Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. for this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. the presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Butantan Inst, Bacteriol Lab, BR-05503900 São Paulo, BrazilSão Paulo Trop Med Inst, Seroepidemiol & Immunol Lab, BR-05403000 São Paulo, BrazilFleury Med & Hlth, BR-04344903 São Paulo, BrazilButantan Inst, Immunopathol Lab, BR-05503900 São Paulo, BrazilButantan Inst, Immunochem Lab, BR-05503900 São Paulo, BrazilAdolfo Lutz Inst, Bacteriol Sect, BR-01246000 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol, BR-04923062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol, BR-04923062 São Paulo, SP, BrazilWeb of Scienc

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities

toxin

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic <i>Escherichia coli</i> Infection in Developing World: Defining the Biomarker, Antibody and Method

<div><p>Background</p><p>Enteropathogenic and enterohemorrhagic <i>Escherichia coli</i> (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.</p><p>Methodology</p><p>First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.</p><p>Principal findings</p><p>EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.</p><p>Conclusion</p><p>RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.</p></div

EspA and EspB production in different culture media.

<p>Atypical EPEC (aEPEC), typical EPEC (tEPEC) and EHEC isolates were cultivated in DMEM or DMEM-T or DMEM-PC. The supernatants were tested by indirect ELISA for EspA detection using anti-EspA IgG-enriched fraction (30 µg/mL) (<b>A</b>) and anti-EspA MAb (5 µg/mL) (<b>B</b>) and for EspB detection using anti-EspB IgG-enriched fraction (30 µg/mL) (<b>C</b>) and anti-EspB MAb (10 µg/mL) (<b>D</b>). The optical densities obtained for the isolates reacted with anti-EspA or anti-EspB polyclonal or monoclonal antibodies were analyzed by GraphPrism 5.01, using Student’s <i>t</i> test and two-away ANOVA. The differences were considered statistically significant when p≤0.05.</p

Rapid agglutination latex test reactivity (%) with bacterial isolates.

<p>tEPEC (typical enteropathogenic <i>E. coli</i>); aEPEC (atypical enteropathogenic <i>E. coli</i>); EHEC (enterohemorrhagic <i>E. coli</i>); DEC/LEE⁻ (LEE-negative diarrheagenic <i>E. coli</i>); NVF <i>E. coli</i> (fecal <i>E. coli</i> negative for DEC virulence factors).</p>a<p><i>Proteus mirabilis</i>.</p><p>Rapid agglutination latex test reactivity (%) with bacterial isolates.</p

EspB production in different culture media.

<p>LEE-positive and LEE-negative isolates were cultivated in DMEM or DMEM-T or DMEM-PC. The supernatants were tested by indirect ELISA for EspB detection using anti-EspB IgG-enriched fraction (30 µg/mL) (<b>A</b>) and anti-EspB MAb (10 µg/mL) (<b>B</b>). The mean optical densities for LEE-negative and LEE-positive isolates were determined. The cut-off obtained by the ROC curve for anti-EspB MAb was 0.027 for DMEM and 0.0145 for DMEM-T and DMEM-PC. For anti-EspB PAb was 0.152 for DMEM, 0.135 for DMEM-T and 0.001 for DMEM-PC.</p

Typical of agglutination latex assay: negative and a semi-quantitative positive (from + to ++++) agglutination pattern with anti-EspB MAb coated beads.

<p>The test control with lysis buffer (B-PER) showed the same pattern as LEE-negative isolates.</p