Role of SdiA on Biofilm Formation by Atypical Enteropathogenic Escherichia coli.

Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E.coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

Late establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli depends on protein expression regulated by Per.

Enteropathogenic Escherichia coli (EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC, a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC (perABC) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains.

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

BACKGROUND: Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (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 (int388-667). 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). FINDINGS: 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 pGEM-T Easy 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. E. coli 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. CONCLUSION: This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.

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

Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichiacoli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea inchildren and adults in many developing and industrialized countries. Considering the fact that antibodies areimportant 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 (int388-667). 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).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 pGEM-T Easy 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. E. coli BL21(DE3)pLys strainwas 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 proteinyield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA andimmunofluorescence 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.

Escherichia coli O125ac:H6 encompasses atypical enteropathogenic E. coli strains that display the aggregative adherence pattern.

O125 is an enteropathogenic Escherichia coli (EPEC) serogroup, which includes the O125ac:H6 serotype, defined as atypical EPEC. Strains of this serotype displayed the aggregative adherence (AA) pattern with HEp-2, Caco-2, T84, and HT-29 cells, possessed all the LEE region genes, and expressed intimin, Tir, and EspABD, although the attaching-effacing lesion was not detected in vitro. These results confirm that E. coli O125ac:H6 is atypical EPEC that displays the AA pattern and indicate the necessity of testing for EPEC genes combined with the determination of the adherence pattern for atypical EPEC identification.