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1.
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
2.
PLoS Negl Trop Dis ; 13(11): e0007825, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31756188

RESUMO

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates. METHODS: Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro. RESULTS: EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules. CONCLUSIONS: EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design.


Assuntos
Antígenos de Bactérias/genética , Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Variação Genética , Glicoproteínas de Membrana/genética , Peptídeo Hidrolases/genética , Antígenos de Bactérias/análise , Escherichia coli Enterotoxigênica/química , Escherichia coli Enterotoxigênica/classificação , Escherichia coli Enterotoxigênica/isolamento & purificação , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/análise , Saúde Global , Humanos , Immunoblotting , Glicoproteínas de Membrana/análise , Peptídeo Hidrolases/análise , Reação em Cadeia da Polimerase , Sequenciamento Completo do Genoma
3.
Vet Microbiol ; 238: 108429, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648721

RESUMO

Viral haemorrhagic disease (VHD) and colibacillosis are common diseases in rabbits that cause economic losses worldwide. The effect of colibacillosis on the immune response of vaccinated rabbits against rabbit haemorrhagic disease virus (RHDV) was studied. Four groups (G1-G4) were included. G1 was the negative control group; G2 was the RHDV vaccine group; G3 was the E. coli-infected group; and G4 was the E. coli-infected + RHDV vaccine group. The E. coli infection and RHDV vaccination were simultaneously performed, with another previous infection, 3 days before vaccination. At 28 days post-vaccination (PV), the rabbits (G2-G4) were challenged intramuscularly with 0.5 ml of RHDV at a dose of 103 50% median lethal dose (LD50)/rabbit. The rabbits were observed for clinical signs, body weight gain and mortality rates. Tissue, blood, serum, and faecal samples and rectal swabs were collected at 3, 5, 7, 14, 21 and 28 days PV. Significant clinical signs and mortality and a decrease in BW were observed in the infected + RHDV vaccine group. On the 3rd day post-infection (PI), compared with all the other groups, the vaccinated group (G2) had significantly upregulated hepatic tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels; however, the infected + RHDV vaccine group had significantly higher intestinal levels of TNF-α and IL-6 than the other groups. Furthermore, E. coli infection in vaccinated rabbits led to immunosuppression, as shown by significant decreases (P < 0.05) in heterophil phagocytic activity, the CD4+/CD8+ ratio, and HI antibody responses to RHDV and a significant increase in the heterophil to lymphocyte (H/L) ratio. In conclusion, colibacillosis leads to immunosuppression involving a shift in the equilibrium of cytokines and reduced weight gain and mortality in vaccinated rabbits and could be a contributing factor in RHDV vaccination failure in rabbit farming.


Assuntos
Infecções por Caliciviridae/veterinária , Infecções por Escherichia coli/veterinária , Coelhos/imunologia , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/mortalidade , Citocinas/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/mortalidade , Infecções por Escherichia coli/fisiopatologia , Regulação da Expressão Gênica/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Coelhos/microbiologia , Coelhos/virologia , Vacinação/normas
5.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31501251

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. Despite the apparent similarities in their known virulence attributes, some ExPEC strains can cross the host species barrier and present a zoonotic potential, whereas other strains exhibit host specificity, suggesting the existence of unknown mechanisms that remain to be identified. We applied a transposon-directed insertion site sequencing (TraDIS) strategy to investigate the ExPEC XM strain, which is capable of crossing the host species barrier, and to screen for virulence-essential genes in both mammalian (mouse) and avian (duck) models of E. coli-related septicemia. We identified 151 genes essential for systemic infection in both mammalian and avian models, 97 required only in the mammalian model, and 280 required only in the avian model. Ten genes/gene clusters were selected for further validation, and their contributions to ExPEC virulence in both mammalian and avian models or mammalian- or avian-only models were confirmed by animal tests. This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPEC-related virulence, host specificity, and adaptation.


