Oral Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

The properties and transport of DNA carried by bacterial outer membrane vesicles (#19)

Natalie Bitto 1 , Adam Costin 2 , Camden Lo 3 , Kate Stacey 4 , Richard Ferrero 1
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. The Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy, Monash University, Clayton, VIC, Australia
  3. Monash Micro Imaging, Monash University, Clayton, VIC, Australia
  4. School of Chemistry and Molecular Biosciences, Faculty of Science, University of Queensland, Brisbane, QLD, Australia

The release of nano-sized membrane vesicles is a conserved phenomenon amongst Gram-negative bacteria. These bi-layered structures, known as outer membrane vesicles (OMVs), are adept at entering eukaryotic cells, facilitating delivery of bacterial factors into host cells. As well as a range of bacterial proteins and glycolipids, OMVs have been shown to contain DNA. Despite its putative importance in both bacterial and immune functions, very little is known about the nature of OMV-associated DNA and its interactions with host cells.

This study investigates the mechanisms by which OMV-associated DNA may play a role in host-pathogen interaction. To this end, we characterised the amount, form and sequence of OMV associated DNA, demonstrated its uptake into eukaryotic cells and determined the destination of OMV-derived DNA once within the cell. To characterise the amount, form and sequence of OMV-associated DNA, we used PicoGreen fluorescent quantitation, Ion Torrent semiconductor sequencing and electron microscopy to show that OMVs are associated with bacterial genomic DNA. This genomic DNA is predominantly bound to the OMV surface, however a small portion is protected within the vesicle lumen. Sequence analysis identified a number of genes that were enriched in the internal and external OMV-derived DNA. Using immunofluorescence, we have shown for the first time the uptake of OMV-associated DNA into eukaryotic cells. Furthermore, we have separated the nuclear and cytosolic fractions of eukaryotic cells treated with OMVs and have shown that OMV-derived DNA localises in the nucleus of host cells. 

Collectively, our findings show that OMVs are associated with bacterial DNA which is carried as cargo into eukaryotic cells and enters the nucleus. The outcomes of this study will give insight into host-pathogen interaction of Gram-negative bacteria, as well as shedding light on the mechanism of action of OMV-based vaccines, such as the current meningococcal vaccine.