Enhanced production of Shiga toxin 1 in enterohaemorrhagic Escherichia coli by oxygen

Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter (P Stx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the P Stx1 region, and an increase in Stx1 production.

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Bile salts and ferric iron-induced PMRAB dependent resistance to camps in EHEC O157:H7

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) colonization of the gastrointestinal tract is critically dependent on its ability to sense and respond to the external environment. This research aims to evaluate the contribution of bile salts- and ferric iron-induced resistance in EHEC to cationic antimicrobial peptide (CAMP) and the roles of pmrAB and am operon in these events. Results showed that EHEC, treatment with either bile salts or ferric iron induced a dose-dependent resistance to Polymyxin B. This resistance phenotype was lost in each of the pmrA and pmrB mutants. PMB resistance in EHEC was also dependent on the concentration of magnesium and on pH, suggesting the involvement of another two component system, PhoPQ. Mutagenesis of the iron-binding site of PmrB abrogated the induced resistance phenotype. The results of this study provide novel insights critical for our understanding of the molecular basis of pathogenesis and may provide new insights toward prevention strategies.

Bile salts and ferric iron-induced PMRAB dependent resistance to camps in EHEC O157:H7

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) colonization of the gastrointestinal tract is critically dependent on its ability to sense and respond to the external environment. This research aims to evaluate the contribution of bile salts- and ferric iron-induced resistance in EHEC to cationic antimicrobial peptide (CAMP) and the roles of pmrAB and am operon in these events. Results showed that EHEC, treatment with either bile salts or ferric iron induced a dose-dependent resistance to Polymyxin B. This resistance phenotype was lost in each of the pmrA and pmrB mutants. PMB resistance in EHEC was also dependent on the concentration of magnesium and on pH, suggesting the involvement of another two component system, PhoPQ. Mutagenesis of the iron-binding site of PmrB abrogated the induced resistance phenotype. The results of this study provide novel insights critical for our understanding of the molecular basis of pathogenesis and may provide new insights toward prevention strategies.

Characterization of the role of yadK in the pathogenesis of enterohaemorrhagic E. coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can use serious diarrhea and haemolytic uremic syndrome. Various factors including adhesins contribute to pathogenesis of EHEC. Previous studies suggested that yadK gene, which encodes a putative fimbrial adhesin in EHEC, may be involved in response of EHEC to acid stress. To characterize role of yadK protein in the pathogenesis of EHEC, recombinant yadK protein was generated and used to immunize rabbit to obtain anti-yadK antiserum, which was able to specifically recognize over-expressed yadK protein in EHEC. Western blotting with anti-yadK revealed a higher level of yadK expression in EHEC under acid adapted-acid stress compared to EHEC under unstressed conditions, which confirmed earlier yadK mRNA studies and indicated that yadK is upregulated in EHEC under acid stress. Finally, we observed that anti-yadK antiserum was able to specifically reduce adhesion of acid stressed EHEC to human epithelial cells compared to adhesion level of unstressed EHEC.

Characterization of the role of yadK in the pathogenesis of enterohaemorrhagic E. coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can use serious diarrhea and haemolytic uremic syndrome. Various factors including adhesins contribute to pathogenesis of EHEC. Previous studies suggested that yadK gene, which encodes a putative fimbrial adhesin in EHEC, may be involved in response of EHEC to acid stress. To characterize role of yadK protein in the pathogenesis of EHEC, recombinant yadK protein was generated and used to immunize rabbit to obtain anti-yadK antiserum, which was able to specifically recognize over-expressed yadK protein in EHEC. Western blotting with anti-yadK revealed a higher level of yadK expression in EHEC under acid adapted-acid stress compared to EHEC under unstressed conditions, which confirmed earlier yadK mRNA studies and indicated that yadK is upregulated in EHEC under acid stress. Finally, we observed that anti-yadK antiserum was able to specifically reduce adhesion of acid stressed EHEC to human epithelial cells compared to adhesion level of unstressed EHEC.

Escherichia coli O157:H7 F9 Fimbriae Recognize Plant Xyloglucan and Elicit a Response in Arabidopsis thaliana

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 °C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.