Abstract
Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.
Highlights
Gram-negative bacteria ubiquitously shed vesicles known as outer membrane vesicles (OMVs) during their normal growth [reviewed in Ref. [1, 2]]
We subsequently confirmed the viability of AGS and HEK293 cells post treatment with each chemical inhibitor, in addition to the inhibitors effectiveness (Figure S1 in Supplementary Material). Both AGS and HEK293 cells were treated for 30 min with either: cytochalasin D or nocodazole, to block macropinocytosis, dynasore monohydrate to inhibit dynamin-dependent endocytosis which is utilized by both clathrin and caveolin-mediated entry, or valinomycin to block clathrin-mediated endocytosis [45]
We found that the entry of fluorescently labeled human transferrin, which enters host cells via clathrin-mediated endocytosis [42], was markedly reduced in cells pre-treated with dynasore or valinomycin as expected, to comparable levels as the positive control chlorpromazine (Figure S2A in Supplementary Material)
Summary
Gram-negative bacteria ubiquitously shed vesicles known as outer membrane vesicles (OMVs) during their normal growth [reviewed in Ref. [1, 2]]. Outer membrane vesicles from a range of bacteria have been identified to have a similar protein [6,7,8] and lipid [9] composition to the outer membranes of their parent bacterium. As the protein composition of OMVs is highly similar to that of their parent bacterium, their use and development as innovative vaccines is being extensively examined [19,20,21,22,23,24,25,26,27]. Due to the similarity of OMVs to their parent bacterium and their highly immunogenic nature, OMV-based vaccines are currently being developed and licensed for human use [reviewed in Ref. Due to the similarity of OMVs to their parent bacterium and their highly immunogenic nature, OMV-based vaccines are currently being developed and licensed for human use [reviewed in Ref. [27, 28]]
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