithelial barrier-strengthening and mucosal injury-alleviating effects of MccJ25 on ETEC infection: increased expression of TJPs by activating the p38/MAPK pathway, balancing the microbiota, and improving short-chain fatty acid concentrations in the cecum of ETEC-infected mice. Although gentamicin and MccJ25 had similar effects in the inflamed gut, MccJ25 was superior to gentamicin with regard to defending the host from ETEC infection. Overall, MccJ25 may be a promising therapeutic drug for treating enteric pathogen-induced intestinal inflammation diseases.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacteriocinas/farmacologia , Epitélio/efeitos dos fármacos , Infecções por Escherichia coli/imunologia , Inflamação/imunologia , Mucosa Intestinal/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Células CACO-2 , Citocinas/metabolismo , Escherichia coli Enterotoxigênica , Feminino , Microbioma Gastrointestinal , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologia
11.
Nature ; 577(7791): 543-548, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31915378

RESUMO

Although maternal antibodies protect newborn babies from infection1,2, little is known about how protective antibodies are induced without prior pathogen exposure. Here we show that neonatal mice that lack the capacity to produce IgG are protected from infection with the enteric pathogen enterotoxigenic Escherichia coli by maternal natural IgG antibodies against the maternal microbiota when antibodies are delivered either across the placenta or through breast milk. By challenging pups that were fostered by either maternal antibody-sufficient or antibody-deficient dams, we found that IgG derived from breast milk was crucial for protection against mucosal disease induced by enterotoxigenic E. coli. IgG also provides protection against systemic infection by E. coli. Pups used the neonatal Fc receptor to transfer IgG from milk into serum. The maternal commensal microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic E. coli and other Enterobacteriaceae species. Induction of maternal antibodies against a commensal Pantoea species confers protection against enterotoxigenic E. coli in pups. This role of the microbiota in eliciting protective antibodies to a specific neonatal pathogen represents an important host defence mechanism against infection in neonates.


Assuntos
Anticorpos/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Imunidade Materno-Adquirida/imunologia , Recém-Nascido/imunologia , Microbiota/imunologia , Leite Humano/imunologia , Animais , Anticorpos/sangue , Anticorpos/metabolismo , Aleitamento Materno , Reações Cruzadas/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Masculino , Camundongos , Mães , Pantoea/imunologia , Receptores Fc/imunologia , Receptores Fc/metabolismo , Simbiose/imunologia
12.
Res Microbiol ; 171(2): 99-101, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31982499

RESUMO

Many septicemic Escherichia coli strains produce polysaccharide capsules, which are important for survival in serum. Here we show that a septicemic E. coli strain of serotype O78 produce an O-antigen capsule (group 4 capsule) and we show that this capsule is essential for serum survival.


Assuntos
Cápsulas Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/imunologia , Antígenos O/imunologia , Óperon , Virulência
13.
Sci Rep ; 10(1): 499, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949265

RESUMO

This study examined the protective effects of citrulline enriched-fermented milk with live Lactobacillus helveticus ASCC 511 (LH511) on intestinal epithelial barrier function and inflammatory response in IPEC-J2 cells caused by pathogenic Escherichia coli. Five percent (v/v) of fermented milk with live LH511 and 4 mM citrulline (5%LHFM_Cit-4mM) significantly stimulated the population of IPEC-J2 cells by 36% as determined by MTT assay. Adhesion level of LH511 was significantly increased by 9.2% when incubated with 5%LHFM_Cit-4mM and 5%LHFM_Cit-4mM reduced the adhesion of enterohemorrhagic (EHEC) and entero-invasive (EIEC) E. coli in IPEC-J2 cells by 35.79% and 42.74%, respectively. Treatment with 5%LHFM_Cit-4mM ameliorated lipopolysaccharide (LPS) from E. coli O55:B5 induced activated inflammatory cytokines expression (TNF-α, IL-6 and IL-8) and concentration (IL-6 and IL-8) and early apoptosis. It restored the transepithelial electrical resistance (TEER) and regulated the expression and distribution of tight junction (TJ) proteins (zonula occluden-1 (ZO-1), occludin and claudin-1), toll-like receptors (TLRs) (TLR2 and TLR4) and negative regulators of TLRs signalling pathway (A20 and IRAK-M). In conclusion, our findings suggested that 5%LHFM_Cit-4mM might have the positive effects on improving and maintaining the intestinal epithelial cell integrity and inflammatory response under both normal and pathogenic LPS-stimulated conditions.


Assuntos
Citrulina/farmacologia , Infecções por Escherichia coli/prevenção & controle , Lactobacillus helveticus/fisiologia , Leite/química , Animais , Aderência Bacteriana , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Escherichia coli Êntero-Hemorrágica/crescimento & desenvolvimento , Infecções por Escherichia coli/imunologia , Fermentação , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lipopolissacarídeos/efeitos adversos , Leite/microbiologia , Suínos
14.
Genes Dev ; 34(3-4): 149-165, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31919189

RESUMO

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.


Assuntos
Núcleo Celular/imunologia , Cromatina/imunologia , Infecções por Escherichia coli/imunologia , Neutrófilos/imunologia , Ativação Transcricional/genética , Ativação Transcricional/imunologia , Células Cultivadas , Escherichia coli , Histonas/metabolismo , Humanos
15.
J Clin Invest ; 130(1): 359-373, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31793912

RESUMO

N-3 docosapentaenoic acid-derived resolvin D5 (RvD5n-3 DPA) is diurnally regulated in peripheral blood and exerts tissue-protective actions during inflammatory arthritis. Here, using an orphan GPCR screening approach coupled with functional readouts, we investigated the receptor(s) involved in mediating the leukocyte-directed actions of RvD5n-3 DPA and identified GPR101 as the top candidate. RvD5n-3 DPA bound to GPR101 with high selectivity and stereospecificity, as demonstrated by a calculated KD of approximately 6.9 nM. In macrophages, GPR101 knockdown limited the ability of RvD5n-3 DPA to upregulate cyclic adenosine monophosphate, phagocytosis of bacteria, and efferocytosis. Inhibition of this receptor in mouse and human leukocytes abrogated the pro-resolving actions of RvD5n-3 DPA, including the regulation of bacterial phagocytosis in neutrophils. Knockdown of the receptor in vivo reversed the protective actions of RvD5n-3 DPA in limiting joint and gut inflammation during inflammatory arthritis. Administration of RvD5n-3 DPA during E. coli-initiated inflammation regulated neutrophil trafficking to the site of inflammation, increased bacterial phagocytosis by neutrophils and macrophages, and accelerated the resolution of infectious inflammation. These in vivo protective actions of RvD5n-3 DPA were limited when Gpr101 was knocked down. Together, our findings demonstrate a fundamental role for GPR101 in mediating the leukocyte-directed actions of RvD5n-3 DPA.


Assuntos
Artrite/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores Acoplados a Proteínas-G/agonistas , Animais , Artrite/genética , Artrite/imunologia , Artrite/patologia , Células CHO , Cricetulus , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/patologia , Masculino , Camundongos , Neutrófilos/patologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia
16.
Inflammation ; 43(1): 179-190, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31758425

RESUMO

Sepsis remains a leading cause of mortality worldwide and is characterized by sustained inflammatory responses, reflected as changes in the expression profile of cytokines with time. The aim of the present study was to investigate the dynamic changes in complete blood count, serum chemistry, procalcitonin (PCT), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in Escherichia coli, Staphylococcus aureus, and Candida albicans bacteremia. Study subjects were 32 healthy male Landrace-Large White pigs, aged 10-15 weeks and of average weight 19 ± 2 kg. Bacteremia was induced by continuous intravenous infusion of microbial suspensions during a period of 8 h. E. coli and S. aureus bacteremia were associated with a significant gradual decrease in white blood cells and platelets, respectively (p = 0.002 and p = 0.004), while candidemia was characterized by a significant gradual decrease in lymphocytes (p = 0.009). Serum PCT levels were either undetectable or very low, with no significant changes with time in all groups. E. coli bacteremia elicited a strong pro-inflammatory response, characterized by a significant increase in TNF-α expression from the onset of bacteremia (p = 0.042). C. albicans exhibited a different profile with an early, moderate increase in TNF-α followed by a subsequent marked increase in IL-6 levels (p = 0.03). The differential regulation of inflammatory and hematological responses depending on the pathogenic agent can reveal differences in the underlying inflammatory mechanisms, which may assist in the ongoing quest for the identification of a panel of circulating biomarkers during bacteremia.


Assuntos
Bacteriemia/imunologia , Candidíase/imunologia , Infecções por Escherichia coli/imunologia , Infecções Estafilocócicas/imunologia , Animais , Bacteriemia/sangue , Bacteriemia/microbiologia , Candidíase/sangue , Candidíase/microbiologia , Modelos Animais de Doenças , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/sangue , Interleucina-6/sangue , Contagem de Leucócitos , Masculino , Contagem de Plaquetas , Pró-Calcitonina/sangue , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Sus scrofa , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue
17.
Am J Physiol Renal Physiol ; 318(2): F468-F474, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841391

RESUMO

Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and ß-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.


Assuntos
Acidose/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Escherichia coli/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Coletores/metabolismo , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/patogenicidade , Acidose/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Túbulos Renais Coletores/imunologia , Túbulos Renais Coletores/microbiologia , Camundongos , Prolil Hidroxilases/metabolismo , Estabilidade Proteica , Transdução de Sinais , Regulação para Cima , Infecções Urinárias/imunologia , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologia , beta-Defensinas/metabolismo
18.
Fish Shellfish Immunol ; 97: 235-247, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31863902

RESUMO

Lipopolysaccharide (LPS) is a classical pathogen-associated molecular pattern that can trigger strong inflammatory response mainly by TLR4-mediated signaling pathway in mammals, but the molecular mechanism of anti-LPS immunity is unclear in teleost fishes. In this study, we analyzed the gene expression features based on transcriptome analysis in Schizothorax prenanti (S. prenanti), after stimulation with two sources of LPS from Aeromonas hydrophila and Escherichia coli (Ah. LPS and Ecoli. LPS). 921 different expression genes (DEGs) after Ah. LPS stimulation and 975 DEGs after Ecoli.LPS stimulation were acquired, but only 706 and 750 DEGs were successfully annotated into the databases, respectively. Both of two groups of DGEs were significantly enriched into immune-related pathways by KEGG enrichment analysis, such as "Toll-like receptor signaling pathway", "Cytokine-cytokine receptor interaction" and "JAK-STAT signaling pathway". The annotated DEGs from Ah. LPS and Ecoli. LPS stimulation shared 470 DEGs, including 88 immune-related DEGs (IRGs) identified mainly by KEGG enrichment to immune-related signaling pathways. Among the shared IRGs, four pattern-recognition genes (TLR5, TLR25, PTX3 and C1q) were induced with high expression foldchange, and IFN-γ and relative genes also showed higher expression levels than control. Meanwhile, inflammatory signals were highlighted by upregulating the expression of inflammatory cytokines (IL-1ß, IL-10 and IL-8). Moreover, some non-shared IRGs (including TLR2 and TLR4) were identified, suggesting that different sources of LPS own different potentials for the induction of immune gene expression. In conclusion, TLR5, TLR25, PTX3 and C1q may function as the sensing molecules to catch the invasion signal of LPS. The anti-LPS immune response may be involved into TLR25/TLR5-mediated inflammatory signals that regulate subsequently the activation of PTX3/C1q-modulated complement pathway upon the induction of PTX3 expression, and the crosstalk between IFN-γ and TLR signaling pathways in teleost fishes. This study will contribute to further explore the molecular mechanism of LPS-induced immunity in teleost fishes.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Lipopolissacarídeos/efeitos adversos , Substâncias Protetoras/farmacologia , Aeromonas hydrophila/fisiologia , Animais , Escherichia coli/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
19.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31818960

RESUMO

Neonates are at increased risk for bacterial sepsis. We established that the immune-suppressive cytokine interleukin-27 (IL-27) is elevated in neonatal mice. Similarly, human cord blood-derived macrophages express IL-27 genes and secrete more cytokine than macrophages from adults. In the present work, we hypothesized that increased levels of IL-27 predispose neonatal mice to more severe infection during Gram-negative sepsis. Serum IL-27 levels continued to rise during infection. Peripheral tissue analysis revealed systemic IL-27 expression, while myeloid cell profiling identified Gr-1- and F4/80-expressing cells as the most abundant producers of IL-27 during infection. Increased IL-27 levels were consistent with increased mortality that was improved in IL-27 receptor α (IL-27Rα)-/- mice that lack a functional IL-27 receptor. Infected IL-27Rα-/- pups also exhibited improved weight gain and reduced morbidity. This was consistent with reduced bacterial burdens and more efficient bacterial killing by Ly6B.2+ myeloid cells and macrophages compared to WT neonates. Live animal imaging further supported a more severe and disseminated infection in WT neonates. This is the first report to describe the impact of elevated early-life IL-27 on the host response in a neonatal infection model while also defining the cell and tissue sources of cytokine. IL-27 is frequently associated with suppressed inflammation. In contrast, our findings demonstrate that IL-27 indirectly promotes an inflammatory cytokine response during neonatal sepsis by directly compromising control of bacteria that drive the inflammatory response. Collectively, our results suggest that IL-27 represents a therapeutic target to limit susceptibility and improve infectious outcomes in neonatal sepsis.


Assuntos
Infecções por Escherichia coli/imunologia , Imunidade Ativa/imunologia , Interleucina-27/metabolismo , Sepse Neonatal/imunologia , Animais , Modelos Animais de Doenças , Camundongos
20.
Folia Microbiol (Praha) ; 65(1): 133-142, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31104302

RESUMO

This study aimed to analyze the proinflammatory cytokine mRNA expression in the urinary tract of BALB/c mice infected with bacterial strains with uropathogenic potential. Groups of four 6-week-old female BALB/c mice were intraurethrally inoculated with 5 × 107 colony-forming units (CFU) of P. mirabilis ATCC29906, EAEC O42, P. mirabilis RTX339, or sterile saline (control group) and then sacrificed at 0, 2, 4, 7, or 10 days post-infection (p.i.). Samples were cultured to determine the CFU/mL in urine or CFU/g in the bladders and kidneys. Cytokine expression (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, -6, and -8) was evaluated in the target organs using real-time PCR and immunohistochemistry; histology was examined with hematoxylin and eosin staining. The results are presented as the means and standard deviations and were compared using one-way ANOVA, with p < 0.05 indicating significant differences. Bacteriuria was not detected in the infected groups; bacterial colonization occurred in the target organs at all time points, but was higher in mice infected with EAEC O42 or P. mirabilis RTX339 at 7 days p.i. The expression of all cytokine mRNAs was seen, but only the levels of the IL-8 protein increased in situ at 7 days p.i. in the P. mirabilis RTX339 and EAEC O42 groups in both organs. Morphological alterations, observed in all of the infected groups, were more prominent in the EAEC O42 and P. mirabilis RTX339 groups. The findings provide insights into the uropathogenicity and inflammatory cytokine expression in the urinary tract of mice infected with three previously untested bacterial strains.


Assuntos
Citocinas/genética , Infecções por Escherichia coli/imunologia , Inflamação/imunologia , Infecções por Proteus/imunologia , Infecções Urinárias/imunologia , Animais , Citocinas/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/urina , Feminino , Inflamação/microbiologia , Rim/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Proteus/urina , Proteus mirabilis/patogenicidade , Bexiga Urinária/microbiologia , Sistema Urinário/imunologia , Sistema Urinário/microbiologia , Infecções Urinárias/microbiologia
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