Discovery and characterization of bacterial cell envelope assembly and remodeling networks that modulate tolerance to antibiotics

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Abstract The bacterial cell envelope is a complex and dynamic multilayered structure essential for cell growth and division. The structural layer of the envelope, known as the cell wall or peptidoglycan (PG), determines cell shape and is essential for survival because it protects bacteria from osmotic lysis. The action of PG synthases, which add new material for the enlargement of the cell wall, and PG hydrolases, which create space for expansion of the PG mesh-like structure, are both necessary for growth. Some of our most powerful and successful antibiotics target PG synthases and derive their efficacy from not only inhibiting cell wall assembly, but also by causing cell lysis through the active destruction of the cell wall by PG hydrolases. Because of their potential to cause cell lysis, it has long been appreciated that bacteria must possess robust mechanisms to control when and where PG hydrolases are activated. However, the molecular details underlying these regulatory processes are lacking. Research in my laboratory focuses on uncovering and characterizing regulatory systems controlling PG hydrolase activity during normal growth, and how antibiotics short-circuit this regulation to trigger cell lysis. Using the human respiratory pathogen Streptococcus pneumoniae as a model organism, we found that PG hydrolases are controlled by two cell envelope polymers known as teichoic acids (TAs): membrane-linked lipoteichoic acids (LTAs) and cell wall-anchored teichoic acids (WTAs). Characterization of novel enzymes involved in TA synthesis and remodeling revealed that cell-wall targeting antibiotics hyperactivate PG hydrolases by disrupting the normal mechanisms that balance the levels of WTAs and LTAs in the cell envelope. Current studies in my laboratory indicate that the levels of TAs are controlled by a complex regulatory network involving post- translational modifications and targeted proteolysis. We also discovered that the modulation of the TA levels in the cell envelope has significant impacts on cell morphology, growth, and tolerance to antibiotics. However, many aspects of this regulation and synthesis and remodeling pathways remain unknown. Therefore, the goals of this proposal are to (i) identify signals and pathways that modulate LTA biogenesis and characterize how antibiotics subvert them; (ii) determine the physiological roles and regulation of a widely-conserved protease and characterize how antibiotics hyperactivate its activity to disrupt LTA biogenesis; (iii) characterize the regulation of a novel WTA remodeling enzyme, and uncover how S. pneumoniae uses WTA levels to control lysis and promote growth. The results generated by this research will provide fundamental insights into broadly relevant principles for envelope assembly and maintenance in S. pneumoniae and related bacteria. S. pneumoniae has become an alarming multidrug-resistant health threat. Therefore, novel antibiotics that target S. pneumoniae are critically needed. The studies proposed here will reveal general mechanisms by which bacteria remodel their envelopes to survive antibiotic exposure and uncover new targets for therapeutic intervention.