Meta J. Kuehn

The release of extracellular vesicles (EVs) is a universally conserved process. Bacterial EVs package diverse cargo, including proteins and nucleic acids, and influence bacterial adaptation and survival, as well as host-pathogen interactions. Currently, our understanding of the mechanisms underlying global principles of EV biogenesis and release in Staphylococcus aureus and in gram-positive species, in general, is limited. Here, we describe a moderately high-throughput approach to analyze the Nebraska Transposon Mutant Library to broadly identify determinants of agr -dependent EV production in S. aureus . We show that the agr quorum-sensing system dictates EV production in response to nutrient availability, likely through communication with the adaptive stress response. This study demonstrates the contribution of nutritional stress to EV biogenesis and supports a conserved communication strategy that allows metabolic state to influence EV production. IMPORTANCE: Studies have demonstrated a role for gram-positive extracellular vesicles (EVs) in virulence and adaptation; however, mechanistic knowledge of EV biogenesis remains limited. Here, using a screening approach, we demonstrate how the downstream targets of the agr -quorum-sensing system in Staphylococcus aureus , α-PSMs and RNAIII, differentially contribute to and alter EV biogenesis. We show a likely conserved communication strategy that allows nutrient availability to influence EV production. Additionally, we identified numerous candidate genes potentially involved in EV biogenesis and its regulation, providing a valuable resource for future mechanistic studies.

The release of extracellular vesicles (EV) is a universally conserved process. Bacterial EVs package diverse cargo, including proteins and nucleic acids, and influence bacterial adaptation and survival as well as host-pathogen interactions. Currently, our understanding of the mechanisms underlying global principles in Gram-positive EV biogenesis and release is limited, partly due to labor-intensive vesicle isolation and assessment methods. Here, we describe a moderately high-throughput approach to analyze the Nebraska Transposon Mutant Library to identify genetic determinants of EV production in S. aureus . We show that the agr quorum sensing system dictates EV production in response to nutrient availability, likely through communication with the adaptive stress response. This study demonstrates the contribution of nutritional stress to vesiculogenesis and supports a conserved communication strategy that allows metabolic state to influence EV production.

Recent publications have shown that bacterial vesicles play important roles in interkingdom communication between bacteria and plants. Indeed, our recently published data reveal that bacterial vesicles from pathogenic and beneficial strains elicit immune responses in plants that protect against future pathogen challenge.

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Selective cargo packaging into bacterial extracellular vesicles has been reported and implicated in many biological processes, however, the mechanism behind the selectivity has remained largely unexplored. In this study, proteomic analysis of outer membrane (OM) and OM vesicle (OMV) fractions from enterotoxigenic E. coli revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. Selectivity was determined to be independent of transcriptional regulation of the proteins upon oxidative stress and was validated using randomly selected protein candidates from the different cargo classes. Based on these data, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA constructs. Our study reveals a basic mechanism for cargo selectivity into OMVs that may be useful for the engineering of OMVs for future biotechnological applications.

Extracellular vesicles are a ubiquitous but often overlooked biological component of all natural environments. Convergent interdisciplinary research highlights an effective approach to understand their properties and functions.

Bacterial adherence is an important early step in infection and results from the specific binding of a bacterial protein, called an adhesin, to a eukaryotic cell receptor. ^1-3 The specificity of a particular adhesin for a particular glycolipid or glycoprotein receptor on epithelial cells is in part responsible for the tissue and host tropism of pathogenic bacteria. Adherence leads to the colonization of tissues by preventing bacteria from being cleared by the normal flow of bodily fluids. Infection, inflammation, and eventually, bacteremia can result from bacterial colonization. In many cases, bacterial adhesins are associated with rod-like structures called pili which radiate from the surface of bacteria. Pili are protein polymers composed of approximately a thousand copies of a major pilus subunit and several minor pilus subunits that play important roles in adhesion presentation, polymerization initiation, and termination.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such asStaphylococcus aureusand their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells.S. aureusproduce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show thatS. aureussecretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.