Assuntos
Adaptação Fisiológica/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Especificidade de Hospedeiro/genética , Adaptação Fisiológica/imunologia , Animais , Patos , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sepse/imunologia , Sepse/microbiologia
6.
Mol Immunol ; 114: 612-619, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31542606

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) are known as the gastrointestinal pathogens and major causes of enterohemorrhagic colitis since decades ago. There is no efficient approved vaccine against EHEC O157 and non-O157. In the present study, a recombinant candidate vaccine against enterohemorrhagic E. coli (EHEC) O157:H7 entrapped in the sodium alginate and PLGA nanoparticles and the efficiency of the immunization of these formulations were investigated. nanoparticles due to their properties like controlled cargoes release, adjuvanticity, cargo protection, increased bioavailability, etc have been noticed for drug delivery. A chimeric protein composed of HcpA, EspA, Tir and Stx2B antigens was designed, recombinantly expressed, purified and entrapped in nanoparticles. BALB/c mice were administrated with nano-formulated and free proteins. IgG titer, EHEC fecal shedding and the ability of the immune sera to neutralize Stx toxin and inhibit the bacterial attachment to Caco-2 cells were analyzed. Fecal shedding analysis demonstrated that the colonization of the bacteria in the intestine of the mice was reduced significantly (P > 0.01). Immune mice were able to tolerate up to 200 LD50 of the active Stx toxin. About 80% of the bacterial binding capacity to Caco-2 cells was declined, especially in groups immunized with nano-formulations. Considering the importance of EHEC, especially O157 serotype, on public health and the other hand, the lack of an efficient vaccine in this regard, delivery of HETS candidate vaccine with NPs can be applied to prevent the infection by the pathogen.


Assuntos
Alginatos/química , Formação de Anticorpos/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli O157/imunologia , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Células CACO-2 , Linhagem Celular Tumoral , Escherichia coli Êntero-Hemorrágica/imunologia , Proteínas de Escherichia coli/imunologia , Feminino , Humanos , Imunização/métodos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/imunologia , Proteínas Recombinantes de Fusão/química , Vacinas Sintéticas/imunologia
7.
Elife ; 82019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31408005

RESUMO

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.


Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Imunidade Inata , Inflamação/fisiopatologia , Fosfoproteínas Fosfatases/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos
8.
J Dairy Sci ; 102(10): 9107-9116, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400895

RESUMO

The dynamic interaction between the host and pathogens, along with environmental factors, influences the regulation of mammalian immune responses. Therefore, comprehensive in vivo immune-phenotyping during an active response to a pathogen can be complex and prone to confounding effects. Evaluating critical fundamental aspects of the immune system at a cellular level is an alternative approach to reduce this complexity. Therefore, the objective of the current study was to examine an in vitro model for functional phenotyping of bovine monocyte-derived macrophages (MDM), cells which play a crucial role at all phases of inflammation, as well influence downstream immune responses. As indicators of MDM function, phagocytosis and nitric oxide (NO-) production were tested in MDM of 16 cows in response to 2 common bacterial pathogens of dairy cows, Escherichia coli and Staphylococcus aureus. Notable functional variations were observed among the individuals (coefficient of variation: 33% for phagocytosis and 70% in the production of NO-). The rank correlation analysis revealed a significant, positive, and strong correlation (rho = 0.92) between NO- production in response to E. coli and S. aureus, and a positive but moderate correlation (rho = 0.58) between phagocytosis of E. coli and S. aureus. To gain further insight into this trait, another 58 cows were evaluated solely for NO- response against E. coli. The pedigree of the tested animals was added to the statistical model and the heritability was estimated to be 0.776. Overall, the finding of this study showed a strong effect of host genetics on the in vitro activities of MDM and the possibility of ranking Holstein cows based on the in vitro functional variation of MDM.


Assuntos
Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Infecções por Escherichia coli/veterinária , Macrófagos/imunologia , Infecções Estafilocócicas/veterinária , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Feminino , Imunidade/genética , Macrófagos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Linhagem , Fagocitose , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia
9.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295956

RESUMO

CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.


Assuntos
Antibacterianos/farmacologia , Proteínas do Sistema Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/imunologia , Fatores Imunológicos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/química , Infusões Parenterais , Camundongos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia
10.
Poult Sci ; 98(11): 5330-5335, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31289817

