Understanding autolysin function in Gram-negative bacterial growth and morphogenesis

SUMMARY Bacterial growth and morphogenesis must be carefully orchestrated in the face of changing environmental conditions to ensure bacterial survival. The primary structure required for cellular integrity maintenance is the bacterial cell wall, mostly composed of the mesh-like peptidoglycan (PG), which surrounds the cell as a covalently-closed macromolecule. PG is synthesized by the concerted action of several PG synthases, namely the Penicillin-binding proteins (PBPs) and associated proteins, which collectively catalyze the formation of the characteristic PG macrostructure, i.e. a polysaccharide backbone crosslinked by short peptide bridges. However, in addition to synthesizing PG, bacteria must also cleave the PG meshwork, presumably to make space for the insertion of new material to promote growth. This activity is catalyzed by so-called “autolysins”, a group of poorly- understood enzymes, including the endopeptidases (EPs) and lytic transglycosylases (LTGs). EPs and LTGs cleave the PG crosslinks and backbone, respectively, and contribute to growth and morphogenesis in a poorly- understood way. This cleavage activity is exploited by some of our most powerful antibiotics, the b-lactams, because a major consequence of their inhibitory effect on PBPs is autolysin-mediated cell wall breakdown, and consequently growth arrest or lytic death. However, autolysin activity is also essential for growth, positioning these enzymes as unique novel targets for the future development antibiotics, since both inhibition and activation of EP/LTG would stymie bacterial growth. We currently have an incomplete understanding of how EPs and LTGs are regulated, depriving us of novel pathways for the development of antibiotics and their adjuvants. Experiments proposed here will fill this gap and we fundamentally aim to understand how autolysins promote bacterial growth and morphogenesis. We will undertake a thorough interrogation of endopeptidase structure, function and physiological role. We ask which regulatory mechanisms ensure properly directed EP cleavage activity, and how EPs interface with the cell wall synthesis apparatus. We will also interrogate the enigmatic LTGs, whose physiological functions are poorly understood. We will leverage our recent genetic screens that have identified novel LTG-dependent functions collectively required for bacterial growth and morphogenesis to define the collective physiological roles of LTG. Overall, our data will inform new conceptual models of cell wall turnover in bacterial growth and morphogenesis, and uncover new potential targets for antibiotics and their adjuvants. Project Number: 1R35GM161163-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Tobias Doerr | Institution: CORNELL UNIVERSITY, ITHACA, NY | Award Amount: $291,491 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award A Study Section[MRAA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11257575