Uncovering the Hidden Proteome of Streptococcus pneumoniae D39

Microproteins encoded in small open reading frames (sORFs), of less than 50 amino acids, are increasingly recognized as vital components of metabolic pathways, regulatory functions, and virulence. Despite their biological importance, their discovery in Gram-positive pathogens remains limited due to the difficulty of their annotation. Conventional computational algorithms lack the accuracy to predict true protein coding sORFs among many false predictions. Here we present a refined approach to identify actively translated sORFs utilizing two translation initiation inhibitors, retapamulin and lefamulin, in combination with ribosome profiling using the model organisms Streptococcus pneumoniae D39 and Streptococcus pyogenes M1T1 5448. We identified 114 novel sORFs in S. pneumoniae and 120 sORFs in S. pyogenes of which a subset was validated experimentally. In S. pneumoniae, two loci associated with virulence and quorum sensing were examined in deeper details. One such sORF, rio03, overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio03 from srf-02 revealed that rio03 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome.