Antibiotic resistance and tolerance are a major increasing threat to treating infectious diseases. Streptococcus pneumoniae, the leading cause of pneumonia, sepsis, and meningitis worldwide, is a model organism for understanding antibiotic-induced autolysis and failure of therapy due to antibiotic tolerance: a phenotype when bacteria stop growing but are not killed. The main pneumococcal autolysin, LytA, drives autolysis but its mode of downregulation during tolerance is unknown. We have discovered a new LytA activity: capsule shedding and through its analysis have, for the first time, discovered candidates for regulators of LytA that also could explain the modulation of penicillin responses that lead to tolerance and treatment failure. Using the pneumococcus as a model, our lab has revealed that, for capsule shedding, LytA is activated to cleave cell wall without lysis in response to antimicrobial peptides, typified but not limited to LL- 37. Rather, LytA removes surface attached capsule in a protective response to avoid LL-37. We have identified 3 loci that regulate these activities of LytA. Mutation of these LytA modulating (Lym) loci recapitulates the penicillin tolerance phenotype of clinical isolates: production of bioactive LytA, but a failure to trigger autolysis after penicillin treatment. Our discovery provides tools to make important inroads into defining the mechanisms governing antibiotic lytic responses (the first mechanistic discovery of how LytA is controlled). From analysis of genomes of streptococcal pathogens in general, it is apparent that lym loci are widespread and have alleles that cluster with distinct penicillin tolerant phenotypes. New insights into autolysin and penicillin responses are needed to advance both the fields of bacterial physiology and infectious diseases. We propose in Aim 1 to take a combined biochemical, genetic, and microscopic approach to analyze the roles of the Lym proteins and lym loci on LytA regulation. In aim 2, we will exploit our new discovery that 3 Lym proteins are the first bacterial proteins shown to form biomolecular condensates and initiate phase separation. This property is widely used in eukaryotes to regulate complex spatiotemporal multi-protein processes and is thus especially well-suited as a highly novel mechanism to underpin LytA regulation by 3 Lyms. In Aim 3, we will examine lym alleles in tolerant clinical isolates of pneumococcus. These isolates are derived from patients dying of meningitis due to treatment failure that is recapitulated in the animal model of meningitis. We will define Lyms as a cause of failure of potent bactericidal action of antibiotics due to tolerance.

Project Narrative/Relevance: Streptococcus pneumoniae, the leading cause of pneumonia, sepsis, and meningitis worldwide, is a model for the study of antibiotic-induced autolysis and the emerging threat of multidrug tolerance. The main pneumococcal autolysin LytA drives both autolysis and the newly discovered capsule shedding response. New data suggests regulators of LytA, discovered through analysis of capsule shedding, affect both the capsule shedding response and activation of autolysis by antibiotics. These regulators may form membraneless organelles as a novel mechanism of thwarting the bacterial lytic response to antibiotics leading to treatment failure.