The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917

Abstract

BackgroundEscherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells.ResultsHere we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections.ConclusionsSat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0591-5) contains supplementary material, which is available to authorized users.

Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells

“…We previously showed that EcN CF-SN have a positive impact on the TER of epithelial cell monolayers and prevent barrier disruption caused by pathogens (Toloza et al, 2015). To test the ability of tcpC -positive strains ECOR63 and ECOR57 to strength the epithelial barrier, T-84 monolayers were incubated for 24 h with CF-SN (2 mg/ml) collected from LB cultures, and TER was measured before and after the treatment period.…”

“…The tcpC knockout mutant derived from EcN was generated by antibiotic marker exchange using the suicide plasmid pUT-miniTn5 Tc (Biomedal) as described previously (Toloza et al, 2015). This vector contains the R6K origin of replication, which functions only in bacteria that contain the gene pir .…”

“…The resulting recombinant plasmid was propagated into strain EB6193 and introduced into E. coli S17 (λpir) by electroporation, to be used as a donor strain in mating experiments aimed at introducing the tcpC mutation into the EcN chromosome. A Rf-resistant derivative of EcN (Toloza et al, 2015) was used as the recipient. Transconjugants were selected for their resistance to Km and Rf on LB plates and further purified in this medium.…”

“…Cell free supernatants (CF-SN) were obtained from bacterial cultures in LB medium or DMEM supplemented with 1% non-essential aminoacids and 25 mM HEPES as described previously (Toloza et al, 2015). Briefly, bacterial cells were pelleted by centrifugation at 10,000 × g for 20 min at 4°C; the supernatants were filtered through a 0.22 μm-pore-size filter (Merck Millipore) to remove residual bacteria and concentrated using a Centricon ® Plus-70 filter device with a molecular weight cutoff of 10 KDa (Merck Millipore).…”

“…Several facts indicate that the TJ strengthening capacity of EcN is mediated by factors released to the extracellular medium: (i) the TcpC activity on epithelial barrier function is associated with EcN culture supernatants (Hering et al, 2014), and (ii) EcN supernatants prevent barrier disruption by EPEC in polarized Caco-2 cell monolayers and confer protection against deleterious effects on paracellular permeability caused by virulence factors such as the serin protease Sat ( s ecreted a utotransporter t oxin) (Toloza et al, 2015). The aim of this study was to define whether the TJ-barrier strengthening capacity of EcN is mediated by secreted factors or by OMVs using as a model T-84 and Caco-2 polarized monolayers.…”