# Antibiotic tolerance: membraneless organelles and autolysin regulation

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2022 · $455,000

## Abstract

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.

## Key facts

- **NIH application ID:** 10333641
- **Project number:** 1R01AI165426-01
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** Elaine I Tuomanen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $455,000
- **Award type:** 1
- **Project period:** 2022-05-05 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10333641

## Citation

> US National Institutes of Health, RePORTER application 10333641, Antibiotic tolerance: membraneless organelles and autolysin regulation (1R01AI165426-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10333641. Licensed CC0.

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