RESUMO
The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as "silent pathogens" with the capacity to emerge as "pathobionts" for the development of inflammatory bowel disease and intestinal carcinogenesis.
Assuntos
Adesinas de Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/transmissão , Interações Hospedeiro-Patógeno , Humanos , Fatores de Risco , Fatores de Virulência/genéticaRESUMO
A vast and diverse array of microbial species displaying great phylogenic, genomic, and metabolic diversity have colonized the gastrointestinal tract. Resident microbes play a beneficial role by regulating the intestinal immune system, stimulating the maturation of host tissues, and playing a variety of roles in nutrition and in host resistance to gastric and enteric bacterial pathogens. The mechanisms by which the resident microbial species combat gastrointestinal pathogens are complex and include competitive metabolic interactions and the production of antimicrobial molecules. The human intestinal microbiota is a source from which Lactobacillus probiotic strains have often been isolated. Only six probiotic Lactobacillus strains isolated from human intestinal microbiota, i.e., L. rhamnosus GG, L. casei Shirota YIT9029, L. casei DN-114 001, L. johnsonii NCC 533, L. acidophilus LB, and L. reuteri DSM 17938, have been well characterized with regard to their potential antimicrobial effects against the major gastric and enteric bacterial pathogens and rotavirus. In this review, we describe the current knowledge concerning the experimental antibacterial activities, including antibiotic-like and cell-regulating activities, and therapeutic effects demonstrated in well-conducted, placebo-controlled, randomized clinical trials of these probiotic Lactobacillus strains. What is known about the antimicrobial activities supported by the molecules secreted by such probiotic Lactobacillus strains suggests that they constitute a promising new source for the development of innovative anti-infectious agents that act luminally and intracellularly in the gastrointestinal tract.
Assuntos
Antibiose , Terapia Biológica/métodos , Gastroenteropatias/terapia , Trato Gastrointestinal/microbiologia , Lactobacillus/fisiologia , Probióticos , Humanos , Lactobacillus/crescimento & desenvolvimento , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses.
Assuntos
Neoplasias do Colo/microbiologia , Enterovirus/patogenicidade , Células CACO-2 , Linhagem Celular Tumoral , Células HT29 , Humanos , Modelos BiológicosRESUMO
We conducted experiments in order to examine whether the probiotic Lactobacillus casei strain Shirota YIT9029 (LcS) in vitro and in vivo antagonism of Helicobacter pylori and Salmonella, involves inhibition of the swimming motility of these pathogens. We report the irreversible inhibition of the swimming motility of H. pylori strain 1101 and reversible inhibition of Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 by compound(s) secreted by LcS. In H. pylori 1101, irreversible inhibition results in the helical cells being progressively replaced by cells with 'c'-shaped and coccoid morphologies, accompanied by a loss of FlaA and FlaB flagellin expression. In S. Typhimurium SL1344, transient inhibition develops after membrane depolarization and without modification of expression of FliC flagellin. The inhibitory activity of strain LcS against both S. Typhimurium and H. pylori swimming motilities is linked with a small sized, heat-sensitive, and partially trypsin-sensitive, secreted compound(s), and needed the cooperation of the secreted membrane permeabilizing lactic acid metabolite. The inhibition of S. Typhimurium SL1344 swimming motility leads to delayed cell entry into human enterocyte-like Caco-2/TC7 cells and a strong decrease of cell entry into human mucus-secreting HT29-MTX cells.
Assuntos
Antibiose , Fatores Biológicos/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Lacticaseibacillus casei/fisiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/fisiologia , Proteínas de Bactérias/metabolismo , Fatores Biológicos/farmacologia , Linhagem Celular , Meios de Cultura/química , Meios de Cultura/metabolismo , Flagelina/metabolismo , Helicobacter pylori/efeitos dos fármacos , Humanos , Lacticaseibacillus casei/química , Salmonella typhimurium/efeitos dos fármacosRESUMO
We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.