RESUMO

During the hatching process, chicks are exposed to opportunistic and/or pathogenic organisms, such as virulent or avirulent Escherichia coli. Virulent E. coli strains have not been feasible for induction of neonatal colibacillosis via in ovo challenge due to high embryonic mortality. In this manuscript, we describe the addition and co-administration of the bacteriostatic antibiotic tetracycline to a virulent E. coli challenge culture, improving hatchability and livability of seeder chicks while allowing robust horizontal transmission in the hatching cabinet to contact chicks. Experiment 1 consisted of 3 trials. Experiment 1, trial 1 was conducted to determine an effective ratio of E. coli challenge and tetracycline dose to be utilized in the seeder model. Trials 2 and 3 were conducted to evaluate the transmission of E. coli from seeder to contact chicks. Experiment 2 consisted of 3 independent 7-D trials where body weight gain (BWG), mortality, and selected enteric bacterial recovery were evaluated. In trials 1 to 3, significantly (P < 0.05) more Gram-negative bacteria were recovered from whole gut samples (GIT) vs. negative controls on day of hatch, from both seeder and contact chicks. At day 7 in trial 1, contact chicks had significantly (P < 0.05) more Gram-negative bacteria recovered from the GIT than the negative control, but not in trials 2 and 3. Presumptive lactic acid bacterial recovery was elevated in contact and seeder chicks compared to the negative control in all 3 trials. Contact challenge caused a significant (P < 0.05) reduction in BWG in 2 out of 3 trials at day 7, and there was a significant (P < 0.05) increase in mortality as compared to the negative controls in all trials. These data suggest that co-administration of a virulent E. coli strain with tetracycline allows for hatch of direct challenged chicks and effective horizontal transmission to contact chicks during the hatching process, as evidenced by reduced day 7 performance and altered selected enteric bacterial recovery.


Assuntos
Antibacterianos/administração & dosagem , Galinhas , Modelos Animais de Doenças , Infecções por Escherichia coli/veterinária , Escherichia coli/patogenicidade , Doenças das Aves Domésticas/imunologia , Tetraciclina/administração & dosagem , Animais , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Óvulo , Doenças das Aves Domésticas/microbiologia , Virulência
11.
Nat Commun ; 10(1): 3325, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346171

RESUMO

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.


Assuntos
Proteínas do Sistema Complemento/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/metabolismo , Glicina/metabolismo , Serina/metabolismo , Soro/química , Treonina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Ciclo do Ácido Cítrico , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Purinas/biossíntese
12.
Res Vet Sci ; 125: 153-161, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31228739

RESUMO

An Escherichia coli (E. coli) O2:K1 bacterial ghost was produced by controlled expression of bacteriophage PhiX 174 lysis gene E. Temperature controlled expression of this gene caused tunnels and holes in the cell wall of E. coli O2:K1 bacterium, leading to loss of cytoplasmic contents. Formation of E. coli O2:K1 ghost was confirmed by scanning electron microscopy and determination of colony forming units. To evaluate the efficiency of this bacterial ghost vaccine to elicit cellular and humoral immune responses, 85 one day old chickens from Ross 308 breed were divided into the following 5 groups; group 1 (non-immunized control), group 2 (vaccine administered by injection of E. coli O2:K1 killed vaccine), group 3 (vaccine administered by injection of E. coli O2:K1 ghost), group 4 (vaccine administered by inhalation of E. coli O2:K1 ghost), and group 5 (neither immunized, nor challenged as negative control). The groups of 2, 3, and 4 were received vaccines at days 7, 14, and 22. Groups 1 to 4 were challenged with the wild type at day 33. Evaluation of post-mortem lesions and immune responses in all groups showed that chicken injected with the killed vaccine and the bacterial ghost had the best protection. These findings suggest that this bacterial ghost has the potential to be used as a poultry colibacillosis vaccine.


Assuntos
Galinhas , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Escherichia coli/imunologia , Imunidade Celular , Imunidade Humoral , Doenças das Aves Domésticas/prevenção & controle , Adenosina/análogos & derivados , Animais , Colífagos/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fenetilaminas , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Sorogrupo , Vacinas de Produtos Inativados/imunologia
13.
Int Immunopharmacol ; 73: 482-490, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173970

RESUMO

There is currently no specific drug for the treatment of sepsis and antibiotic administration is considered the best option, despite numerous issues. Therefore, the development of drugs to control the pathogen-induced inflammatory responses associated with sepsis is essential. To address this, our study examined the transcriptomes of lipopolysaccharide (LPS)-induced dendritic cells (DCs), identifying TANK-binding kinase1 (Tbk1) as a key factor involved in the inflammatory response. These data suggested drug repositioning of the Tbk1 inhibitor CYT387, currently used for the treatment of myelofibrosis and some cancers, as a candidate for regulating the LPS-induced inflammatory response. CYT387 also inhibited pro-inflammatory cytokine and surface molecule expression by mature DCs after LPS exposure. These effects correlated with both Akt phosphorylation and IκBα degradation. Finally, CYT387 demonstrated therapeutic effects in LPS-induced endotoxemia and Escherichia coli K1-induced mouse models of sepsis and decreased the expression of pro-inflammatory cytokines. In conclusion, our study suggests that drug repositioning of CYT387 may serve as a potential therapeutic for sepsis.


Assuntos
Benzamidas/uso terapêutico , Endotoxemia/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Sepse/tratamento farmacológico , Animais , Benzamidas/farmacologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Reposicionamento de Medicamentos , Endotoxemia/imunologia , Infecções por Escherichia coli/imunologia , Feminino , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Sepse/imunologia , Transcriptoma/efeitos dos fármacos
14.
Microb Pathog ; 134: 103600, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202906

RESUMO

INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Quitosana/farmacologia , Imunização , Nanopartículas/química , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/imunologia , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Regulação Bacteriana da Expressão Gênica , Imunidade nas Mucosas , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Proteínas Recombinantes de Fusão/genética , Toxinas Shiga/genética , Toxinas Shiga/imunologia , Análise de Sobrevida , Vibrio cholerae/genética , Vibrio cholerae/imunologia
15.
J Appl Microbiol ; 127(2): 565-575, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102489

RESUMO

AIMS: To observe the therapeutic effects of vaginal infusion of probiotic Clostridium butyricum WZ001 on bacterial vaginosis (BV) in mice. METHODS AND RESULTS: Female ICR mice were used to establish the model of BV by infecting oestrogen-treated mice with Escherichia coli, and then treated with high- and low dose of C. butyricum. Clinical indexes of mice in the C. butyricum-treated groups were significantly improved and comparable to those in the antibiotic group. Pap staining showed that neutrophil count was significantly increased after modelling and largely decreased after C. butyricum treatment (P < 0·01). Dynamic observation of E. coli and Lactobacillus showed that the number of E. coli significantly decreased in the C. butyricum-treated groups or in the antibiotic group with prolonged treatment (P < 0·01). Besides, the number of E. coli in the low-dose C. butyricum group was higher than that in either its high-dose counterpart or the antibiotic group respectively (P < 0·01). The number of Lactobacillus decreased evidently in the antibiotic group (P < 0·01), while that in the C. butyricum groups remained consistent. Moreover, C. butyricum inhibited the proliferation of E. coli by the experiment in vitro. The phosphorylation of nuclear factor-kappa B (NF-κB) p65 in vaginal tissue and the serum levels of inflammatory cytokines, IL-1ß, TNF-α and IL-6, increased after modelling and significantly decreased after treated with C. butyricum (P < 0·01), with no difference found when compared with the antibiotic group. CONCLUSION: Clostridium butyricum inhibits the growth of pathogenic bacteria as well as the inflammatory response induced by E. coli and promotes the growth of Lactobacillus to maintain the vaginal micro-ecological balance. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that probiobitc C. butyricum WZ001 has a great potential in the clinical treatment of BV.


Assuntos
Clostridium butyricum , Infecções por Escherichia coli/terapia , Probióticos/uso terapêutico , Vaginose Bacteriana/terapia , Animais , Citocinas/sangue , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Lactobacillus/isolamento & purificação , Camundongos , Camundongos Endogâmicos ICR , Fator de Transcrição RelA/metabolismo , Vagina/metabolismo , Vagina/microbiologia , Vaginose Bacteriana/imunologia , Vaginose Bacteriana/metabolismo , Vaginose Bacteriana/microbiologia
16.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061144

RESUMO

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Hormônios Gastrointestinais/imunologia , Peptídeos Natriuréticos/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Reações Cruzadas , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/administração & dosagem , Enterotoxinas/química , Enterotoxinas/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Suínos
17.
Adv Clin Exp Med ; 28(7): 899-905, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31066244

