Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.
Bibliographical note© 2017 O’Donoghue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk/) grants BB/M021513/1 and BB/L007916/1 (to AMK), and a BBSRC MIBTP studentship (to EJO).
- Cell Wall/chemistry
- Gram-Negative Bacteria/chemistry
- Gram-Negative Bacterial Infections/metabolism
- Host-Pathogen Interactions
- Transport Vesicles/metabolism
- Virulence Factors/metabolism