Assuntos
Adesinas de Escherichia coli/metabolismo , Enterócitos/citologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Neutrófilos/fisiologia , alfa-Defensinas/metabolismo , Adesinas de Escherichia coli/genética , Linhagem Celular , Técnicas de Cocultura , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Histonas/metabolismo , Humanos , Peptídeo Hidrolases/metabolismoRESUMO
We report that both culture and the cell-free culture supernatant (CFCS) of Lactobacillus acidophilus strain LB (Lactéol Boucard) have the ability (i) to delay the appearance of Salmonella enterica serovar Typhimurium strain SL1344-induced mobilization of F-actin and, subsequently, (ii) to retard cell entry by S. Typhimurium SL1344. Time-lapse imaging and Western immunoblotting showed that S. Typhimurium SL1344 swimming motility, as represented by cell tracks of various types, was rapidly but temporarily blocked without affecting the expression of FliC flagellar propeller protein. We show that the product(s) secreted by L. acidophilus LB that supports the inhibitory activity is heat stable and of low molecular weight. The product(s) caused rapid depolarization of the S. Typhimurium SL1344 cytoplasmic membrane without affecting bacterial viability. We identified inhibition of swimming motility as a newly discovered mechanism by which the secreted product(s) of L. acidophilus strain LB retards the internalization of the diarrhea-associated pathogen S. enterica serovar Typhimurium within cultured human enterocyte-like cells.
Assuntos
Enterócitos/microbiologia , Flagelos/fisiologia , Lactobacillus acidophilus/fisiologia , Probióticos , Salmonella typhimurium/fisiologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Membrana Celular/fisiologia , Células Cultivadas , Humanos , Viabilidade Microbiana , Movimento , Salmonella typhimurium/patogenicidade , Imagem com Lapso de TempoRESUMO
The secreted autotransporter toxin, Sat, which belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, acts as a virulence factor in extraintestinal and intestinal pathogenic strains of Escherichia coli. We observed that HeLa cells exposed to the cell-free culture supernatant of recombinant strain AAEC185p(Sat-IH11128) producing the Sat toxin (CFCS(Sat) ), displayed dramatic disorganization of the F-actin cytoskeleton before loosening cell-to-cell junctions and detachment. Examination of the effect of Sat on GFP-microtubule-associated protein light chain 3 (LC3) HeLa cells revealed that CFCS(Sat) -induced autophagy follows CFCS(Sat) -induced F-actin cytoskeleton rearrangement. The induced autophagy shows an acceleration of the autophagy flux soon after Sat treatment, followed later by a blockade of the flux leading to the accumulation of large GFP-LC3-positive vacuoles in the cell cytoplasm. CFCS(Sat) did not induce cell detachment in autophagy-deficient mouse embryonic fibroblasts in contrast with wild-type mouse embryonic fibroblasts. The CFCS(Sat) -induced large GFP-LC3 dots do not display the characteristics of autophagolysosomes including expression of cathepsin D and Lamp-1 and 2 proteins, and Lysotracker Red- and DQ-BSA-positive labelling. We provide evidences that CFCS(Sat) -induced autophagy is not a cell response intended to get rid of the intracellular toxin. By a pharmacological blockers approach, we found that the blockade of Erk1/2 and p38 MAPKs, but not JNK, inhibited the CFCS(Sat) -induced autophagy and cell detachment whereas phosphatidylinositol-3 kinase blockers inhibiting canonical autophagy were inactive. When attached CFCS(Sat) -treated cells start to detach they showed caspase-independent cell death and rearrangements of the focal adhesion-associated vinculin and paxillin. Collectively, our results support that Sat triggers autophagy in epithelial cells that relies on its cell-detachment effect.
