RESUMO
AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.
Assuntos
Escherichia coli Êntero-Hemorrágica , Proteínas de Escherichia coli , Proteínas de Escherichia coli/toxicidade , Proteínas de Escherichia coli/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Chaperonas Moleculares , Retículo Endoplasmático/metabolismo , Enterotoxinas , Proteínas de Transporte/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Oxirredução , BiologiaRESUMO
Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.
Assuntos
Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Inflamassomos/metabolismo , Meningite devida a Escherichia coli/metabolismo , Sepse Neonatal/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animais , Células Cultivadas , Escherichia coli K12 , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Macrófagos/metabolismo , Meningite devida a Escherichia coli/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Sepse Neonatal/microbiologia , Receptores Purinérgicos P2X/genéticaRESUMO
Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.
Assuntos
Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Células CACO-2 , Polaridade Celular , Proliferação de Células , Neoplasias do Colo/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Infecções por Escherichia coli/metabolismo , Humanos , Junções Intercelulares , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.
Assuntos
Proteínas de Escherichia coli/toxicidade , Síndrome Hemolítico-Urêmica/etiologia , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Síndrome Hemolítico-Urêmica/patologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Glomérulos Renais/citologia , Túbulos Renais Proximais/citologia , Masculino , Camundongos Endogâmicos BALB CRESUMO
Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.
Assuntos
Toxinas Bacterianas/toxicidade , Escherichia coli Enterotoxigênica , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Animais , Toxinas Bacterianas/farmacocinética , Células CACO-2 , Edema/induzido quimicamente , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Cardiopatias Congênitas/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Humanos , Intestinos/metabolismo , Miocárdio/metabolismo , Saco Vitelino/efeitos dos fármacos , Peixe-Zebra/anormalidades , Peixe-Zebra/metabolismoRESUMO
The Hok/Gef family consists of structurally similar, single-span membrane peptides that all contain a positively charged N-terminal domain, an α-helix and a periplasmic C-terminal domain. Hok/Gef peptides have previously been described to play distinct physiological roles. Indeed, while HokB has been implicated in bacterial persistence, other members of the Hok/Gef family are known to induce cell lysis. However, the generalizability of previously published studies is problematic, as they have all used different expression systems. Therefore, we conducted a systematic study of the nine Hok/Gef peptides of Escherichia coli. We observed rapid cell death following expression of hokA, hokC, hokD, hokE, pndA1, hok or srnB, while expression of hokB or pndA2 does not result in cell lysis. A remarkable feature of Hok/Gef peptides is the presence of conserved periplasmic tyrosine and/or cysteine residues. For the HokB peptide, one of these residues has previously been implicated in intermolecular dimerization, which is essential for HokB to exert its role in persistence. To assess the role of the periplasmic cysteine and tyrosine residues in other Hok/Gef peptides and to decipher whether these residues determine peptide toxicity, an array of substitution mutants were constructed. We found that these residues are important activators of toxicity for Hok, HokA and HokE peptides. Despite the loss of the cell killing phenotype in HokS31_Y48, HokAS29_S46 and HokES29_Y46, these peptides do not exert a persister phenotype. More research is needed to fully comprehend why HokB is the sole peptide of the Hok/Gef family that mediates persistence.
Assuntos
Toxinas Bacterianas/metabolismo , Cisteína/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Cisteína/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/toxicidade , Viabilidade Microbiana , Mutação , Periplasma/metabolismo , Sistemas Toxina-Antitoxina , Tirosina/genética , Tirosina/metabolismoRESUMO
Enterotoxigenic Escherichia coli causes severe infectious diarrhea with high morbidity and mortality in newborn and weanling pigs mainly through the production of heat-stable enterotoxins (STs). However, the precise regulatory mechanisms involved in ST-induced intestinal epithelium injury remain unclear. Consequently, we conducted the experiments in vivo (mice), ex vivo (mouse and porcine enteroids), and in vitro (MODE-K and IPEC-J2 cells) to explore the effect of STp (one type of STa) on the integrity of the intestinal epithelium. The results showed that acute STp exposure led to small intestinal edema, disrupted intestinal integrity, induced crypt cell expansion into spheroids, and downregulated Wnt/ß-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active ß-catenin, ß-catenin, Lgr5, PCNA, and KRT20 were significantly decreased after STp treatment, as determined ex vivo. In addition, STp inhibited cell proliferation, induced cell apoptosis, destroyed cell barriers, and reduced Wnt/ß-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/ß-catenin reactivation protected the IPEC-J2 cells from STp-induced injury. Taking these findings together, we conclude that STp inhibits intestinal stem cell expansion to disrupt the integrity of the intestinal mucosa through the downregulation of the Wnt/ß-catenin signaling pathway.
