Poster Presentation Lorne Infection and Immunity 2022

Methods of vesicle production and quantification can influence immunological outcomes.  (#113)

Natalie Bitto 1 2 , Lauren Zavan 2 3 , Ella L Johnston 2 3 , Tim P Stinear 4 , Andrew F Hill 5 , Maria Kaparakis Liaskos 2 3
  1. Research Centre for Extracellular Vesicles, School of Molecular Sciences, La Trobe University, Bundoora, VIC, Australia
  2. Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Melbourne, Victoria, Australia
  3. Research Centre for Extracellular Vesicles, School of Molecular Sciences, La Trobe University, Bundoora, Victoria, Australia
  4. Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
  5. La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia

Bacterial membrane vesicles (BMVs) are naturally secreted by all bacteria and can facilitate host-microbe interactions in a multitude of ways via their biologically active cargo. Quantification of BMVs is a critical step that underpins all subsequent assays examining their composition and functions. Historically, protein assays have been the preferred method of BMV quantification. However, BMV protein content can varying with bacterial growth stage, growth conditions and BMV size, suggesting that BMV protein amount may not directly correlate with BMV number.

 

To investigate this, we compared the enumeration of BMVs from Gram-positive and Gram-negative bacteria using different protein assays, and nanoparticle tracking analysis (NTA). We showed that different protein assays vary significantly in their quantification of BMVs, and the species from which BMVs originate influence their quantification by different protein assays. Moreover, stimulation of epithelial cells with an equivalent amount of BMVs quantified by different protein assays resulted in significant differences in IL-8 responses. Quantification of BMVs by NTA and normalization of BMV cargo to particle number revealed BMV protein, DNA and RNA content were variable between strains, species and throughout bacterial growth. The effect of strain differences in BMV protein content on their ability to activate NF-κB and induce IL-8 responses were evident when epithelial cell stimulations were based on particle number, but not when stimulations were based on protein amount. Similarly, strain differences in the ability of BMVs to activate TLR7 and 9 by HEKBlue reporter cell lines were evident when stimulations were based on particle number but not protein amount.

 

In summary, our findings show that standardization of BMVs by protein amount can reduce the ability to distinguish differences in their immunological functions. In contrast, species, strain and growth stage-dependent differences in BMV cargo content and immunogenic functions are evident when BMVs are enumerated by particle number. These findings highlight that standardization of BMV enumeration is required in the BMV field to bring uniformity and comparability, and ultimately advance our understanding of BMV functions.