Assuntos
Autofagia , Adesão Celular , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Fatores de Virulência/metabolismo , Animais , Toxinas Bacterianas , Citoesqueleto/metabolismo , Fibroblastos/microbiologia , Células HeLa , Humanos , Camundongos , Transdução de SinaisRESUMO
CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Rim/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Linhagem Celular , Polaridade Celular , Cães , Escherichia coli/química , Engenharia Genética , Humanos , Lipídeo A , Lipopolissacarídeos/imunologia , Microdomínios da Membrana/metabolismo , Mucosa/metabolismo , Mucosa/fisiologia , Fosforilação , Isoformas de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno , Ressonância de Plasmônio de Superfície , Receptor 4 Toll-Like/genéticaRESUMO
The mechanism underlying the killing activity of Lactobacillus strains against bacterial pathogens appears to be multifactorial. Here, we investigate the respective contributions of hydrogen peroxide and lactic acid in killing bacterial pathogens associated with the human vagina, urinary tract or intestine by two hydrogen peroxide-producing strains. In co-culture, the human intestinal strain Lactobacillus johnsonii NCC933 and human vaginal strain Lactobacillus gasseri KS120.1 strains killed enteric Salmonella enterica serovar Typhimurium SL1344, vaginal Gardnerella vaginalis DSM 4944 and urinary tract Escherichia coli CFT073 pathogens. The cell-free culture supernatants (CFCSs) produced the same reduction in SL1344, DSM 4944 and CFT073 viability, whereas isolated bacteria had no effect. The killing activity of CFCSs was heat-stable. In the presence of Dulbecco's modified Eagle's minimum essential medium inhibiting the lactic acid-dependent killing activity, CFCSs were less effective at killing of the pathogens. Catalase-treated CFCSs displayed a strong decreased activity. Tested alone, hydrogen peroxide triggered a concentration-dependent killing activity against all three pathogens. Lactic acid alone developed a killing activity only at concentrations higher than that present in CFCSs. In the presence of lactic acid at a concentration present in Lactobacillus CFCSs, hydrogen peroxide displayed enhanced killing activity. Collectively, these results demonstrate that for hydrogen peroxide-producing Lactobacillus strains, the main metabolites of Lactobacillus, lactic acid and hydrogen peroxide, act co-operatively to kill enteric, vaginosis-associated and uropathogenic pathogens.
Assuntos
Antibiose , Peróxido de Hidrogênio , Ácido Láctico , Lactobacillus , Salmonella typhimurium , Escherichia coli Uropatogênica , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sinergismo Farmacológico , Enterobacteriaceae/classificação , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/metabolismo , Feminino , Gardnerella vaginalis/efeitos dos fármacos , Gardnerella vaginalis/crescimento & desenvolvimento , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Lactobacillus/classificação , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Infecções Urinárias/microbiologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Vagina/microbiologia , Vaginose Bacteriana/microbiologia , Vaginose Bacteriana/prevenção & controleRESUMO
In rabbit ligated ileal loops, two atypical enteropathogenic Escherichia coli (aEPEC) strains, 3991-1 and 0421-1, intimately associated with the cell membrane, forming the characteristic EPEC attachment and effacement lesion of the brush border, induced a mucous hypersecretion, whereas typical EPEC (tEPEC) strain E2348/69 did not. Using cultured human mucin-secreting intestinal HT29-MTX cells, we demonstrate that apically aEPEC infection is followed by increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins. The transcription of the MUC5AC and MUC4 genes was transiently upregulated after aEPEC infection. We provide evidence that the apically adhering aEPEC cells exploit the mucins' increased production since they grew in the presence of membrane-bound mucins, whereas tEPEC did not. The data described herein report a putative new virulence phenomenon in aEPEC.
Assuntos
Aderência Bacteriana , Enterócitos/metabolismo , Enterócitos/microbiologia , Escherichia coli Enteropatogênica/patogenicidade , Mucinas/biossíntese , Animais , Linhagem Celular , Modelos Animais de Doenças , Escherichia coli Enteropatogênica/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Íleo/microbiologia , Íleo/patologia , Coelhos , Regulação para Cima , VirulênciaRESUMO
The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.
Assuntos
Citocinas/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Norepinefrina/metabolismo , Adesinas de Escherichia coli/biossíntese , Adesinas de Escherichia coli/imunologia , Adulto , Aderência Bacteriana/efeitos dos fármacos , Células CACO-2 , Contagem de Colônia Microbiana , Escherichia coli/fisiologia , Humanos , Lactente , Fosforilação , Adulto JovemRESUMO
We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the alpha5 or beta1 integrin subunits nor alpha5beta1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.