Assuntos
Toxinas Bacterianas/toxicidade , Edema/genética , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Receptores Frizzled/genética , Mucosa Intestinal/efeitos dos fármacos , Organoides/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , beta Catenina/genética , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Edema/induzido quimicamente , Edema/metabolismo , Edema/patologia , Escherichia coli Enterotoxigênica/química , Escherichia coli Enterotoxigênica/patogenicidade , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Queratina-20/genética , Queratina-20/metabolismo , Camundongos , Organoides/citologia , Organoides/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Suínos , beta Catenina/metabolismoRESUMO
α-Hemolysin (HlyA) is an extracellular protein toxin secreted by uropathogenic strains of Escherichia coli that inserts into membranes of eukaryotic cells. The main goal of this work was to investigate the involvement of tryptophan (W) residues in the hemolytic activity of HlyA. We investigated the hemolytic activity of six single-point mutant proteins, in which one of the four Ws was replaced by cysteine (C) or leucine (L). We also analyzed the photoinactivation of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation was analyzed by ultraviolet-visible spectrophotometry, hemolytic activity measurement, fluorescence spectroscopy, and electrophoretic analysis. The results indicate that Ws are important in the hemolytic process. Specifically, the chemical structure of the amino acid at position 578 is important for the acylation of HlyA at residue K563. Furthermore, the exposure of HlyA to ultraviolet radiation, with energy similar to that experienced under sun exposure, in the presence of Ptr induces the inactivation of the toxin, causing chemical changes in, at least, W and Y, the rate of damage to W residues being faster than that observed for Y residues. This work not only deepens our understanding of the structure-function relationship of the toxin but also introduces the possibility of using photoinactivation of HlyA for potential applications such as obtaining innocuous molecules for vaccine production and the elimination of the toxin from contaminated surfaces and drinking water.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Luz , Triptofano , Substituição de Aminoácidos , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Hemólise/efeitos dos fármacos , Hemólise/efeitos da radiaçãoRESUMO
The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.
Assuntos
Membrana Celular/metabolismo , Colestanos/farmacologia , Dobramento de Proteína , Multimerização Proteica , Espermina/análogos & derivados , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Fenômenos Biofísicos/efeitos dos fármacos , Carboxil e Carbamoil Transferases/química , Carboxil e Carbamoil Transferases/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/toxicidade , Humanos , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Espermina/farmacologia , alfa-Sinucleína/química , alfa-Sinucleína/toxicidadeRESUMO
Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dexametasona/toxicidade , Diarreia/etiologia , Dimetil Sulfóxido/toxicidade , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diarreia/induzido quimicamente , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Masculino , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismoRESUMO
Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Isomerases de Dissulfetos de Proteínas/metabolismo , Subtilisinas/toxicidade , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Proteínas de Choque Térmico/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Humanos , MAP Quinase Quinase 4/metabolismo , Fosforilação , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , RNA Interferente PequenoRESUMO
Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Toxinas Bacterianas/toxicidade , Células CACO-2 , Citotoxinas/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Humanos , Serina Endopeptidases/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Fatores de Virulência/metabolismoRESUMO
The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 µmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.
Assuntos
Proteínas de Escherichia coli , Subunidades Proteicas , Subtilisinas , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/toxicidade , Células HeLa , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/toxicidade , Escherichia coli Shiga Toxigênica , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/toxicidadeRESUMO
Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.
