Abstract
Gram-negative bacteria ubiquitously produce and release nano-size, non-replicative outer membrane vesicles (OMVs). In the gastrointestinal (GI-) tract, OMVs generated by members of the intestinal microbiota are believed to contribute to maintaining the intestinal microbial ecosystem and mediating bacteria–host interactions, including the delivery of bacterial effector molecules to host cells to modulate their physiology. Bacterial OMVs have also been found in the bloodstream although their origin and fate are unclear. Here we have investigated the interactions between OMVs produced by the major human gut commensal bacterium, Bacteroides thetaiotaomicron (Bt), with cells of the GI-tract. Using a combination of in vitro culture systems including intestinal epithelial organoids and in vivo imaging we show that intestinal epithelial cells principally acquire Bt OMVs via dynamin-dependent endocytosis followed by intracellular trafficking to LAMP-1 expressing endo-lysosomal vesicles and co-localization with the perinuclear membrane. We observed that Bt OMVs can also transmigrate through epithelial cells via a paracellular route with in vivo imaging demonstrating that within hours of oral administration Bt OMVs can be detected in systemic tissues and in particular, the liver. Our findings raise the intriguing possibility that OMVs may act as a long-distance microbiota–host communication system.
Highlights
The mammalian GI-tract is home to a vast number of microbes that make up the intestinal microbiota which has co-evolved with the host to establish a mutualistic relationship (Round and Mazmanian, 2009)
To determine the kinetics of outer membrane vesicle (OMV) uptake, DiO-labeled Bacteroides thetaiotaomicron (Bt) OMVs were incubated with Caco-2 monolayers and intracellular fluorescence intensity quantified at various timepoints over a 48 h period
To confirm the intracellular fluorescence was attributable to Bt OMVs and not free dye, OMV–Caco2 co-cultures were stained with an in-house generated rabbit anti-Bt outer membrane protein-A (OmpA) (BT_3852) antisera
Summary
The mammalian GI-tract is home to a vast number of microbes that make up the intestinal microbiota which has co-evolved with the host to establish a mutualistic relationship (Round and Mazmanian, 2009). The mucus layer that coats the entirety of the intestinal epithelium prevents direct contact of luminal microbes with host cells, bacterial products such as metabolites or bacterial OMVs, can access and cross the epithelial barrier (Fateh et al, 2015) to influence both local and systemic host responses (Bomberger et al, 2009; Stentz et al, 2014). Gram-negative bacteria ubiquitously shed bilayer OMVs into their external environment (Brown et al, 2015; Schwechheimer and Kuehn, 2015). These non-replicative spherical vesicles bud from the bacterial outer membrane and range in size from 20 to 400 nm (Toyofuku et al, 2019). Bacterial DNA of potential OMV origin has been detected in human blood and urine (Yoo et al, 2016; Lee et al, 2017; Park et al, 2017) as well as in body compartments previously thought to be sterile, such as the heart (Svennerholm et al, 2017), suggesting OMVs can reach distant sites from their site of origin and production, including the lumen of the GI-tract (Stentz et al, 2018)
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