Role of Prp protease in the S. pneumoniae ribosome

PROJECT SUMMARY The Streptococcus genus contains many human pathogens of medical significance. S. pneumoniae is one of the most important human respiratory pathogens worldwide, and the increased incidence of multidrug resistance in streptococci is a significant public health concern. Unfortunately, antibiotic discovery and development has slowed down in recent years, and there is an urgent need for novel antibiotics against streptococci and other pathogens. The eubacterial ribosome is an essential organelle, and a target for numerous antibiotics. The ribosome consists of three RNAs and 56 proteins. One of these proteins, L27, product of the rpmA gene, is a component of the ribosomal large subunit that does not have an equivalent in eukaryotes and archaea. In Firmicutes and related bacteria, L27 has an additional 9–12 amino acid N-terminal extension compared to their counterparts in Gram-negative bacteria like Escherichia coli. We previously described a novel cysteine protease, named Prp, that cleaves the N-terminal extension of L27, and showed that this cleavage step was essential for cell viability in Staphylococcus aureus. Here, we show that, in S. pneumoniae, L27 cleavage by Prp plays a role in ribosome activation. The overall objective of this exploratory/developmental project is to understand the roles of Prp and L27 cleavage in S. pneumoniae. Why do S. pneumoniae, S. aureus and related bacteria employ this additional Prp-mediated L27 cleavage step? Our hypothesis is that cleavage of L27 by Prp serves as a checkpoint required for full activation of the ribosome and may be associated with dormancy, persistence and survival under certain conditions. In this project, we will explore the role of Prp and L27 cleavage using a combination of genetics, functional assays, quantitative mass spectrometry and cryo- electron microscopy. Our specific aims are: Aim 1: Define the role of Prp and L27 cleavage in ribosome function; Aim 2: Examine the structural consequences of L27 cleavage. Our project addresses a gap in knowledge about streptococcal ribosomal structure, assembly and regulation and targets a hitherto undescribed step in ribosomal assembly—processing of ribosomal protein L27 by the Prp protease—that appears to be unique to Firmicutes and related bacteria. The role of L27 in translation is controversial and the role of Prp in ribosomal assembly and function is still unknown. A better understanding of these processes may lead to more effective therapeutics against S. pneumoniae and other Gram-positive pathogens that target these mechanisms.