RESUMO

BACKGROUND: The relationship of diffusely adherent Escherichia coli (DAEC) with pediatric inflammatory bowel disease (IBD) has not been previously studied. Diffusely adherent E. coli are a common cause of long-lasting childhood diarrhea and we postulated that they may induce inflammation of the intestinal mucosa, contributing to the development of IBD in susceptible children. OBJECTIVES: The aim of the study was to investigate the relationship between DAEC and pediatric IBD, including Crohn's disease (CD) and ulcerative colitis (UC). Diffusely adherent E. coli isolates were also assessed regarding their pathogenicity. MATERIAL AND METHODS: Diffusely adherent E. coli were screened among 130 E. coli strains isolated from intestinal biopsy specimens from 26 children with IBD using polymerase chain reaction (PCR) with primers specific to the pathotype and adherence assays to HEp-2 cells. Diffusely adherent E. coli were further analyzed for their ability to adhere to and invade polarized Caco-2 cells. The immunomodulatory effect of DAEC on the secretion of tumor necrosis factor α (TNF-α) by human monocyte-derived macrophages (MDM) was assessed using an immunoenzymatic assay. RESULTS: Diffusely adherent E. coli were recovered from 18 (69.2%) of the 26 intestinal biopsy specimens from both CD and UC patients. Most DAEC isolates carried AfaE3 adhesin, adhered to and were internalized by Caco-2 cells, and induced secretion of elevated levels of TNF-α. CONCLUSIONS: The study demonstrated the internalization of DAEC by intestinal epithelial cells and their ability to induce secretion of increased level of TNF-α in a Caco-2/macrophage compartmentalized culture. This indicated that the pathovar should be considered a pathobiont inducing inflammation of the intestinal mucosa in pediatric patients with IBD.


Assuntos
Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , Infecções por Escherichia coli/epidemiologia , Escherichia coli/isolamento & purificação , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/microbiologia , Adesinas de Escherichia coli , Células CACO-2 , Moléculas de Adesão Celular/genética , Criança , Colite Ulcerativa/epidemiologia , Colite Ulcerativa/imunologia , Doença de Crohn/epidemiologia , Doença de Crohn/imunologia , Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Doenças Inflamatórias Intestinais/epidemiologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Polônia/epidemiologia , Prevalência
18.
Mol Immunol ; 112: 131-139, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31102985

RESUMO

BACKGROUND: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. METHODS: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. RESULTS: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-ß. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. CONCLUSION: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.


Assuntos
Escherichia coli/imunologia , Receptores de Lipopolissacarídeos/antagonistas & inibidores , Fagocitose/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Citocinas/imunologia , Infecções por Escherichia coli/imunologia , Granulócitos/imunologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Interferon beta/imunologia , Receptores de Lipopolissacarídeos/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Peptídeos Cíclicos/imunologia , Receptor da Anafilatoxina C5a/imunologia , Sepse/imunologia , Sepse/microbiologia
19.
Microbes Infect ; 21(10): 464-474, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31085336

RESUMO

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.


Assuntos
Carboxipeptidases/imunologia , Endopeptidases/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/enzimologia , Imunidade Inata , Animais , Carboxipeptidases/genética , Citocinas/metabolismo , Endopeptidases/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Óxido Nítrico/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/imunologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Deleção de Sequência
20.
J Appl Microbiol ; 127(2): 396-405, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075179

RESUMO

AIMS: This study was designed to investigate, in an in vivo setting, the effects of single and combined infections with either Mycoplasma gallisepticum (MG) and/or Escherichia coli on the chicken immune response induced by Newcastle disease virus (NDV) vaccine. METHODS AND RESULTS: Humoral immunity was measured through detection of NDV antibody and anti-NDV IgG titres using haemagglutination-inhibition test and enzyme-linked immunosorbent assay, respectively. In addition, the expression levels of pro-inflammatory cytokines' genes (interleukin (IL) 6, IL4 and interferon (IFN) γ) were analysed using quantitative reverse transcription PCR. Significant (P < 0·05) results in all immunological parameters were detected in the vaccinated noninfected chicken group in comparison with those in groups exposed to bacterial infections. Bacterial infection along with vaccination hampered the NDV antibodies production and reduced the vaccine upregulated cytokine genes. The vaccinated mixed infection group reported lower antibody titres and cytokines expression levels compared to those in the single infection groups. All the previously enhanced immunological parameters reflected the maximum protection post challenge with velogenic viscerotropic NDV in the vaccinated noninfected chicken group. CONCLUSIONS: These findings provide novel insights into the immunosuppression activities of MG and E. coli infection in chickens vaccinated against NDV. SIGNIFICANCE AND IMPACT OF THE STUDY: This study hopes to provide a better insight to the immunosuppressive action of bacterial pathogens in chickens. This will help to improve biosecurity strategies during NDV vaccination in the future.


Assuntos
Galinhas/imunologia , Infecções por Escherichia coli/veterinária , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Coinfecção/veterinária , Citocinas/genética , Citocinas/metabolismo , Infecções por Escherichia coli/imunologia , Imunidade Humoral , Infecções por Mycoplasma/imunologia , Doenças das Aves Domésticas/microbiologia
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