RESUMO
AIMS: To use the phage display technique to develop peptides with the capability to neutralize the cytotoxicity induced by Stx1 and Stx2 toxins produced by Shiga toxin-producing Escherichia coli (STEC). METHODS AND RESULTS: The phage display technique permitted the development of three peptides, named PC7-12, P12-26 and PC7-30, which bind to the globotriaosylceramide (Gb3) receptor for Shiga toxins produced by STEC. Moreover, these peptides were capable of competing efficiently with the Shiga toxins for binding to Gb3. The peptides described herein partially inhibited the Stx-induced cytotoxicity of cell-free filtrates of STEC O157 : H7 and purified Stx toxins in Vero cells. The inhibition of lethality induced by Stx toxins in mice indicated that peptide PC7-30 inhibited the lethality caused by Stx1 (2LD50) in mice. CONCLUSIONS: The phage display technique permitted the development of peptides that inhibited the cytotoxicity induced by Stx toxins in vitro. Peptide PC7-30 inhibited the lethality of Stx1 in vivo; this molecule would be a promising candidate for the development of therapeutic agents for STEC-related diseases in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of Gb3, the common receptor for Stx1 and Stx2, may contribute to the development of efficient neutralizers for both toxins, and our approach would be an interesting alternative for the development of therapeutic molecules for the treatment of diseases caused by STEC strains.
Assuntos
Peptídeos/farmacologia , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Animais , Chlorocebus aethiops , Humanos , Camundongos , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/metabolismo , Triexosilceramidas/metabolismo , Células VeroRESUMO
Escherichia coli Vacuolating Factor (ECVF) is a heat-labile, vacuolating cytotoxin produced by avian pathogenic E. coli (APEC) isolated from avian cellulitis lesions. In this report, we intend to demonstrate that purified ECVF induces the inflammatory process of cellulitis. Our group is the first to demonstrate the effect of ECVF in a histological analysis by in situ inoculation of broiler chickens with purified ECVF. The animals were inoculated with the APEC AC53 and with purified ECVF subcutaneously on their ventral surface (in the sternum region). The histological analysis showed different grades of an acute inflammatory response in the epidermis, dermis and panniculus. An increase in mRNA expression of the proinflammatory cytokine TNF-α was also demonstrated in the inflamed tissue. When ECVF was systemically administered, increased levels of TNF-α and IL-10 were observed in the serum. These results suggest that ECVF plays a key role in the inflammatory process associated with cellulitis that is mainly mediated by TNF-α. In addition, this inflammation can be downregulated by the anti-inflammatory cytokine IL-10.
Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/toxicidade , Celulite (Flegmão)/veterinária , Infecções por Escherichia coli/veterinária , Escherichia coli/metabolismo , Doenças das Aves Domésticas/induzido quimicamente , Doenças das Aves Domésticas/microbiologia , Animais , Celulite (Flegmão)/sangue , Celulite (Flegmão)/induzido quimicamente , Celulite (Flegmão)/microbiologia , Embrião de Galinha , Galinhas , Escherichia coli/genética , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/induzido quimicamente , Infecções por Escherichia coli/microbiologia , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-10/genética , Masculino , Doenças das Aves Domésticas/sangue , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.
Assuntos
Adulto , Animais , Humanos , Adesinas Bacterianas/fisiologia , Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Sepse/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/ultraestrutura , Aderência Bacteriana/genética , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Genótipo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Células VeroRESUMO
The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Sepse/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/ultraestrutura , Adulto , Animais , Aderência Bacteriana/genética , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Genótipo , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Células VeroRESUMO
Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60ºC and 100ºC for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for αHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.(AU)
Assuntos
Humanos , Escherichia coli/virologia , Água Potável/análise , Enterotoxinas/efeitos adversos , Citotoxinas , Infecções por Escherichia coli/etiologia , Água Potável/efeitos adversos , Citotoxinas/efeitos adversosRESUMO
Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60¨¬C and 100¨¬C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for ¥áHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.
Assuntos
Animais , Camundongos , Água Potável/análise , Citotoxinas , Escherichia coli Enterotoxigênica , Enterotoxinas , Escherichia coli/isolamento & purificaçãoRESUMO
Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6%), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60% of strains vs 11.7% of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75% for fimH, fyuA, kpsMTII and iucD, and between 35-65% for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.
Assuntos
Escherichia coli/patogenicidade , Sepse/microbiologia , Fatores de Virulência/genética , Adulto , Animais , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Humanos , Reação em Cadeia da Polimerase , Células Vero/microbiologia , Virulência/genéticaRESUMO
Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6 percent), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60 percent of strains vs 11.7 percent of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75 percent for fimH, fyuA, kpsMTII and iucD, and between 35-65 percent for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.
