Browsing by Subject "STEC"

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Characterization of function and regulation of the subtilase cytotoxin and Shiga toxin of pathogenic Escherichia coli
(2021) Heinisch, Laura; Schmidt, Herbert
Food-borne diseases caused by enterohemorrhagic Escherichia coli (EHEC) constitute a great threat to human health worldwide. Pathogenicity of EHEC strongly depends on the ability to produce virulence factors such as amongst others bacterial toxins. One of these toxins are the so-called Shiga toxins (Stx), which is why EHEC are assigned to the group of Shiga toxin-producing Escherichia coli (STEC). Stx belong to the family of AB5 protein toxins consisting of two subunits. One of them, the StxA-subunit causes depurination of the 28S rRNA in eukaryotic ribosomes by exhibiting N-glycosidase activity subsequently leading to inhibition of the protein biosynthesis followed by apoptosis of the host cell. The second one is the homopentameric B-subunit, which mediates binding to the host cell surface via the receptor glycolipid globotriaosylceramide (Gb3). Besides Stx, the subtilase cytotoxin (SubAB) has been described in STEC in recent years. SubAB, also assigned to the family of AB5 toxins, generates its cytotoxic activity via cleavage of the endoplasmic chaperone binding immunoglobulin protein (BiP) by its A-subunit. This cleavage leads to an unfolded protein response, resulting in apoptosis of the host cell. The B-subunit forms a ring-like homopentameric structure which is responsible for the binding to the receptor N-glycolylneuraminic acid (Neu5Gc) and other O-glycans. Although the mode of cytotoxicity of AB5 toxins have been studied extensively, some mechanisms remain unsolved. The scope of this thesis was to analyze further the mode of action of AB5 toxins and the gene regulation of stx and subAB. Both publications included in this thesis combine the characterization of the cytotoxic activity of AB5 toxins, the regulation of their genes, their subunits, and the combination of subunits of Stx and SubAB. In the first publication the regulation of gene expression of AB5 toxins was investigated in more detail. In this study, the gene expression of subAB1 was analyzed with a luciferase reporter gene assay and by quantitative real-time polymerase chain reaction. To unravel the regulatory mechanisms, both the laboratory E. coli strain DH5α and the STEC O113:H21 strain TS18/08 were used. Expression of subAB1 and promoter activity was studied using standard cultivation methods. Moreover, this work shed light on the impact of the global regulatory proteins host factor of bacteriophage Qβ (Hfq) and histone-like nucleoid structuring protein (H-NS) on subAB1 gene expression. Therefore, isogenic deletion mutants of hfq and hns gene were generated in the respective strains. Afterwards, plasmid-based complementation was conducted to verify that the observed effects were due to the deletion. Analysis of subAB1 promoter activity revealed impact of both Hfq and H-NS during different growth phases in both strains. In addition, the influence of both regulatory proteins on the expression toxin genes in STEC strain TS18/08 was investigated. This study did not only focus on the expression of stx2a and subAB1, but also the gene expression of the gene of the cytolethal distending toxin V (cdtV) was analyzed. Interestingly, all three toxin genes studied were upregulated in the deletion mutants of Δhfq and Δhns. Those results demonstrate the impact of global regulatory proteins on AB5 toxin gene expression and show that all three toxin genes investigated are integrated into the same regulatory network. In the second publication, the mode of action of AB5 toxins on the example of Stx2a was analyzed in more detail. The paradigm of AB5 toxin was known as the receptor binding B-subunit which mediates uptake of the enzymatic A-subunit and the subsequent cytotoxic activity. Previous studies have questioned this paradigm by showing cytotoxic effects of the SubA-subunit in absence of its corresponding B-subunit. This work analyzed whether this cytotoxic effect of the A-subunit is not only true for SubAB, but also for Stx. Thus, seperate recombinant expression of StxA2a subunits and subsequent His tag-based purification was performed. Both StxA2a-His and StxB2a-His were analyzed on cytotoxicity separately or in combination with the other subunit. Strikingly, cytotoxic effects of the StxA2a-His was observed in the absence of its corresponding B-subunit cell-type independently on HeLa, Vero B4, and HCT-116 cells. Studies on the B-subunit revealed no cytotoxicity on all cell lines. Additionally, combinations of different A- and B-subunits of Stx2a and SubAB1 proteins were analyzed. The hybrid combination showed that the cytotoxic effect of StxA2a-His on HeLa and HCT-116 cells could be reduced in the presence of the SubB1-His. Contrary, the cytotoxic effects of SubA1- His were unaltered in combination with StxB2a-His. Those results give the assumption that the Stx2aA-subunit binds to a target cell receptor blocked by SubB1-His. Additional experiments on the binding capacity of the Stx2a-subunits to Gb3 revealed that while StxB2a-His was able to bind to the receptor, no binding of the recombinant A-subunit was observed. The results indicate a cytotoxic effect of StxA2a on different cell types in absence of its corresponding B-subunit, which is designated as “single-A” effect in this work. The role of this effect in STEC pathogenicity, the uptake mechanism and subsequent transport inside the host cells of StxA-subunit need to be further analyzed in the future.
