AU richness within the 5′ coding region of the Escherichia coli heat-stable enterotoxin b mRNA affects toxin secretion

Abstract

The majority of protein secretion in bacteria is mediated by the T2SS pathway. Substrates processed through this pathway are guided by the N-terminal signal sequence within the nascent polypeptide. Recent experimental evidence suggests that in similar secretory pathways, such as the T3SS, information in the 5′ coding region of the mRNA affects secretion and may also participate in mRNA localization. The majority of studies on the effects of AU richness on translation have focussed on the 5′ UTR in mRNAs. To look at the effects of AU richness within the coding region of mRNA on secretion, we have generated several silent mutations within the 5′ coding region of the E. coli heat-stable enterotoxin b (STb). This toxin is a well studied T2SS substrate. The mutations were generated such that AU richness within the 5′ coding region (corresponding to the N-terminal signal sequence) was gradually reduced. Reduction of AU richness within the first 15 codons resulted in reduced secretion of the toxin as the AU/GC ratio was reduced from 2.13 for the WT STb to 1.65 (S-I) and subsequently to 1.30 (S-II). This reduction did not correlate with mRNA accumulation and decreased stability of the transcripts could not account for the reduced secretion observed. Reduction of AU richness beyond the first 15 codons recovered secretion efficiency of the toxin (S-III). To validate the experimental approach, a positive control was used in which a mutation involving the insertion of a positive charge within the hydrophobic domain of the N-terminal signal sequence was constructed. As expected, this mutation abolished secretion of the toxin. In conclusion, reducing AU richness within the 5′coding region in the STb mRNA reduces toxin secretion but other factors, such as formation of hairpins, must also be taken into consideration. This will have implications for both homologous and heterologous expression of STb for biological studies and for toxin production.