Translation control by altered start codon usage as a means of modulating the general stress response and virulence in Listeria monocytogenes.

Abstract In the food-borne pathogen Listeria monocytogenes, SigB is the central regulator of general stress response (GSR) and it mediates host entry by promoting acid resistance and epithelial cell attachment. The post-translational regulation of SigB activity is complex and is controlled by multiple genes. There is significant evidence that mutations can readily arise in these regulatory genes, or in sigB itself, leading to reduced SigB activity, which suggests that there is considerable genetic plasticity In the GSR. To further investigate this, we defined the complete genome sequence of a clinical isolate with attenuated SigB activity and investigated how it adapts to lethal acidic challenge (mimicking the selective pressure encountered during entry into the host). Acid resistance developed rapidly and 6 acid resistant derivatives (ARDs) were selected for further investigation. All 6 ARDs restored SigB activity due to mutations acquired in rsbW, which encodes an antagonist of SigB. These mutations resulted in non-canonical start codons (rsbWATG to rsbWATA or rsbWATT) or premature translation termination (rsbW-). A translational reporter assay demonstrated distinct differences in translation efficiency between three start codons: ATG>ATA>ATT, suggesting that a perturbation of RsbW:SigB stoichiometry alters SigB activity. We then analysed start codon usage for all conserved genes in 60,692 L. monocytogenes genomes available in the NCBI database. This analysis revealed flexible usage of start codons associated with genetic clades in 39 conserved genes, 13 of which are involved in virulence and stress response. Further, we show that flexible use of canonical start codons (ATG and GTG) also mediates different levels of expression of virulence and stress response genes. Taken together, we show the genetic plasticity of GSR regulation in a model pathogen, and highlight the importance of translational control as a means of fine-tuning gene expression during short-term adaptation and long-term evolution for optimal fitness. Author Summary The general stress response (GSR) in foodborne pathogen Listeria monocytogenes is important for environmental stress response and for host entry, but GSR is also highly variable across wild isolates. In this study, we analysed the evolutionary trajectory of a clinical isolate with attenuated GSR and characterized the adaption by this strain to a host-mimicking stress (acidic condition). Under extreme selective pressure, mutations disabling a negative regulator of the GSR were enriched. Interestingly, several independently occurring mutations negatively affect the translation initiation by using non-canonical start codons. Prompted by this, we analysed the population-wide start codon usage by examining all available L. monocytogenes genomes available (n = 60,690). This analysis revealed differential start codon usage in 39 conserved genes that are associated with different genetic clades. Furthermore, we demonstrated differential translation efficiencies between the different canonical start codons. This work highlights the genetic plasticity of GSR in the important food-borne pathogen L. monocytogenes and shows that altered translational start codon can be used as means of regulatory control. Together the data suggest that genetic changes in the regulation of the GSR might confer niche-specific fitness advantages.