Abstract

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

Highlights

  • Release of extracellular vesicles is a highly conserved and natural process known in all kingdoms of life: in Archaea, Bacteria, and Eukarya (Gill et al, 2018)

  • We focused on the effects on the host cells of outer membrane vesicles (OMV) released by the virulent F. tularensis subsp. holarctica strain FSC200

  • Outer Membrane Vesicles Enter and Accumulate in Macrophages, but Not Epithelial Cell Line A549 The direct interaction or internalization of OMV derived from pathogenic bacteria has been reported in a number of studies

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Summary

Introduction

Release of extracellular vesicles is a highly conserved and natural process known in all kingdoms of life: in Archaea, Bacteria, and Eukarya (Gill et al, 2018). The vesicle release serves the bacteria for interaction with the environment, for communication with other bacteria in their community, and in the case of pathogenic bacteria, for interaction with the host (Ellis and Kuehn, 2010; Avila-Calderón et al, 2014; Jan, 2017). OMV are known to enhance the invasiveness and survival of bacteria inside the host (Jan, 2017). OMV can increase the bacterial adherence to various surfaces (Ellis and Kuehn, 2010), they are part of the membrane stress response to hostile environments (MacDonald and Kuehn, 2013; Klimentova et al, 2019), and they can modulate the host immune response (Kaparakis-Liaskos and Ferrero, 2015). Great attention is currently focused on the vaccine potential of OMV because of their strong immunomodulatory effect, self-adjuvant characteristic, and the ease of genetic modifications (Acevedo et al, 2014; Bitto and Kaparakis-Liaskos, 2017; Yang et al, 2018; Gerritzen et al, 2019)

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