Assuntos
Adulto , Animais , Humanos , Escherichia coli/patogenicidade , Sepse/microbiologia , Fatores de Virulência/genética , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Reação em Cadeia da Polimerase , Células Vero/microbiologia , Virulência/genéticaRESUMO
AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.
Assuntos
Apoptose , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Mucosa Intestinal/microbiologia , Fosfatase Alcalina/metabolismo , Células CACO-2 , Sobrevivência Celular , Diarreia/metabolismo , Diarreia/microbiologia , Escherichia coli/química , Células HT29 , Humanos , Mucosa Intestinal/citologia , L-Lactato Desidrogenase/metabolismoRESUMO
AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.
Assuntos
Citotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Stenotrophomonas maltophilia/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citotoxinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Ferro/farmacologia , Ovinos , Células Vero , Zinco/farmacologiaRESUMO
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 æg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 æg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Assuntos
Animais , Masculino , Camundongos , Toxinas Bacterianas , Eritrócitos , Escherichia coli , Células Eucarióticas , Toxinas Bacterianas , Membrana Celular , Colesterol , Eletroforese em Gel de Poliacrilamida , Hemólise , Ligação ProteicaRESUMO
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 microg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 microg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Eritrócitos/efeitos dos fármacos , Escherichia coli/química , Células Eucarióticas/efeitos dos fármacos , Proteínas Hemolisinas/isolamento & purificação , Animais , Toxinas Bacterianas/toxicidade , Membrana Celular , Colesterol/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli , Proteínas Hemolisinas/toxicidade , Hemólise , Masculino , Camundongos , Ligação ProteicaRESUMO
Seven of 50 Enterobacter cloacae strains from clinical isolates produced small turbid zones of hemolysis in horse and sheep blood agar plates, and the culture supernatants were also positive for hemolytic activity. The hemolysin was partially purified from the culture supernatant of E. cloacae by ultrafiltration (PM-10 membrane) and extraction with acetone. Semipurified hemolysin was stable to heating (100 degrees C, 30 min) and was soluble in organic solvents (acetone, ethanol, and methanol). The toxin showed no loss of biological activity after treatment with trypsin and was stable to acid treatment at pH 2.0 but not at a pH greater than 7.0. In the rat intestinal loop assay, the hemolysin caused hemorrhagic fluid accumulation and severe histological alterations. These findings indicate that this hemolysin may be a putative virulence factor in E. cloacae infections.
Assuntos
Enterobacter cloacae/patogenicidade , Proteínas Hemolisinas , Intestinos/efeitos dos fármacos , Ágar , Animais , Enterobacter cloacae/metabolismo , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/química , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/toxicidade , Hemólise , Cavalos , Humanos , Peso Molecular , Ratos , Ovinos , VirulênciaRESUMO
AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.
Assuntos
Plesiomonas/patogenicidade , Microbiologia da Água , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/toxicidade , Citotoxinas/biossíntese , Citotoxinas/toxicidade , Proteínas Hemolisinas/biossíntese , Hemólise , Humanos , Vacúolos/efeitos dos fármacos , VirulênciaRESUMO
Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.
Assuntos
Citotoxinas/isolamento & purificação , Citotoxinas/toxicidade , Serratia marcescens/química , Animais , Linhagem Celular/efeitos dos fármacos , Cricetinae , Eletroforese em Gel de Poliacrilamida , Haplorrinos , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Peso MolecularRESUMO
Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 æg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli
Assuntos
Animais , Cricetinae , Humanos , Camundongos , Citotoxinas , Serratia marcescens , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Haplorrinos , Hemólise , Peso MolecularRESUMO
The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen. In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA. Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate. Cytoplasmic blebbing and nuclear condensation also occurred. DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis. These results show that the cytotoxic enterotoxin of A. hydrophila can induce apoptosis in human intestinal cells in culture.
Assuntos
Aeromonas hydrophila/patogenicidade , Apoptose/efeitos dos fármacos , Proteínas de Bactérias , Enterotoxinas/toxicidade , Mucosa Intestinal/microbiologia , Aeromonas hydrophila/metabolismo , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Microscopia EletrônicaRESUMO
Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 microg/ml and 50 microg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 microg/ml and 1250 microg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.
Assuntos
Adjuvantes Imunológicos/farmacologia , Escherichia coli/citologia , Fímbrias Bacterianas/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Mucoproteínas/farmacologia , Fagocitose/efeitos dos fármacos , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , UromodulinaRESUMO
Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 æg/ml and 50 æg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 æg/ml and 1250 æg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages
Assuntos
Humanos , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Escherichia coli/citologia , Fímbrias Bacterianas/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacosRESUMO
The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.