Role of Bacterial Extracellular Vesicles in Manipulating Infection.

Mammalian-cell-derived extracellular vesicles, such as exosomes, have been a key focal point for investigating host-pathogen interactions and are major facilitators in modulating both bacterial and viral infection. However, in recent years, increasing attention has been given to extracellular vesicles produced by bacteria and the role they play in regulating infection and disease. Extracellular vesicles produced by pathogenic bacteria employ a myriad of strategies to assist in bacterial virulence or divert antibacterial responses away from the parental bacterium to promote infection by and survival of the parental bacterium. Commensal bacteria also produce extracellular vesicles. These vesicles can play a variety of roles during infection, depending on the bacterium, but have been primarily shown to aid the host by stimulating innate immune responses to control infection by both bacteria and viruses. This article will review the activities of bacterial extracellular vesicles known to modulate infection by bacterial and viral pathogens.

Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

Vesicular Transmitter Content in Chromaffin Cells Can Be Regulated via Extracellular ATP.

The energy carrying molecule adenosine triphosphate (ATP) has been implicated for its role in modulation of chemical signaling for some time. Despite this, the precise effects and mechanisms of action of ATP on secretory cells are not well-known. Here, bovine chromaffin cells have been used as a model system to study the effects of extracellular ATP in combination with the catecholamine transmitter norepinephrine (NE). Both transmitter storage and exocytotic release were quantified using complementary amperometric techniques. Although incubation with NE alone did not cause any changes to either transmitter storage or release, coincubation with NE and ATP resulted in a significant increase that was concentration dependent. To probe the potential mechanisms of action, a slowly hydrolyzable version of ATP, ATP-γ-S, was used either alone or together with NE. The result implicates two different behaviors of ATP acting on both the purinergic autoreceptors and as a source of the energy needed to load chromaffin cell vesicles.

Altered Lipid Composition of Secretory Cells Following Exposure to Zinc Can Be Correlated to Changes in Exocytosis.

A micromolar concentration of zinc has been shown to significantly change the dynamics of exocytosis as well as the vesicle contents in a model cell line, providing direct evidence that zinc regulates neurotransmitter release. To provide insight into how zinc modulates these exocytotic processes, neurotransmitter release and vesicle content were compared with single cell amperometry and intracellular impact vesicle cytometry with a range of zinc concentrations. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) images of lipid distributions in the cell membrane after zinc treatment correlate to changes in exocytosis. By combining electrochemical techniques and mass spectrometry imaging, we proposed a mechanism by which zinc changes the fusion pore and the rate of neurotransmitter release by changing lipid distributions and results in the modulation of synaptic strength and plasticity.

Anticancer Drug Tamoxifen Affects Catecholamine Transmitter Release and Storage from Single Cells.

Electrochemical measurements of exocytosis combined with intracellular vesicle impact electrochemical cytometry have been used to evaluate the effect of an anticancer drug, tamoxifen, on catecholamine release at the single-cell level. Tamoxifen has been used for over 40 years to treat estrogen receptor-positive breast cancers during both early stages of the disease and in the adjuvant setting. Tamoxifen causes memory and cognitive dysfunction, but the reasons for the cognitive impairment and memory problems induced by this anticancer drug are not well-known. We show that tamoxifen, through a nongenomic mechanism, can modulate both exocytosis and vesicle catecholamine storage in a model cell line. The results indicate that exocytosis is inhibited at high concentrations of tamoxifen and is stimulated at low levels. Tamoxifen also elicits a significant concentration-dependent change in total catecholamine content of single vesicles, while sub-nanomolar concentrations of the drug have stimulatory activity on the catecholamine content of vesicles. In addition, it has profound effects on storage at higher concentrations. Tamoxifen also reduces the intracellular free Ca2+ but only at micromolar concentration, by acting on voltage-gated Ca2+ channels, which likely affects neurotransmitter secretion.

Using Single-Cell Amperometry and Intracellular Vesicle Impact Electrochemical Cytometry To Shed Light on the Biphasic Effects of Lidocaine on Exocytosis.

Single cell amperometry and intracellular vesicle impact electrochemical cytometry were used to examine whether lidocaine can regulate neurotransmitter release or storage for PC12 cells to explain the biphasic effects whereby it can protect neurons and improve cognitive outcome at low concentration, but can cause neurotoxicity at high concentration. We show that lidocaine affects the behavior of PC12 cell exocytosis in a concentration dependent way, which exactly corresponds to its biphasic effects. At a relatively high concentration, it shows a much narrower pore size and a longer-duration fusion pore with less monoamine released than control cells. However, at a relatively low concentration, the fusion pore is open even longer than at high concentration, and with more monoamine released than control cells. Furthermore, intracellular vesicle impact electrochemical cytometry was used to confirm that lidocaine did not change the catecholamine content of the vesicles. These data provide a mechanism for the observed biphasic effects of the drug and suggest that lidocaine influences exocytosis through multiple mechanisms.

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Zinc Regulates Chemical-Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry.

We applied electrochemical techniques with nano-tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 µm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc-treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles.

We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.

The diagnostic and prognostic potential of plasma extracellular vesicles for cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of death worldwide and its prevalence is expected to rise rapidly worldwide in the coming decades. Atherosclerosis, the syndrome underlying CVD, is a chronic progressive disease of the arteries already present at a young age. Strokes, heart attacks and heart failure are acute CVD events that occur after decades, however, and require timely diagnosis and treatment. Plasma extracellular vesicles (EVs) are microstructures with a lipid bilayer membrane involved in hemostasis, inflammation and injury. Both EV-counts and EV-content are associated with CVD and the identification of plasma EVs is a novel source of blood-based biomarkers with the potential to improve diagnosis and prognosis of CVD. Presented in this review is an overview of the current use of EVs in CVD and a discussion of the need for robust and easy isolation technologies for plasma EV subsets. This is needed to bring this promising field towards clinical application in the patient.