Molekulargenetische Untersuchungen zur Expression des Typ III Effektors NleA 4795 von Shiga Toxin-produzierenden Escherichia coli
(2010) Schwidder, Maike; Schmidt, Herbert
Shiga toxin-producing E. coli (STEC) are the causative agents of foodborne infections in many countries and can lead to severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The bacteria colonize the human intestine where they normally cause the formation of characteristic ?attaching and effacing?-lesions. Essential for this effect is a pathogenicity island, termed as ?locus of enterocyte effacement? (LEE), that encodes the components of a type III secretion system and several effector proteins, which are translocated directly into the host cells by the TTSS machinery. In addition to the LEE-encoded effectors a large number of effector proteins have been identified which are encoded outside of the pathogenicity island. Among these is the ?non-LEE encoded effector A? (NleA), which is encoded on cryptic or inducible prophages and is widely distributed among pathogenic E. coli strains. In the present study, the expression and regulation of the nleA-variant nleA4795 of E. coli O84:H4 strain 4795/97 was investigated, which is located on the Shiga toxin-converting bacteriophage BP-4795. Therefore, different environmental conditions as well as certain regulatorproteins were tested on their influence on nleA4795-expression using a luciferase-reportersystem and the quantitative real-time PCR. Among the analyzed environmental factors, certain concentrations of NaCl and KCl were identified to activate nleA4795-expression, indicating an osmotic-based influence. The suggested induction of nleA4795 in preconditioned medium due to quorum sensing could not be confirmed, since none of the so far known autoinducers showed a positive influence on the expression. The increased expression of nleA4795 could be associated with a reduced amount of nutrients in subsequent investigations and therefore demonstrated a relation between nleA4795-expression and bacterial stress-response-systems. Furthermore, a possible correlation of nleA4795-expression with the induction of phage BP 4795 and Shiga toxin-expression was analyzed. Different from the expression of Shiga toxin, induction-experiments with norfloxacin showed no activation, but a strong repression of nleA4795-expression. Analysis of the regulatory level demonstrated that the expression of nleA4795 depends on the three LEE-encoded regulators Ler, GrlA und GrlR as well as on the Pch-regulators, which are encoded outside of the LEE. The non-LEE encoded regulator EtrA showed no influence on the expression of nleA4795. In addition, the regulator proteins Ler, GrlA and PchA were tested for direct binding to the nleA4795-promoterregion. Regulators GrlA and PchA showed no specific binding and were therefore classified as indirect regulators of nleA4795-expression. In contrast, regulator Ler showed a specific binding to different areas of the nleA4795-promoter region and thereby confirmed the integration of nleA4795 in the Ler-mediated circuit of LEE-regulation.
