Post-transcriptional regulation of a micro-injection system
Wawszczyk, Agata Barbara
Enterohaemorrhagic Escherichia coli (EHEC) is a food-borne pathogen associated with outbreaks of bloody diarrhoea and haemolytic uraemic syndrome in humans. It originates from asymptomatic carriage in cattle and other ruminants. A critical step in EHEC infection of the human or ruminant host is attachment and effacement of the intestinal epithelium, which is dependent on production of a Type 3 Secretion System (T3SS). This is a molecular syringe encoded on the Locus of Enterocyte Effacement (LEE) composed of five operons LEE1,2,3,5 and 4. The expression of the T3SS is thought to be staged with a checkpoint after production of the membrane-embedded basal apparatus and before production of the LEE4-encoded translocon filament (EspABD) that contacts the host cell. The first protein encoded on the LEE4 operon is SepL and its expression is coupled to translocon proteins EspABD. SepL has been established to be essential for production of surface translocon filaments, although its mechanism of action is still unknown. A recent study by Wang et al. provided evidence that the conformation of the sepL mRNA determined its translation and the RNA-binding protein Hfq was shown to negatively regulate SepL expression. There is evidence for a LEE1-encoded factor required to enable LEE4 translation. Together, the sepL mRNA secondary structure, Hfq and LEE1-encoded components are hypothesised to form a regulatory interplay that determines SepL expression. The research described in this thesis aimed to further characterise the regulatory factors and unravel the mechanism behind the proposed post-transcriptional control of sepL mRNA. Investigation into the LEE4 mRNA expression revealed a disconnect between the transcriptional activation and LEE4 transcript abundance. This was concluded to be due to rapid degradation of the LEE4 mRNA. The secondary structure of sepL mRNA was mapped using a chemical probing technique called selective 2′-hydroxyl acylation analysed by primer extension (SHAPE). It revealed that the sepL transcript adopts a ‘closed’ conformation that likely obstructs the Shine-Dalgarno region from the ribosomal access. Previous research had shown that reporter fusions to truncated sepL mRNAs had high expression levels compared to fusions to the full length sepL mRNA. Those truncated sepL mRNA constructs were computationally predicted to form an ‘open’ secondary structure enabling ribosome binding. The ‘closed’ sepL mRNA conformation was linked to a higher rate of transcript decay in comparison to the sepL mRNA constructs predicted to form an ‘open’ secondary structure. I propose a model in which the ‘unfolding’ of the sepL mRNA leads to translation and protection from degradation. This was indicated to be EHEC background specific. While Hfq had a clear negative impact on sepL mRNA stability, deletion and complementation analyses demonstrated that the LEE1-encoded T3SS chaperone CesAB opposed the Hfqmediated degradation of sepL mRNA and is a post-transcriptional regulator of SepL and other LEE4-encoded proteins. CesAB was shown to directly interact with the sepL transcript and the mRNA signatures involved in the interaction were investigated. The positive impact of CesAB on sepL mRNA maintenance was diverted by expression of CesAB’s protein cargo, the LEE4-encoded T3SS filament protein EspA. This indicates a hierarchical feedback loop controlling expression of the LEE4-encoded proteins. To our knowledge this is the first reported instance of a T3SS protein chaperone also regulating the expression of its subsequent protein cargo at the mRNA level. Therefore, it acts first to stabilise the LEE4 transcript followed by interaction with two of LEE4 mRNA-encoded proteins. The switch of CesAB from protein-RNA interaction to protein-protein interactions would naturally lead to degradation of the translocon mRNA. The duality of substrates may be a paradigm common to other substrates and this needs evaluation. The conducted study aimed to widen the knowledge of the mechanisms that have evolved to stage the construction of complex bacterial organelles. In turn this can lead to a better understanding of the processes underlying potentially lethal infections and indicate new anti-virulence drug targets.