Poster Presentation 50th Lorne Proteins Conference 2025

A structural perspective on pore formation and regulation of Bacteroides fragilis toxins (#317)

Riya Joseph 1 2 , Michelle Christie 1 , Hunter Abrahamsen 3 , Bronte Johnstone 1 , Craig Morton 1 4 , Rodney Tweten 3 , Michael Parker 1 2
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria,, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  3. Department of Microbiology and Immunology, University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma , USA
  4. CSIRO Biomedical Manufacturing Program, Clayton, Victoria, Australia

Bacteroides fragilis, a Gram-negative anaerobic bacterium, is typically a commensal in the human gut but can become pathogenic under conditions of intestinal dysbiosis. A critical factor in its pathogenicity is the production of Cholesterol-Dependent Cytolysin-Like proteins (CDCLs), a novel class of pore-forming toxins that enable B. fragilis to thrive by targeting and disrupting competing microorganisms in the intestinal environment. Despite their importance, several aspects of CDCL pore formation and regulation remain poorly understood. (1,2)

Our study investigates the structural and functional properties of two CDCL proteins, Bf long and Bf short, from B. fragilis. Interestingly, in addition to the pore-forming proteins, B. fragilis produces BcdI, a surface lipoprotein that confers immunity against its own toxins. A central question is: how do these CDCLs assemble into large, multi-component pores upon proteolytic activation, and what role does BcdI play in regulating their pore-forming activity? (3)

Using an integrated approach of X-ray crystallography, small-angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM), we present the first high-resolution crystal structures of Bf short and BcdI, along with SAXS and cryo-EM models of the CDCL pore complex. Furthermore, we conduct protein functional analysis using liposome rupture and liposome pull-down experiments. These techniques provide critical insights into the interactions between CDCLs and lipid membranes.

Our findings represent a significant advance in understanding the molecular mechanisms underlying pore formation in bacterial toxins and their regulation. Together with our ongoing research, these results contribute to a deeper understanding of the mechanisms behind CDCL pore formation and regulation in B. fragilis, shedding light on how the bacterium exploits these virulence factors to its advantage. This work not only deepens our knowledge of CDCL toxins but also highlights the potential for targeting these mechanisms in therapeutic strategies for microbial diseases.

  1. Evans JC, Johnstone BA, Lawrence SL, Morton CJ, Christie MP, Parker MW, et al. A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins. mBio. 2020;11(5).
  2. Johnstone BA, Joseph R, Christie MP, Morton CJ, McGuiness C, Walsh JC, et al. Cholesterol-dependent cytolysins: The outstanding questions. IUBMB Life. 2022.
  3. Abrahamsen HL, Sanford TC, Collamore CE, et al. Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria. Nat Commun. 2024;15(1):5028. Published 2024 Jun 12. doi:10.1038/s41467-024-49103-5