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1.
Emerg Microbes Infect ; 8(1): 734-748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130074

RESUMO

Many pathogens infect hosts through various immune evasion strategies. However, the molecular mechanisms by which pathogen proteins modulate and evade the host immune response remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a pathological strain that can induce mitogen-activated protein (MAP) kinase (Erk, Jnk and p38 MAPK) and NF-κB pathway activation and proinflammatory cytokine production, which then causes diarrheal diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Transforming growth factor ß-activated kinase-1 (TAK1) is a key regulator involved in distinct innate immune signalling pathways. Here we report that EHEC translocated intimin receptor (Tir) protein inhibits the expression of EHEC-induced proinflammatory cytokines by interacting with the host tyrosine phosphatase SHP-1, which is dependent on the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). Mechanistically, the association of EHEC Tir with SHP-1 facilitated the recruitment of SHP-1 to TAK1 and inhibited TAK1 phosphorylation, which then negatively regulated K63-linked polyubiquitination of TAK1 and downstream signal transduction. Taken together, these results suggest that EHEC Tir negatively regulates proinflammatory responses by inhibiting the activation of TAK1, which is essential for immune evasion and could be a potential target for the treatment of bacterial infection.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , MAP Quinase Quinase Quinases/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Fatores de Virulência/metabolismo , Animais , Infecções por Escherichia coli/microbiologia , Células HEK293 , Humanos , Macrófagos Peritoneais , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Células RAW 264.7
2.
Microbiome ; 7(1): 43, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30890187

RESUMO

BACKGROUND: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. RESULTS: We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites-4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid-that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. CONCLUSION: Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.


Assuntos
Bactérias/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/patologia , Intestinos/citologia , Técnicas de Cultura de Órgãos/métodos , Animais , Benzoatos/farmacologia , Caproatos/farmacologia , Células Cultivadas , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Microbioma Gastrointestinal , Ácidos Heptanoicos/farmacologia , Humanos , Intestinos/microbiologia , Masculino , Camundongos , Procedimentos Analíticos em Microchip , Especificidade da Espécie
3.
Virulence ; 10(1): 180-193, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30806162

RESUMO

Enterohaemorrhagic Escherichia coli (EHEC) are food-borne pathogens responsible for bloody diarrhoea and renal failure in humans. While Shiga toxin (Stx) is the cardinal virulence factor of EHEC, its production by E. coli is not sufficient to cause disease and many Shiga-toxin producing E. coli (STEC) strains have never been implicated in human infection. So far, the pathophysiology of EHEC infection is not fully understood and more knowledge is needed to characterize the "auxiliary" factors that enable a STEC strain to cause disease in humans. In this study, we applied a recombinase-based in vivo expression technology (RIVET) to the EHEC reference strain EDL933 in order to identify genes specifically induced during the infectious process, using mouse as an infection model. We identified 31 in vivo-induced (ivi) genes having functions related to metabolism, stress adaptive response and bacterial virulence or fitness. Eight of the 31 ivi genes were found to be heterogeneously distributed in EHEC strains circulating in France these last years. In addition, they are more prevalent in strains from the TOP seven priority serotypes and particularly strains carrying significant virulence determinants such as Stx2 and intimin adhesin. This work sheds further light on bacterial determinants over-expressed in vivo during infection that may contribute to the potential of STEC strains to cause disease in humans.


Assuntos
Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Animais , Escherichia coli Êntero-Hemorrágica/patogenicidade , Expressão Gênica , Camundongos , Reação em Cadeia da Polimerase , Prevalência , Sorogrupo , Estresse Fisiológico , Virulência , Fatores de Virulência/genética
4.
PLoS Pathog ; 15(1): e1007494, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629725

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.


Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Prófagos/metabolismo , Ativação Viral/fisiologia , Animais , Bacteriófagos/metabolismo , Bacteriófagos/patogenicidade , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Feminino , Mucosa Intestinal/microbiologia , Lisogenia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Resposta SOS (Genética)/fisiologia , Toxina Shiga II/genética , Toxina Shiga II/metabolismo
5.
Biochem Soc Trans ; 47(1): 229-238, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30559275

RESUMO

Enterohaemorrhagic Escherichia coli (EHEC) mediates disease using a type 3 secretion system (T3SS), which is encoded on the locus of enterocyte effacement (LEE) and is tightly controlled by master regulators. This system is further modulated by a number of signals that help to fine-tune virulence, including metabolic, environmental and chemical signals. Since the LEE and its master regulator, Ler, were established, there have been numerous scientific advancements in understanding the regulation and expression of virulence factors in EHEC. This review will discuss the recent advancements in this field since our previous review, with a focus on the transcriptional regulation of the LEE.


Assuntos
Aderência Bacteriana , Enterócitos/microbiologia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Transdução de Sinais , Enterócitos/metabolismo , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/metabolismo , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Transcrição Genética , Virulência
6.
Cell Host Microbe ; 23(5): 607-617.e6, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746832

RESUMO

Bacteriophage-encoded genetic elements control bacterial biological functions. Enterohemorrhagic Escherichia coli (EHEC) strains harbor lambda-phages encoding the Shiga-toxin (Stx), which is expressed during the phage lytic cycle and associated with exacerbated disease. Phages also reside dormant within bacterial chromosomes through their lysogenic cycle, but how this impacts EHEC virulence remains unknown. We find that during lysogeny the phage transcription factor Cro activates the EHEC type III secretion system (T3SS). EHEC lambdoid phages are lysogenic under anaerobic conditions when Cro binds to and activates the promoters of T3SS genes. Interestingly, the Cro sequence varies among phages carried by different EHEC outbreak strains, and these changes affect Cro-dependent T3SS regulation. Additionally, infecting mice with the related pathogen C. rodentium harboring the bacteriophage cro from EHEC results in greater T3SS gene expression and enhanced virulence. Collectively, these findings reveal the role of phages in impacting EHEC virulence and their potential to affect outbreak strains.


Assuntos
Colífagos/metabolismo , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Repressoras/farmacologia , Proteínas Virais Reguladoras e Acessórias/farmacologia , Fatores de Virulência/genética , Animais , Cromossomos Bacterianos/efeitos dos fármacos , Citrobacter rodentium/patogenicidade , Colífagos/genética , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/patologia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Êntero-Hemorrágica/virologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Genes Bacterianos/efeitos dos fármacos , Células HeLa , Humanos , Lipídeos , Lisogenia , Camundongos , Camundongos Endogâmicos C3H , Proteínas Repressoras/genética , Toxina Shiga/genética , Fatores de Transcrição , Sistemas de Secreção Tipo III/efeitos dos fármacos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Ensaio de Placa Viral , Proteínas Virais Reguladoras e Acessórias/genética , Virulência/efeitos dos fármacos , Virulência/genética
7.
PLoS One ; 13(2): e0191544, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29420567

RESUMO

OBJECTIVES: Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli [STEC/EHEC] are the most common cause of Haemolytic Uraemic Syndrome [HUS] related to infectious haemorrhagic colitis. Nearly all recommendations on long term treatment of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first of this dimension to be caused by the serotype O104:H4. We report on the 3.5 year follow up of 61 patients diagnosed with symptomatic EHEC O104:H4 infection in spring 2011. METHODS: Patients with EHEC O104 infection were followed in a monocentric, prospective observational study at four time points: 4, 12, 24 and 36 months. These data include the patients' histories, clinical findings, and complications. RESULTS: Sixty-one patients suffering from EHEC O104:H4 associated enterocolitis participated in the study at the time of hospital discharge. The mean age of patients was 43 ± 2 years, 37 females and 24 males. 48 patients participated in follow up 1 [FU 1], 34 patients in follow up 2 [FU 2], 23 patients in follow up 3 [FU 3] and 18 patients in follow up 4 [FU 4]. Out of 61 patients discharged from the hospital and included in the study, 54 [84%] were examined at least at one additional follow up. Serum creatinine decreased significantly between discharge and FU 1 from 1.3 ± 0.1 mg/dl to 0.7 ± 0.1 mg/dl [p = 0.0045]. From FU 1 until FU 4, no further change in creatinine levels could be observed. The patients need of antihypertensive medications decreased significantly [p = 0.0005] between discharge and FU 1 after four months. From FU 1 until FU 3, 24 months later, no further significant change in antihypertensive treatment was observed. CONCLUSIONS: Our findings suggest that patients free of pathological findings at time of discharge do not need a specific follow up. Patients with persistent health problems at hospital discharge should be clinically monitored over four months to evaluate chronic organ damage. Progressive or new emerging renal damage could not be observed over time in any patient.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/terapia , Adulto , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento
8.
Transbound Emerg Dis ; 65 Suppl 1: 49-71, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29369531

