Students' attitudes towards somatic genome editing versus genome editing of the germline using an example of familial leukemia.

Although the discussion on possibilities and pitfalls of genome editing is ever present, limited qualitative data on the attitudes of students, who will come into contact with this technology within a social and professional context, is available. The attitude of 97 medical students and 103 students of other subjects from Hannover and Oldenburg, Germany, was analyzed in winter 2017/18. For this purpose, two dilemmas on somatic and germline genome editing concerning familial leukemia were developed. After reading the dilemmas, the students filled out a paper-and-pencil test with five open questions. The qualitative evaluation of the answers was carried by a deductive-inductive procedure of content analysis. There was a high approval for the use of somatic genome editing. When it came to germline genome editing, concerns were raised regarding enhancement, interventions in nature, and loss of uniqueness. The students recognized that somatic genome editing and germline genome editing prove different ethical challenges and need to be judged separately. Many students expressed not feeling fully informed. The results of this project show the importance of educating the public about the possibilities, limitations, and risks of somatic and germline genome editing. We recommend that this should already be addressed in schools in order to optimally prepare students and adults for participation in public discourse. Especially for patients affected by genetic diseases, it is of great importance that the treating physicians and geneticists are sufficiently informed about the method of genome editing to ensure good counseling.

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin.

The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin.

Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns.

Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coliIMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.