The development of phenotypic protocols and adjustment of experimental designs in Pelargonium zonale breeding
(2018) Molenaar, Heike; Piepho, Hans-Peter
Ornamental plant variety improvement is limited by current phenotyping approaches and the lack of use of experimental designs. Robust phenotypic data obtained from experiments laid out to best control local variation by blocking allow adequate statistical analysis and are crucial for any breeding purpose, including MAS. Often experiments consist of multiple phases like in P. zonale breeding, where in the first phase stock plants are cultivated to obtain the stem cutting count and in the second phase the stem cuttings are further assess for root formation. The first analyses of rooting experiments raised questions regarding options for improving the two-phase experimental layout, for example whether there is a disadvantage to using exactly the same design in both phases. The other question was, whether a design can be optimized across both phases, such that the MVD can be decreased. Instead of generating a separate layout for each phase. Moreover, optimal selection methods that maximize selection gain in P. zonale breeding based on available data collected from unreplicated trials and containing pedigree information were sought. This thesis was conducted to evaluate the benefits of using two-phase experimental designs and corresponding analysis in P. zonale for production related traits, for which it was necessary to establish phenotyping protocols. To optimize the rooting experiments with their two-phase nature, alternative approaches were explored involving two-phase design generation either in phase wise order or across phases. Furthermore, selection methods considering pedigreeinformation (family-index selection) or not (individual selection), were evaluated to enhance selection efficiency in P. zonale breeding. The benefits of using experimental designs in P. zonale breeding was shown by the simulated response to selection. Alternative designs were evaluated by the MVD obtained by the intrablock analysis and the joint inter-block-intra-block analysis. The efficiency of individual and family-index selection was evaluated in terms of heritability obtained from linear mixed models implementing the selection methods. Simulated response to selection varied greatly, depending on the genotypic variances of the breeding population and traits. However, by using efficient designs allowing adequate analysis, a varietal improvement of over 20% of stock plant reduction is possible for stem cutting count, root formation, branch count and flower count. The smallest MVD for alternative designs was most frequently obtained for designs generated across phases rather than for each phase separately, in particular when both phases of the design were separated with a single pseudolevel. Family-index selection was superior to individual selection in P. zonale indicating that the pedigree-based BLUP procedure can further enhance selection efficiency in productionrelated traits in P. zonale. The quantification of genotypic variation by phenotypic protocols and the optimized two-phase designs for estimating genotypic values were necessary and successful steps in laying the foundation for effective MAS. Phenotypic protocols effectively characterized the genetic material on an observational unit level, while the two-phase experimental designs enabled effective characterization on a genotype level by adjusting entry means using linear mixed models. The resulting adjusted entry means are the basis for future genotype phenotype association for MAS.
Untersuchungen zur spezifischen Genexpression von enterohämorrhagischen Escherichia coli (EHEC) in der Lebensmittelmatrix
(2012) Kroj, Andrea; Schmidt, Herbert
Ground beef as a high risk food is known to be an cause of human infection with Shiga toxin-producing E. coli (STEC). The pathogens infect humans by the ingestion of undercooked ground meat and cause severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. E. coli O157:H7 strain EDL933 as a representative of enterohemorrhagic E. coli (EHEC), a subgroup of STEC, was analysed for in vivo induced genes in ground beef with the help of the in vivo expression technology. It could be demonstrated that the promoter selection vector pKK232-8, which contains a promoterless chloramphenicol resistance gene, is not a suitable vector for a study of gene expression in this matrix. The detection of in vivo expressed genes using the alcohol-soluble and bacteriostatic antibiotic was not possible. Therefore, the promoter selection vector pAK-1 was developed. The new vector system was based on a water-soluble and bactericidal kanamycin resistance gene for selection. In the present study, the vector was established and used for analysis of the gene expression in ground meat. 20 in vivo induced genes that were expressed during growth in ground meat under elevated temperature conditions at 42°C could be detected. Eight genes were associated with energy and nucleotide metabolism, macromolecule synthesis, transport and stress response of the cell. The major part of 12 genes was attributed to a putative or unknown function. Predominantly, identified genes could not be associated with virulence or stress response of the cell. The results of this study, using the in vivo expression technology, showed that genes which are expressed under specific conditions in ground meat could be detected with the help of the chosen method. A first insight into the gene expression of strain EDL933 in ground beef could be acquired. During further investigations a comparison of the fitness of 23 E. coli strains belonging to serogroups O26, O103 and O157 was realized. The isolates originating from foods, patients with HUS and animals were compared in ground beef. The determined differences showed strain-specificity and temperature-specificty. The fitness of the strains varied dependent on the chosen temperatures at 15, 20 and 37 degrees. The analysis of the strains based on ten virulence factors showed that the observed differences could not be attributed to the presence or the number of virulence genes. A correlation between the fitness and the production of a bacteriocin could not be found.