Assuntos
Aderência Bacteriana , Citoplasma/microbiologia , Escherichia coli/fisiologia , Receptores de Superfície Celular/fisiologia , Adesinas de Escherichia coli/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos CD55/metabolismo , Células CHO , Moléculas de Adesão Celular/metabolismo , Cricetinae , Cricetulus , Células Epiteliais/microbiologia , Proteínas Ligadas por GPI , Células HeLa , Humanos , Viabilidade Microbiana , Receptores de Superfície Celular/metabolismo , Vacúolos/microbiologiaRESUMO
Until recently, intermediate filaments (IF) were thought to be only involved in resistance to physical stress and mechanical integrity of cells and tissues. Recent data indicate that IF play a much more important role in cellular physiology including organelle structure and positioning within the cell. Here, we show that Salmonella enterica serovar Typhimurium (S. typhimurium) induces in epithelial cells and macrophages the formation of an aggresome-like structure with a dramatic remodelling of cytoplasmic IF (vimentin and cytokeratin) networks and the adaptor proteins 14-3-3 which are recruited around intracellular S. typhimurium microcolonies. These rearrangements are not necessary for bacterial replication. Depletion of vimentin and cytokeratin by siRNA indicates that IF remodelling is required to maintain Salmonella microcolonies in the juxtanuclear area.
Assuntos
Núcleo Celular/microbiologia , Filamentos Intermediários/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/fisiologia , Vacúolos/microbiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/química , Filamentos Intermediários/microbiologia , Camundongos , Biossíntese de Proteínas , Infecções por Salmonella/microbiologia , Vacúolos/metabolismoRESUMO
Human decay accelerating factor (hDAF, CD55) and members of the carcinoembryonic-antigen-related cell-adhesion molecules (hCEACAMs) family are recognized as receptors by Gram-negative, diffusely adhering Escherichia coli (DAEC) strains expressing Afa/Dr adhesins. We report here that hCEACAM1-4L has a key function in downregulating the protein tyrosine Src kinase associated with hDAF signalling. After infecting HeLa epithelial cells stably transfected with hCEACAM1-4L cDNA with Dr adhesin-positive E. coli, the amount of the pTyr(416)-active form of the Src protein decreased, whereas that of the pTyr(527)-inactive form of Src protein did not increase. This downregulation of the Src protein implies that part of the hCEACAM1-4L protein had been translocated into lipid rafts, the protein was phosphorylated at Tyr residues in the cytoplasmic domain, and it was physically associated with the protein tyrosine phosphatase, SHP-2. Finally, we found that the hCEACAM1-4L-associated SHP-2 was not phosphorylated and lacked phosphatase activity, suggesting that the downregulation of Src protein associated with hDAF signalling results from the absence of dephosphorylation of the pTyr(527)-inactive form necessary for Src kinase activation.
Assuntos
Antígenos CD/metabolismo , Antígenos CD55/metabolismo , Moléculas de Adesão Celular/metabolismo , Escherichia coli/fisiologia , Quinases da Família src/metabolismo , Adesinas de Escherichia coli/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Microdomínios da Membrana/química , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
BACKGROUND: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). METHODOLOGY: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. PRINCIPAL FINDINGS: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. CONCLUSIONS: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.
