L-amino acid oxidase activity present in fractions of Bothrops jararaca venom is responsible for the induction of programmed cell death in Trypanosoma cruzi.

Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti-T. cruzi activity was associated with fractions that present L-amino acid oxidase (L-AAO) activity. L-AAO produces H(2)O(2), which is highly toxic. The addition of catalase to the medium, a H(2)O(2) scavenger, reverted the killing capacity of venom fractions. The anti-T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for L-AAO activity. These results were confirmed with a commercial purified L-AAO. Treatment for 24 h with fractions that present L-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H(2)O(2). These results indicate that H(2)O(2), the product of L-AAO reaction, induces programmed cell death explaining the anti-T. cruzi activity of B. jararaca venom.

Synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities of thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles.

In this work we reported the synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities in vitro of three new compound series obtained from ethyl(5-methyl-1-H-imidazole-4-carboxylate): acylthiosemicarbazide analogues 3a-d, 4-thiazolidinone analogues 4a-d and 1,3,4-thiadiazole analogues 5a-d. All synthesized compounds were characterized by IR, (1)H, (13)C NMR and HRMS. The majority of the tested compounds show excellent anti-T. gondii activity when compared to hydroxyurea and sulfadiazine. In addition it was also shown that most of the compounds in this study have a better performance against intracellular tachyzoites. The results for antimicrobial activity evaluation showed weak antibacterial and antifungal activities for all the tested molecules, when compared with the standard drugs (chloramphenicol and rifampicin for antibacterial activity; nistatin and ketoconazole for antifungal activity).

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression.

AIMS: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. METHODS AND RESULTS: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco's Modified Eagle's Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl(2) , they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. CONCLUSION: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin.

AIMS: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. METHODS AND RESULTS: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1.3x10(-8) mol l(-1), failed in the detection of some of these isolates. CONCLUSION: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. SIGNIFICANCE AND IMPACT OF THE STUDY: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression

The aim of study was to develop a colony immunoblot assay to differentiatetypical from atypical enteropathogenic Escherichia coli (EPEC) by detectionof bundle-forming pilus (BFP) expression. Anti-BFP antiserum was raised in rabbits and itsreactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. The assay enables reliable identification of BFP-expressing isolatesand contributes to the differentiation of typical and atypical EPEC.The colony immunoblot for BFP detectiondeveloped in this study combines the simplicity of an immunoserologicalassay with the high efficiency of testing a large number of EPECcolonies.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

To evaluate the sensitivity and specificity of polyclonal and monoclonalantibodies (Mabs) against intimin in the detection of enteropathogenic andenterohaemorrhagic Escherichia coli isolates using immunoblotting.Polyclonal and Mabs against the intimin-conservedregion were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of1Æ3 · 10)8 mol l)1, failed in the detection of some of these isolates. All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract.

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin â, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom.

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50 percent growth inhibition was obtained with 10 æg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Virulence factors of Escherichia coli strains belonging to serogroups O127 and O142.

A total of 102 Escherichia coli strains belonging to serogroups O127 and O142 were examined for genotypic and phenotypic characteristics. The most frequent serotypes found were O127:H21, O127:H40 and O142:H34. The virulence properties were evaluated by adhesion to HeLa cells and hybridization with gene probes for diarrhoeagenic E. coli. Most strains in the two serogroups were categorized as enteropathogenic E. coli, but enteroaggregative E. coli was also detected in both serogroups. All strains that carried the eae sequence presented the LEE region inserted in selC. Five ribotypes were detected in serogroup O127 and four in serogroup O142 and a correlation between serotypes and ribotypes was observed mainly in serogroup O142.

A 2S albumin-homologous protein from passion fruit seeds inhibits the fungal growth and acidification of the medium by Fusarium oxysporum.

Antimicrobial proteins have been isolated from a wide range of plant species. More recently, it has become increasingly clear that these types of proteins play an important role in the protection of plants. In this study, we investigate the presence of defense-related proteins from passion fruit (Passiflora edulis f. flavicarpa) seeds. Initially, seed flour was extracted for 2h (at 4 degrees C) with phosphate buffer, pH 5.5. The precipitate obtained between 0 and 70% relative ammonium sulfate saturation was re-dissolved in distilled water and heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant (F/0-70) was extensively dialyzed. A Sephadex G-50 size exclusion column was employed for further separation of proteins. The fraction with antifungal activity was pooled and submitted to CM-Sepharose cation exchange. Two proteins, named Pf1 and Pf2, were eluted in 0.1 and 0.2M of salt, respectively, and submitted to reverse-phase chromatography in HPLC. This fraction inhibited the growth, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae and strongly inhibited glucose-stimulated acidification of the medium by F. oxysporum in a dose-dependent manner. The molecular masses of these proteins, referred to now as Pf1-RP and Pf2-RP, were obtained by MALDI-TOF spectrometry and corresponded to 12,088 Da for Pf1-RP and 11,930 Da for Pf2-RP. These proteins were also subjected to automated N-terminal amino acid sequencing. Sequence comparisons for the heavy subunit of Pf2-RP showed the presence of a protein with a high degree of homology to storage 2S albumins.

