Muruzabal-Galarza_2025_mBio__e0185625

Small proteins encoded by small open reading frames are often overlooked by annotation algorithms and consequently missing from genome databases. Here, by combining multiomic approaches, we found 27 novel small proteins in Staphylococcus aureus, one of the most relevant pathogens worldwide. To better understand the importance of small proteins associated with S. aureus virulence factors, we focused on two small proteins encoded in the former 5'- and 3'-untranslated regions of the sal1 mRNA, respectively. SAL1 is one of the two extracellular lipases involved in S. aureus pathogenesis. Both small proteins were predicted as amphipathic helices similar to phenol-soluble modulins (PSMs) and were highly conserved in members of the genus Staphylococcus. Our results showed that the sal1 upstream small protein, LspU, was exported through the same transporters as PSMs, positioning LspU as a putative novel member of the PSM family. Interestingly, constitutive LspU expression induced biofilm structures and produced extracellular amyloidogenic protein aggregates composed of LspU, lipases, and PSMs. Although mutating the PSM-encoding genes reduced said aggregates, it had no impact on lipase trapping at cell surfaces. Altogether, these findings suggest that S. aureus may be using LspU to avoid dispersion of lipases during infections and help localize efforts at the infection site.IMPORTANCEBacterial small proteins shorter than 50 amino acids are becoming increasingly recognized as key players for many cellular processes. However, since computational algorithms commonly disregard proteins of this size, most small proteins are not found in genome annotations. In this study, we used genome-scale multiomic analyses to unveil more than 30 novel small proteins, updating the catalog of these proteins in Staphylococcus aureus, one of the most relevant bacterial pathogens in hospitals. Among them, we functionally characterized the small protein LspU as a novel member of the phenol-soluble modulin (PSM) family. PSMs are involved in cell toxicity, biofilm structure, and virulence. We showed that LspU induces the formation of extracellular protein aggregates that trap lipases at cell surfaces, which may be a useful strategy to avoid dispersion of extracellular lipases and provide lipase activity at the infection site.