Assuntos
Aderência Bacteriana , Escherichia coli/fisiologia , Mucosa Intestinal/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adesinas Bacterianas/fisiologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Intestinos/enzimologia , Intestinos/microbiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: Previous studies have shown that selected strains of Lactobacillus have the capacity to antagonize rotavirus-induced diarrhea. However, only a few reports have documented their efficacy against nonrotavirus diarrhea. This study involved an experimental investigation and a clinical trial of the antisecretory activity of Lactobacillus acidophilus strain LB in the context of nonrotavirus diarrhea. METHODS: The activity of a culture of L. acidophilus LB or of the lyophilized, heat-killed L. acidophilus LB bacteria plus their spent culture medium was tested in inhibiting the formation of fluid-formed domes in cultured human intestinal Caco-2/TC7 cell monolayers infected with diarrheagenic, diffusely adhering Afa/Dr Escherichia coli C1845 bacteria. A randomized, double-blind, placebo-controlled clinical trial of male or female children who were 10 months of age and presented with nonrotavirus, well-established diarrhea was conducted to evaluate the therapeutic efficacy of a pharmaceutical preparation that contains 10 billion heat-killed L. acidophilus LB plus 160 mg of spent culture medium. RESULTS: Infection of the cells with C1845 bacteria that were treated with L. acidophilus LB culture or the lyophilized, heat-killed L. acidophilus LB bacteria plus their culture medium produced a dosage-dependent decrease in the number of fluid-formed domes as compared with cells that were infected with untreated C1845 bacteria. The clinical results show that in selected and controlled homogeneous groups of children with well-established, nonrotavirus diarrhea, adding lyophilized, heat-killed L. acidophilus LB bacteria plus their culture medium to a solution of oral rehydration solution shortened by 1 day the recovery time (ie, the time until the first normal stool was passed) as compared with children who received placebo oral rehydration solution. CONCLUSIONS: Heat-killed L. acidophilus LB plus its culture medium antagonizes the C1845-induced increase in paracellular permeability in intestinal Caco-2/TC7 cells and produces a clinically significant benefit in the management of children with nonrotavirus, well-established diarrhea.
Assuntos
Diarreia Infantil/terapia , Lactobacillus acidophilus , Probióticos/uso terapêutico , Células Cultivadas , Método Duplo-Cego , Escherichia coli , Feminino , Humanos , Lactente , Masculino , Probióticos/farmacologia , Soluções para Reidratação/uso terapêutico , Resultado do TratamentoRESUMO
We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Intestinos/virologia , Proteínas de Membrana/metabolismo , Rotavirus/fisiologia , Junções Íntimas/virologia , Animais , Células CACO-2 , Carbazóis/farmacologia , Fracionamento Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Humanos , Indóis/farmacologia , Intestinos/citologia , Intestinos/enzimologia , Isoquinolinas/farmacologia , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia Confocal , Ocludina , Octoxinol/química , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Tionucleotídeos/farmacologia , Junções Íntimas/química , Junções Íntimas/enzimologia , Transcrição Gênica , Proteína da Zônula de Oclusão-1 , Proteína da Zônula de Oclusão-2RESUMO
Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is a brush border membrane (BBM)-associated enzyme in intestinal cells that hydrolyse lactose, the most important sugar in milk. Impairing in lactase activity during rotavirus infection has been described in diseased infants but the mechanism by which the functional lesion occurs remains unknown. We undertook a study to elucidate whether rotavirus impairs the lactase enzymatic activity in BBM of human enterocyte cells. In this study we use cultured human intestinal fully differentiated enterocyte-like Caco-2 cells to demonstrate how the lactase enzymatic activity at BBM is significantly decreased in rhesus monkey rotavirus (RRV)-infected cells. We found that the decrease in enzyme activity is not dependent of the Ca(2+)- and cAMP-dependent signalling events triggered by the virus. The LPH biosynthesis, stability, and expression of the protein at the BBM of infected cells were not modified. We provide evidence that in RRV-infected cells the kinetic of lactase enzymatic activity present at the BBM was modified. Both BBM(control) and BBM(RRV) have identical K(m) values, but hydrolyse the substrate at different rates. Thus, the BBM(RRV) exhibits almost a 1.5-fold decreased V(max) than that of BBM(control) and is therefore enzymatically less active than the latter. Our study demonstrate conclusively that the impairment of lactase enzymatic activity at the BBM of the enterocyte-like Caco-2 cells observed during rotavirus infection results from an inhibitory action of the secreted non-structural rotavirus protein NSP4.
Assuntos
Antígenos Virais/metabolismo , Células CACO-2/enzimologia , Glicoproteínas/metabolismo , Lactase-Florizina Hidrolase/metabolismo , Microvilosidades/enzimologia , Rotavirus/metabolismo , Toxinas Biológicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Antígenos Virais/genética , Células CACO-2/citologia , Células CACO-2/microbiologia , Glicoproteínas/genética , Humanos , Microvilosidades/microbiologia , Receptores Virais/genética , Receptores Virais/metabolismo , Infecções por Rotavirus/enzimologia , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).