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

Abstract

BackgroundDiarrhea 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.Methods and FindingsRecombinant 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.ConclusionThe developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

Presence and characterization of Escherichia coli virulence genes isolated from diseased pigs in the central region of Argentina

“…Porcine diarrhea is a disease that causes death and economic loss in swine production [ 1 - 5 ]. Enterotoxigenic, enteropathogenic, and shiga toxigenic (ETEC, EPEC, and STEC, respectively) pathotypes of Escherichia coli are the major pathogens responsible for neonatal diarrhea, post weaning diarrhea and edema disease (ED) in pigs [ 1 ], affecting almost all stages of production [ 2 - 4 ].…”

“…ETEC enterocyte adhesion is mediated by fimbriae (F) such as F4 (K88), F5 (K99), F6 (P987), F18, and F41 and is capable of producing enterotoxins which act on enterocytes. According to their thermal stability, enterotoxins are classified as heat-stable ( STa , STb , and EAST1 ) and heat-labile ( LTI and LTII ) [ 5 ]. STEC strains produce shiga toxin ( STx1 and STx2 ) and an adhesion protein called intimin (encoded by the eae gene) which is responsible for microvilli attaching and effacing on enterocytes surface [ 6 ], this protein is also important in EPEC pathotype.…”

Development and Validation of Shiga Toxin-Producing Escherichia coli Immunodiagnostic Assay

“…We used in this study a collection of 96 Shiga toxin-producing E. coli (STEC) strains belonging to different serotypes and stx subtypes (Table 1). We also included for the ELISA (EIA) cut-off definition and specificity of the latex agglutination (LA) and lateral flow assay (LFA), 12 typical enteropathogenic E. coli (tEPEC) [36,37], 11 atypical enteropathogenic E. coli (aEPEC) [38], 45 enterotoxigenic E. coli (ETEC) [39,40], nine enteroaggregative E. coli (EAEC) [41], eight enteroinvasive E. coli (EIEC) [42], 14 diffusely-adherent E. coli (DAEC) [42], three fecal E. coli negative for DEC virulence factors (NVF E. coli ), four microbiota E. coli isolates and 19 Enterobacteriaceae isolates ( Citrobacter freundii , Edwardsiella tarda , Enterobacter cloacae , Klebsiella pneumoniae , K. oxitoca , Morganella morganii , Proteus mirabilis , Providencia spp ., Salmonella Agona, S. Enteritidis, S. Infantis, S. Newport, S. Typhimurium, Serratia marcescens , Shigella boydii , S. flexneri , and S. sonnei ) from our laboratory collection. The prototype EHEC EDL933 [43] was included in the assay as a positive control for the Stx 1 /Stx 2 producing strain.…”

TCR-pMHC bond length controls TCR ligand discrimination

“…Bacteria were spun down, and cell pellets were resuspended with B-PER (Bacterial Protein Extraction Reagent, Thermo Fisher), lysozyme, and DNase, followed by washing with inclusion wash buffer (100 mM NaCl, 50 mM Tris Base, 0.05% (volume) Triton X-100) 32 . Refolding and purification of recombinant scFvs were performed using a modified method based on previous publications 33,34 . In brief, scFvs were unfolded in the presence of 10 mM β-mercaptoethanol for 2 hours at 25 °C (in 100 mL of 100 mM Tris-HCl buffer with pH = 8.0, 6 M GuHCl and 200 mM NaCl), so that it could be refolded in correct conformations by step-wise dialysis methods without causing protein oxidation.…”