Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Developing Dual proteomic Approaches to Study Burkholderia cenocepacia Infections in Macrophage Cells (#96)

Michael G. Bacus 1 , Nichollas E. Scott 1
  1. Department of Microbiology and Immunology, University of Melbourne, Melbourne, VICTORIA, Australia

The opportunistic pathogen Burkholderia cenocepacia is an environmental Gram-negative species associated with severe and persistent infections in individuals with Cystic Fibrosis (CF). Within CF patients, B. cenocepacia colonizes the lungs, establishing an intracellular replicative niche within macrophages 1. While the significance of macrophages for persistent infections is established, the mechanisms by which B. cenocepacia manipulates macrophages and the changes within B. cenocepacia required for intracellular replication remain unclear. A key limitation in understanding the intracellular replication of B. cenocepacia has been its low infectivity2,3, resulting in highly heterogeneous infections dominated by uninfected cells in in vitro models. Employing opsonization2 to enhance the uptake of B. cenocepacia into THP-1 macrophages, we have developed a model to enhance the uniformity of B. cenocepacia internalization, allowing for proteomic analysis of B. cenocepacia infections. Utilizing Data-Independent Acquisition (DIA) proteomics, we compare the impact of opsonization on the host response and our ability to monitor intracellular B. cenocepacia. Our findings indicate that opsonization improves the detection of proteomic changes in response to B. cenocepacia compared to non-opsonized infections. Excitingly, opsonization not only improves the detection of host changes but also dramatically enhances the detection of B. cenocepacia proteins, revealing significant changes in metabolic proteins in response to intracellular replication. To further understand these changes, we have coupled the crude isolation of intracellular bacteria to our opsonization and DIA workflow allowing the quantification of more than 2000 bacterial proteins from intracellular B. cenocepacia, revealing changes in the B. cenocepacia proteome from early (3hr) to late (24hr) stages of infection. Our study is the first to provide a comprehensive profile of both host and pathogen proteome changes during B. cenocepacia infection in macrophage cells, shedding light on the fine-tuning of bacterial metabolism employed by intracellular B. cenocepacia.

  1. 1 Mesureur, J. et al. Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation. PLoS pathogens 13, e1006437 (2017). https://doi.org:10.1371/journal.ppat.1006437 2 Huynh, K. K., Plumb, J. D., Downey, G. P., Valvano, M. A. & Grinstein, S. Inactivation of macrophage Rab7 by Burkholderia cenocepacia. J Innate Immun 2, 522-533 (2010). https://doi.org:10.1159/000319864 3 Lamothe, J., Huynh, K. K., Grinstein, S. & Valvano, M. A. Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles. Cellular microbiology 9, 40-53 (2007). https://doi.org:10.1111/j.1462-5822.2006.00766.x