Poster Presentation 50th Lorne Proteins Conference 2025

Using minicells to examine the β-barrel protein arrays of Klebsiella pneumoniae (#225)

Cara Press 1 , Matthew Johnson 2 , Richard Morris 3 , Debnath Ghosal 2 , Trevor Lithgow 1
  1. Monash University, Melbourne, Victoria, Australia
  2. University of Melbourne, Melbourne, Victoria, Australia
  3. University of New South Wales, Sydney, New South Wales, Australia

In recent years the model of the Gram-negative bacterial cell envelope has changed. It is now understood that the outer membrane proteins exist within β-barrel protein arrays (Lithgow, Stubenrauch and Stumpf, 2023). For the antimicrobial resistant pathogen Klebsiella pneumoniae the cell surface is an important contributor to its virulence and adaptability. This study sought to further our understanding of the importance of outer membrane protein organisation in K. pneumoniae. Minicells are small anucleate cells produced following aberrant cell division. The Min system is responsible for the positioning of the cell division machinery at the mid-cell. Deletion or mutation of the minCDE operon that encodes the Min machinery results cell division that occurs at the cell pole, producing minicells. In recent years E. coli minicells have proven a useful tool in cryo-electron tomography (cryoET) as the thinner cells allow for improved resolution (Farley et al., 2016). Here, we present the development and characterisation of K. pneumoniae minicells using an interdisciplinary approach: Following the generation of the ΔminCDE mutant in K. pneumoniae, biochemical techniques and proteomics are used to examine the protein composition of the outer membrane in minicells. This protein composition is then examined in situ using cryoET to visualise the cellular and membrane components of the minicells. Alongside this analysis, the inter- and intramolecular forces exerted upon the β-barrel outer membrane proteins in the context of a minicell is interrogated using mathematical modelling.

 

  1. Lithgow, T., Stubenrauch, C. J. & Stumpf, M. P. H. Surveying membrane landscapes: a new look at the bacterial cell surface. Nat Rev Microbiol 21, 502-518 (2023). https://doi.org/10.1038/s41579-023-00862-w
  2. Farley, M. M., Hu, B., Margolin, W. & Liu, J. Minicells, Back in Fashion. J Bacteriol 198, 1186-1195 (2016). https://doi.org/10.1128/JB.00901-15