Assuntos
Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Sinergismo Farmacológico , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Síndrome Hemolítico-Urêmica , Humanos , Rim/irrigação sanguíneaRESUMO
Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter â¼200â nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Eritrócitos/efeitos dos fármacos , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Micropartículas Derivadas de Células/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismoRESUMO
Transcription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX and DNAse I footprinting in vitro. All these approaches require large amounts of purified proteins. However, overproduction of transcription factors leading to their extensive binding to the regulatory elements on the DNA make them toxic to a bacterial cell thus significantly complicating production of a soluble protein. Here, on the example of three regulators from Escherichia coli, UxuR, ExuR, and LeuO, we show that stable production of toxic transcription factors in a soluble fraction can be significantly enhanced by holding the expression of a recombinant protein back at the early stages of bacterial growth. This can be achieved by cloning genes together with their regulatory regions containing repressor sites, with subsequent growth in a very rich media where activity of excessive regulators is not crucial, followed by induction with a very low concentration of an inducer. Schemes of further purification of these proteins were developed, and functional activity was confirmed.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/toxicidade , Regulação Bacteriana da Expressão Gênica , Óperon , Fatores de Transcrição/metabolismo , Fatores de Transcrição/toxicidadeRESUMO
EAST1 is produced by a subset of enteroaggregative Escherichia coli strains. This toxin is a 38-amino acid peptide of 4100 Da. It shares 50% homology with the enterotoxic domain of STa and interacts with the same receptor. The mechanism of action of EAST1is proposed to be identical to that of STa eliciting a cGMP increase. EAST1 is associated with diarrheal disease in Man and various animal species including cattle and swine. Nevertheless, as EAST1-positive strains as well as culture supernatants did not provoke unequivocally diarrhea either in animal models or in human volunteers, the role of this toxin in disease is today still debated. This review intent is to examine the role of EAST1 toxin in diarrheal illnesses.
Assuntos
Diarreia , Enterotoxinas/fisiologia , Infecções por Escherichia coli , Proteínas de Escherichia coli/fisiologia , Escherichia coli/patogenicidade , Animais , Toxinas Bacterianas/toxicidade , Bovinos , Diarreia/microbiologia , Diarreia/veterinária , Enterotoxinas/toxicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/toxicidade , Humanos , SuínosAssuntos
Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Fatores de Virulência , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Humanos , Escherichia coli Shiga Toxigênica/patogenicidade , Estados Unidos , United States Food and Drug AdministrationRESUMO
The present study was carried out to evaluate the effect of dietary supplementation of Scutellaria baicalensis extracts (SBE) on intestinal health in terms of morphology, barrier integrity and immune responses in weaned piglets challenged with Escherichia coli K88. A total of seventy-two weaned piglets were assigned into two groups to receive a basal diet without including antibiotic additives or the basal diet supplemented 1000 mg SBE/kg diet for 14 d. On day 15, twelve healthy piglets from each group were selected to expose to oral administration of either 10 ml 1 × 109 colony-forming units of E. coli K88 or the vehicle control. After 48 h of E.coli K88 challenge, blood was sampled, and then all piglets were killed humanely for harvesting jejunal and ileal samples. Dietary supplementation of SBE significantly decreased diarrhoea frequency and improved feed conversion ratio (P < 0·05). SBE supplementation to E.coli K88-challenged piglets improved villous height and villous height/crypt depth (P < 0·05), recovered the protein expression of occludin and zonula occludens-2 in both the jejunum and ileum (P < 0·05), and mitigated the increases in plasma IL-1ß, TNF-α, IL-6, IgA and IgG (P < 0·05). Meanwhile, dietary SBE effectively inhibited the stimulation of NF-κB, P38 and TNF-α as well as IL-1ß in the small intestine of piglets challenged by E. coli K88 and prevented the activation of NF-κB/P38 signalling pathways (P < 0·05). Collectively, SBE supplementation can potently attenuate diarrhoea in weaning piglets and decrease inflammatory cytokine expressions through inhibiting the NF-κB and P38 signalling pathways.
Assuntos
Antígenos de Bactérias/toxicidade , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/toxicidade , Proteínas de Fímbrias/toxicidade , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/fisiologia , Scutellaria baicalensis/química , Doenças dos Suínos/prevenção & controle , Animais , Diarreia/microbiologia , Diarreia/prevenção & controle , Diarreia/veterinária , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Expressão Gênica , Íleo/imunologia , Íleo/metabolismo , Íleo/patologia , Jejuno/imunologia , Jejuno/metabolismo , Jejuno/patologia , Extratos Vegetais/administração & dosagem , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Proteínas de Junções Íntimas/genética , DesmameRESUMO
One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.