Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli

Enteroaggregative Escherichia eoli (EAEC) is an emerging cause of pediatric and adult travellers diarrhea. the mechanism by which EAEC induce diarrhea is not completely known. Two serine protease autotransporter proteins, named Pet and Pic have been identified in EAEC strains. Pet has enterotoxic and cytotoxic activities, while the role of Pic in pathogenesis may lie on its mucinolytic activity. Little is known about Pet and Pic biological activities in vivo. in this study the antibody responses against these autotransporter proteins in convalescent children is investigated. Fifteen (83%) children showed specific antibodies against Pet or Pic in their sera. IgG and IgM antibodies were the main isotype found. Specific antibodies against Pic, but not against Pet, were detected in sera from age-matched control group. These data show that specific anti-Pet and anti-Pic antibodies are produced during the course of a natural EAEC infection in children. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.Univ São Paulo, Dept Microbiol, Inst Ciencias Biomed, São Paulo, BrazilInst Butantan, Lab Especial Microbiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, BR-05508900 São Paulo, BrazilCINVESTAV, IPN, Dept Cell Biol, Mexico City 14000, DF, MexicoUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

<p>Abstract</p> <p>Background</p> <p>Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic <it>Escherichia coli </it>(EPEC) and enterohemorrhagic <it>Escherichia coli </it>(EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int_388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).</p> <p>Findings</p> <p>Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into <it>pGEM-T Easy </it>vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. <it>E. coli </it>BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.</p> <p>Conclusion</p> <p>This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int_388-667) in purified form and the EPEC isolate.</p

Novel Hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) Emerging From Pet Birds

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

AAF/II fimbriae biogenesis in enteroaggregative Escherichia coli

Escherichia coli enteroagregativa (EAEC) e um patogeno emergente, associado a doenca diarreica, que apresenta como caracteristica o padrao de adesao agregativa em celulas HEp-2. Varios fatores de virulencia ja foram descritos em amostras de EAEC, utilizando como modelo a amostra 042, comprovadamente virulenta para humanos. Dentre esses fatores estao as enterotoxinas PET e EAST1, alem da fimbria denominada AAF/II. Todos esses fatores sao codificados pelo plasmidios de 65 MDa (pAA2). Embora seu papel na virulencia tenha sido demonstrado, os determinantes geneticos da biogenese de AAF/II nao eram conhecidos. Para tanto, no presente trabalho foram realizados experimentos para caracterizacao da regiao de pAA2 relacionada a biogenese de AAF/II. Atraves da analise das sequencias de nucleotideos de uma regiao de 25 kb do plasmidio pAA2, foi demonstrado que a mesma alberga os, genes das enterotoxinas (pet e astA), flanqueados por duas regioes relacionadas com a biogenese de AAF/II (regiao l e 2), alem do ativador de transcricao (aggm relacionado a sua expressao. A organizacao genetica da biogenese de AAF/II identificada na amostra 042 e unica, uma vez que na familia Dr de adesinas, da qual AAF/II e membro, esses genes estao organizados em um operon. Nessa organizacao exclusiva, a regiao l apresenta o cluster chaperonina-pilina-ativador de transcricao (aafD-aafA-aggR, enquanto a regiao 2, distante em 12 kb, apresenta o cluster chaperonina nao funcional-usher-invasina. As duas regioes sao flanqueadas por Sequencias de Insercao sugerindo que essa regiao seja um local em pAA2 de alta taxa de recombinacao. A analise de homologias a nivel de aminoacidos indicaram que a regiao l deriva do operon da fimbria AAF/I, enquanto a regiao 2 deriva do operon das adesinas AfaE de E. coli uropatogenicas. Os dados de ativacao da transcricao atraves da regulacao por aggR tambem corroboram com essa hipotese, pois apenas os genes da regiao l sao fortemente regulados pelo mesmo, como ocorre em AAF/I. Atraves da mutagenese nao-polar, foi demonstrado que os genes essenciais para a expressao de AAF/II sao: aafA, aafC, aafD e aggR, os quais correspondem a pilina, usher, chaperonina e ativador de transcricao, respectivamente. Esse locus descrito em pAA2 deve um representar um importante set de determinantes de virulencia em outras amostras de EAEC, uma vez que apresenta os potenciais fatores e ate entao descritos na amostra 042. Alem de AAF/II, a fimbria AAF/I esta...(au)BV UNIFESP: Teses e dissertaçõe

Aeromonas: prevalencia, avaliacao do fenomeno suicida e dos marcadores de virulencia em especies isoladas de criancas com gastroenterite aguda e controles

BV UNIFESP: Teses e dissertaçõe

A sensitive and specific molecular tool for detection of both typical and atypical enteroaggregative Escherichia coli

A multiplex PCR has been standardized and evaluated for the identification of both typical and atypical enteroaggregative Escherichia coli (EAEC). the assay detecting aaiA, aaiG, aggR and aatA presented 94.8% of sensitivity, 94.3% of specificity and was able to efficiently detect both groups of EAEC among E. coli isolated from stool cultures. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Inst Butantan, Bacteriol Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 2010/19515-2Web of Scienc