RESUMO

Escherichia coli comprises a highly diverse group of Gram-negative bacteria and is a common member of the intestinal microflora of humans and animals. Generally, such colonization is asymptomatic; however, some E. coli strains have evolved to become pathogenic and thus cause clinical disease in susceptible hosts. One pathotype, the Shiga toxigenic E. coli (STEC) comprising strains expressing a Shiga-like toxin is an important foodborne pathogen. A subset of STEC are the enterohaemorrhagic E. coli (EHEC), which can cause serious human disease, including haemolytic uraemic syndrome (HUS). The diagnosis of EHEC infections and the surveillance of STEC in the food chain and the environment require accurate, cost-effective and timely tests. In this review, we describe and evaluate tests now in routine use, as well as upcoming test technologies for pathogen detection, including loop-mediated isothermal amplification (LAMP) and whole-genome sequencing (WGS). We have considered the need for improved diagnostic tools in current strategies for the control and prevention of these pathogens in humans, the food chain and the environment. We conclude that although significant progress has been made, STEC still remains an important zoonotic issue worldwide. Substantial reductions in the public health burden due to this infection will require a multipronged approach, including ongoing surveillance with high-resolution diagnostic techniques currently being developed and integrated into the routine investigations of public health laboratories. However, additional research requirements may be needed before such high-resolution diagnostic tools can be used to enable the development of appropriate interventions, such as vaccines and decontamination strategies.


Assuntos
Controle de Doenças Transmissíveis/métodos , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/prevenção & controle , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Escherichia coli Êntero-Hemorrágica/patogenicidade , Humanos , Escherichia coli Shiga Toxigênica/patogenicidade , Zoonoses/diagnóstico , Zoonoses/prevenção & controle
9.
Rev Med Interne ; 38(12): 833-839, 2017 Dec.
Artigo em Francês | MEDLINE | ID: mdl-28947259

RESUMO

Major achievements in the understanding of thrombotic microangiopathies (TMA) have not only resulted in a reclassification of TMA but most of all they have culminated in the design of new treatments and have enabled clinicians to better delineate their prognosis. Recent multicenter studies have improved our understanding of the prognosis of atypical hemolytic and uremic syndromes (aHUS). More specifically, they have highlighted the role of genetic testing on predicting the recurrence of aHUS, the risk of chronic kidney disease and the recurrence following kidney transplantation. A major advance consisted of the identification of the alternative complement pathway in the pathogenesis of aHUS, thus paving the way for the use of the C5a inhibitor eculizumab in this indication. Eculizumab has thereafter dramatically improved the management of patients affected with aHUS. During spring 2011, a great epidemic of entero-hemorrhagic Escherichia coli (EHEC) associated HUS occurred in Germany, providing clinicians the opportunity to examine the relevance of antibiotic prophylaxis, plasma exchange and eculizumab in EHEC-associated HUS. In this work, we herein present advances achieved in the setting of therapeutic management and prognosis in HUS and other related TMA syndromes.


Assuntos
Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/terapia , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/terapia , Disenteria Bacilar/complicações , Disenteria Bacilar/diagnóstico , Disenteria Bacilar/terapia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/complicações , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/terapia , Síndrome Hemolítico-Urêmica/epidemiologia , Humanos , Infecções Pneumocócicas/complicações , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/terapia , Prognóstico , Shigella dysenteriae/patogenicidade , Microangiopatias Trombóticas/epidemiologia , Deficiência de Vitamina B 12/complicações , Deficiência de Vitamina B 12/diagnóstico , Deficiência de Vitamina B 12/terapia
10.
Biotechnol J ; 12(10)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28869356

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.


Assuntos
Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/biossíntese , Plantas Geneticamente Modificadas/genética , Ruminantes/microbiologia , Tabaco/genética , Vacinas de Subunidades/biossíntese , Administração Oral , Animais , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Vacinas contra Escherichia coli/imunologia , Fezes/microbiologia , Regulação da Expressão Gênica de Plantas , Imunização , Masculino , Folhas de Planta/química , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes , Ovinos , Toxina Shiga II/genética , Tabaco/química , Vacinação , Fatores de Virulência/genética
11.
PLoS Pathog ; 13(8): e1006501, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28771584

RESUMO

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely-related pathogens that attach tightly to intestinal epithelial cells, efface microvilli, and promote cytoskeletal rearrangements into protrusions called actin pedestals. To trigger pedestal formation, EPEC employs the tyrosine phosphorylated transmembrane receptor Tir, while EHEC relies on the multivalent scaffolding protein EspFU. The ability to generate these structures correlates with bacterial colonization in several animal models, but the precise function of pedestals in infection remains unclear. To address this uncertainty, we characterized the colonization properties of EPEC and EHEC during infection of polarized epithelial cells. We found that EPEC and EHEC both formed distinct bacterial communities, or "macrocolonies," that encompassed multiple host cells. Tir and EspFU, as well as the host Arp2/3 complex, were all critical for the expansion of macrocolonies over time. Unexpectedly, EspFU accelerated the formation of larger macrocolonies compared to EPEC Tir, as EspFU-mediated actin assembly drove faster bacterial motility to cell junctions, where bacteria formed a secondary pedestal on a neighboring cell and divided, allowing one of the daughters to disengage and infect the second cell. Collectively, these data reveal that EspFU enhances epithelial colonization by increasing actin-based motility and promoting an efficient method of cell-to-cell transmission.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Actinas/metabolismo , Células CACO-2 , Proteínas de Transporte/metabolismo , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência
12.
Infect Immun ; 85(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28717028

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) is a commonly occurring foodborne pathogen responsible for numerous multistate outbreaks in the United States. It is known to infect the host gastrointestinal tract, specifically, in locations associated with lymphoid tissue. These niches serve as sources of enteric neurotransmitters, such as epinephrine and norepinephrine, that are known to increase virulence in several pathogens, including enterohemorrhagic E. coli The mechanisms that allow pathogens to target these niches are poorly understood. We previously reported that 3,4-dihydroxymandelic acid (DHMA), a metabolite of norepinephrine produced by E. coli, is a chemoattractant for the nonpathogenic E. coli RP437 strain. Here we report that DHMA is also a chemoattractant for EHEC. In addition, DHMA induces the expression of EHEC virulence genes and increases attachment to intestinal epithelial cells in vitro in a QseC-dependent manner. We also show that DHMA is present in murine gut fecal contents and that its production requires the presence of the commensal microbiota. On the basis of its ability to both attract and induce virulence gene expression in EHEC, we propose that DHMA acts as a molecular beacon to target pathogens to their preferred sites of infection in vivo.


Assuntos
Quimiotaxia , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Ácidos Mandélicos/metabolismo , Microbiota/fisiologia , Simbiose , Fatores de Virulência/genética , Animais , Aderência Bacteriana , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Fezes/química , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Virulência
13.
Infect Immun ; 85(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28630074

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.


