Decoding bacterial virulence with high-definition ribosome profiling
Decades of research have provided significant insight into the physiology, metabolism and virulence of the model intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium).  The use of global RNA quantification studies has provided important information on the transcript behaviour of bacterial pathogens. However, despite transcription and translation being closely linked in prokaryotes, transcriptional levels are often not well correlated with protein abundances. Ribosome profiling (RiboSeq), which involves the deep-sequencing of ribosome protected fragments, is a powerful method to monitor in vivo global translation.  Here, we performed high definition ribosome profiling of S. Typhimurium. We introduced methodological modifications to further decrease rRNA contamination and, importantly obtained clear triplet phasing in read mapping positions, reflecting the triplet periodicity of decoding. We showed that a substantial proportion of S. Typhimurium protein synthesis is dedicated to virulence factor expression, and translational control plays a fundamental role in ensuring appropriate components are expressed at the correct stoichiometric ratios. We found that translational efficiency is mainly determined at the level of initiation by the strength of the Shine-Dalgano sequence's interaction with 16S rRNA and its distance to the initiation codon, the presence of an adenosine 3 nt upstream of the initiation codon, and a lack of RNA structure around the initiation site. Our study identified the underlying importance of translational regulation in bacterial gene expression and virulence.