Browsing by Subject "Subtilase Cytotoxin"

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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.