Combinations of putative virulence markers in typical and variant enteroaggregative Escherichia coli strains from children with and without diarrhoea.

Enteroaggregative Escherichia coli (EAEC) is defined by the ability to produce aggregative adherence (AA) to cultured cells. We analysed 128 EAEC strains, isolated from children with and without diarrhoea, regarding the presence of 11 EAEC virulence genes. Seventy strains carried and 58 lacked the EAEC probe sequence; 17 probe positive and 31 probe negative strains showed variations in the AA pattern. All EAEC probe positive strains carried at least one EAEC marker; aspU (94.3%), irp2 (91.4%), and aggR (74.3%) were the most prevalent. Conversely, among the EAEC probe negative strains, 41.4% were devoid of any marker and astA predominated (44.8%). No significant statistical difference in the prevalence of any marker between cases and controls in both EAEC probe groups or AA variants was found. We suggest that the EAEC probe positive strains may have a higher pathogenic potential or alternatively, EAEC probe negative strains may harbour virulence factors as yet undescribed.

Biochemical and biological properties of phospholipases A(2) from Bothrops atrox snake venom.

Phospholipases A(2) (PLA(2)s), of molecular mass 13-15kDa, are commonly isolated from snake venom. Two myotoxins with PLA(2) activity, BaPLA(2)I and BaPLA(2)III, with estimated molecular masses of 15kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA(2)I was basic, with a pI of 9.1, while BaPLA(2)III was neutral with a pI of 6.9. On a molecular basis, BaPLA(2)III exhibited higher catalytic activity on synthetic substrates than BaPLA(2)I. Comparison of the N-terminal residues of BaPLA(2)I with other PLA(2) proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA(2) Lys(49) from B. atrox (89%). In contrast, BaPLA(2)III demonstrated 75, 72, and 71% homology with PLA(2) from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA(2)I and BaPLA(2)III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA(2)s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA(2)I- and BaPLA(2)III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA(2) edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA(2) enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA(2) enzyme.

Ultrastructural alterations and growth inhibition of Trypanosoma cruzi and Leishmania major induced by Bothrops jararaca venom.

Snake venom can affect the growth of Trypanosoma cruzi and Leishmania spp. As new classes of therapeutic drugs against protozoan parasites could be derived from snake venom, alterations in the ultrastructure and growth of the epimastigotes, trypomastigotes and amastigotes of T. cruzi, as well as the promastigotes of Leishmania major, were analyzed after treatment with crude venom from Bothrops jararaca. Parasite growth (epimastigotes and promastigotes) of venom treated cultures showed a negative correlation between cell growth and venom concentration. No growth occurred at a dose of 100 microg/ml of venom, while 50% growth inhibition was obtained in the range 0.1-0.3 microg/ml. Ultrastructural observations of treated bloodstream trypomastigotes, intracellular amastigotes, as well as axenic cultures of epimastigotes and promastigotes, demonstrated mitochondrial swelling and kinetoplast disorganization. Our data show that B. jararaca venom effectively inhibited the growth of T. cruziand L. major parasites. Growth inhibition was probably related to mitochondrial impairment.

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil.

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke fluid accumulation in rabbit ileal loops.

Production of Shiga toxin (Stx) in Escherichia coli strains belonging to serogroups O26, O111, and O157 was evaluated in the rabbit ileal loop assay and results were compared to those using tissue culture assays and DNA hybridization with specific probes for Stx1 and Stx2. All 14 Shiga toxin-producing E. coli strains tested provoked fluid accumulation in the rabbit intestinal loop. Eleven strains hybridized with Stx1 probe, one strain with Stx2 and two strains with both probes. Filtered culture supernatants of all E. coli strains presented cytotoxic effects in both HeLa and Vero cells. In this study, we found a strong association between the production of Stx and its effect in an animal model. This is the first description of high-level Stx-producing E. coli O111ac isolated in Brazil.