Assuntos
Actinas/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Humanos , Cinesina/metabolismo , Camundongos , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido
14.
EcoSal Plus ; 7(2)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28635589

RESUMO

σN (also σ54) is an alternative sigma factor subunit of the RNA polymerase complex that regulates the expression of genes from many different ontological groups. It is broadly conserved in the Eubacteria with major roles in nitrogen metabolism, membrane biogenesis, and motility. σN is encoded as the first gene of a five-gene operon including rpoN (σN), ptsN, hpf, rapZ, and npr that has been genetically retained among species of Escherichia, Shigella, and Salmonella. In an increasing number of bacteria, σN has been implicated in the control of genes essential to pathogenic behavior, including those involved in adherence, secretion, immune subversion, biofilm formation, toxin production, and resistance to both antimicrobials and biological stressors. For most pathogens how this is achieved is unknown. In enterohemorrhagic Escherichia coli (EHEC) O157, Salmonella enterica, and Borrelia burgdorferi, regulation of virulence by σN requires another alternative sigma factor, σS, yet the model by which σN-σS virulence regulation is predicted to occur is varied in each of these pathogens. In this review, the importance of σN to bacterial pathogenesis is introduced, and common features of σN-dependent virulence regulation discussed. Emphasis is placed on the molecular mechanisms underlying σN virulence regulation in E. coli O157. This includes a review of the structure and function of regulatory pathways connecting σN to virulence expression, predicted input signals for pathway stimulation, and the role for cognate σN activators in initiation of gene systems determining pathogenic behavior.


Assuntos
Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Proteínas de Escherichia coli/genética , Regiões Promotoras Genéticas , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Transativadores , Virulência/genética
15.
FEMS Microbiol Lett ; 364(13)2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28651361

RESUMO

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, inhibits the type III secretion system (T3SS) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Salmonella enterica serovar Typhimurium, and Yersinia pseudotuberculosis. The inhibitory effect causes the inhibition of hemolysis, cell invasion, cell adhesion and apoptosis, which are functions of the type III secretion device. In the case of EPEC, EspB accumulates in the cells. RT-PCR showed that the translation of EspB was not blocked. The transcription of escN, which supplies energy for the injection of the effector factor into the host cells, was also not inhibited. EGCG does not suppress the transcription and translation of T3SS constitutive protein in bacterial cells, but it seems to suppress the normal construction or secretion of T3SS. When Luria-Bertani (LB) medium was used to visualize the EGCG-induced inhibition of T3SS, the inhibitory effect disappeared. The inhibition of T3SS was partially canceled when the T3SS inhibitory potency of EGCG was examined by adding yeast extract, which is a component of LB medium, to DMEM. These results suggest that EGCG probably inhibits secretion by suppressing some metabolic mechanisms of T3SS.


Assuntos
Catequina/análogos & derivados , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Enteropatogênica/efeitos dos fármacos , Salmonella typhi/efeitos dos fármacos , Sistemas de Secreção Tipo III/efeitos dos fármacos , Yersinia pseudotuberculosis/efeitos dos fármacos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Catequina/farmacologia , Linhagem Celular , Meios de Cultura/farmacologia , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microbiologia de Alimentos , Humanos , Salmonella typhi/patogenicidade , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/patogenicidade
16.
Artigo em Inglês | MEDLINE | ID: mdl-28469995

