Introduction and Aim: The production of Shiga toxins is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC). Shiga toxins are AB5 protein toxins consisting of a single enzymatically active A-subunit and a pentamer of non-covalently linked B-subunits. The genes encoding the Stx2 subunits, stxA2 and stxB2, are located in the late-regulated phage region within the genome of lambdoid prophages, downstream of the antiterminator gene Q and upstream of the genes encoding the phage lysis cassette. The aim of this study was to quantitatively analyze the relation of the transcription of stxA2 and stxB2 in five EHEC wildtype strains. Due to the operon structure of the stx genes, we hypothesized either a 1:1 transcription ratio, or due to the AB5 structure of the toxin, a 1:5 ratio.
Material and Methods: To perform quantitative transcriptional analysis, total RNA was isolated and purified from E. coli O157:H7 strain HUSEC003, E. coli O157:H- strain HUSEC004, E. coli O103:H- strains HUSEC008, E. coli O26:H11 strain HUSEC018 and E. coli O104:H4 strain LB226692. Subsequently, qRT-PCR was performed to determine the transcript levels for stxA2, stxB2, and the housekeeping gene rrsB, which was used as an internal control.
Results and Summary: Surprisingly, stxA2 was expressed approximately ~ 1.3 - 2.5 times stronger than stxB2 in all investigated EHEC wildtype strains. The analyzed genes were not expressed in the expected ratios, indicating that free A-subunits might circulate in the bacterial environment which do not find a B-pentamer for holotoxin formation. Further analysis on translational and protein level should help to clarify this phenomenon.