A global data-driven census of <i>Salmonella</i> small proteins and their potential functions in bacterial virulence

Abstract

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than <i>Escherichia coli</i>. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in <i>Salmonella</i> Typhimurium. We merged the annotated small proteome of <i>Salmonella</i> with new small proteins predicted with <i>in silico</i> and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a <i>Salmonella</i> Δ<i>mgrB</i> mutant and reveal an effect of MgrB in regulating the <i>Salmonella</i> transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available 'omics' datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.