RESUMO

Serotype O157:H7, an enterohemorrhagic Escherichia coli (EHEC), is known to cause gastrointestinal and systemic illnesses ranging from diarrhea and hemorrhagic colitis to potentially fatal hemolytic uremic syndrome. Specific genetic factors like ompA, nsrR, and LEE genes are known to play roles in EHEC pathogenesis. However, these factors are not specific to EHEC and their presence in several non-pathogenic strains indicates that additional factors are involved in pathogenicity. We propose a comprehensive effort to screen for such potential genetic elements, through investigation of biomolecular interactions between E. coli and their host. In this work, an in silico investigation of the protein-protein interactions (PPIs) between human cells and four EHEC strains (viz., EDL933, Sakai, EC4115, and TW14359) was performed in order to understand the virulence and host-colonization strategies of these strains. Potential host-pathogen interactions (HPIs) between human cells and the "non-pathogenic" E. coli strain MG1655 were also probed to evaluate whether and how the variations in the genomes could translate into altered virulence and host-colonization capabilities of the studied bacterial strains. Results indicate that a small subset of HPIs are unique to the studied pathogens and can be implicated in virulence. This subset of interactions involved E. coli proteins like YhdW, ChuT, EivG, and HlyA. These proteins have previously been reported to be involved in bacterial virulence. In addition, clear differences in lineage and clade-specific HPI profiles could be identified. Furthermore, available gene expression profiles of the HPI-proteins were utilized to estimate the proportion of proteins which may be involved in interactions. We hypothesized that a cumulative score of the ratios of bound:unbound proteins (involved in HPIs) would indicate the extent of colonization. Thus, we designed the Host Colonization Index (HCI) measure to determine the host colonization potential of the E. coli strains. Pathogenic strains of E. coli were observed to have higher HCIs as compared to a non-pathogenic laboratory strain. However, no significant differences among the HCIs of the two pathogenic groups were observed. Overall, our findings are expected to provide additional insights into EHEC pathogenesis and are likely to aid in designing alternate preventive and therapeutic strategies.


Assuntos
Simulação por Computador , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas/fisiologia , Animais , Bovinos , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Células Epiteliais , Escherichia coli/genética , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Virulência/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-28484684

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are human pathogens responsible for bloody diarrhea and renal failures. EHEC employ a type 3 secretion system to attach directly to the human colonic epithelium. This structure is encoded by the locus of enterocyte effacement (LEE) whose expression is regulated in response to specific nutrients. In this study, we show that the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA) inhibit EHEC adhesion to epithelial cells through down-regulation of LEE expression. The effect of NAG and NANA is dependent on NagC, a transcriptional repressor of the NAG catabolism in E. coli. We show that NagC is an activator of the LEE1 operon and a critical regulator for the colonization of mice intestine by EHEC. Finally, we demonstrate that NAG and NANA as well as the metabolic activity of Bacteroides thetaiotaomicron affect the in vivo fitness of EHEC in a NagC-dependent manner. This study highlights the role of NagC in coordinating metabolism and LEE expression in EHEC and in promoting EHEC colonization in vivo.


Assuntos
Acetilglucosamina/antagonistas & inibidores , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas/genética , Proteínas Repressoras/genética , Animais , Bacteroides thetaiotaomicron/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Células Epiteliais/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Células HCT116 , Células HeLa , Humanos , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Ácido N-Acetilneuramínico/antagonistas & inibidores , Óperon , Fosfoproteínas/metabolismo , Proteínas Repressoras/fisiologia
18.
J Biol Chem ; 292(27): 11423-11430, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28522607

RESUMO

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.


Assuntos
Proteínas de Bactérias/metabolismo , Citrobacter rodentium/enzimologia , Escherichia coli Êntero-Hemorrágica/enzimologia , Escherichia coli Enteropatogênica/enzimologia , Proteínas de Escherichia coli/metabolismo , Salmonella enterica/enzimologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Citrobacter rodentium/genética , Citrobacter rodentium/patogenicidade , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/genética , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicosilação , Camundongos , Células RAW 264.7 , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Fatores de Virulência/genética
19.
PLoS Pathog ; 13(3): e1006246, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28288207

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.


Assuntos
Catalase/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Sistemas de Secreção Tipo VI/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Virulência/fisiologia
20.
Sci Rep ; 7: 44655, 2017 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-28317910

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Fímbrias Bacterianas/metabolismo , Nódulos Linfáticos Agregados/microbiologia , Nódulos Linfáticos Agregados/patologia , Animais , Aderência Bacteriana/genética , Translocação Bacteriana , Células CACO-2 , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/genética , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Escherichia coli O157 , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Intestino Delgado/microbiologia , Intestino Delgado/patologia , Masculino , Camundongos , Modelos Biológicos , Óperon/genética , Sorotipagem , Estômago/microbiologia , Estômago/patologia , Virulência
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