Capturing of extracellular vesicles derived from single cells of Escherichia coli.

Bacteria secrete extracellular vesicles (EVs), also referred to as bacterial membrane vesicles, which carry, among other compounds, lipids, nucleic acids and virulence factors. Recent studies highlight the role of EVs in the emergence of antibiotic resistance, e.g. as carrier and absorbent particles of the drug to protect the cells, or as a pathway to disseminate resistance elements. In this study, we are interested in characterizing the secretion of EVs at the single bacterial level to ultimately understand how cells respond to antibiotic treatment. We introduce a microfluidic device that enables culture of single bacterial cells and capture of EVs secreted from these individuals. The device incorporates parallel, narrow winding channels to trap single rod-shaped E. coli cells at their entrances. The daughter cells are immediately removed by continuous flow on the open side of the trap, so that the trap contains always only a single cell. Cells grew in these traps over 24 h with a doubling time of 25 minutes. Under antibiotic treatment, the doubling time did not change, but we observed small changes in the cell length of the trapped cells (decrease from 4.0 µm to 3.6 µm for 0 and 250 ng mL-1 polymyxin B, respectively), and cells stopped growing within hours, depending on the drug concentration. Compared to bulk culture, the results indicate a higher susceptibility of on-chip-cultured cells (250 ng mL-1vs. >500 ng mL-1 in bulk), which may be caused, among other reasons, by the space limitation in the cell trap and shear forces. During the culture, EVs secreted by the trapped cells entered the winding channel. We developed a procedure to selectively coat these channels with poly-L-lysine resulting in a positively charged surface, which enabled electrostatic capture of negatively charged EVs. Subsequently, the immobilized EVs were stained with a lipophilic dye and detected by fluorescence microscopy. Our findings confirm large variations of EV secretion among individual bacteria and indicate a relative high rate of EV secretion under antibiotic treatment. The proposed method can be extended to the detection of other secreted substances of interest and may facilitate the elucidation of unknown heterogeneities in bacteria.

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains.

Extracellular vesicles (EVs) have attracted an attention as important targets in the fields of biology and medical science because they contain physiologically active molecules. Curvature-sensing peptides are currently used as novel tools for marker-independent EV detection techniques. A structure-activity correlation study demonstrated that the α-helicity of the peptides is prominently involved in peptide binding to vesicles. However, whether a flexible structure changing from a random coil to an α-helix upon binding to vesicles or a restricted α-helical structure is an important factor in the detection of biogenic vesicles is still unclear. To address this issue, we compared the binding affinities of stapled and unstapled peptides for bacterial EVs with different surface polysaccharide chains. We found that unstapled peptides showed similar binding affinities for bacterial EVs regardless of surface polysaccharide chains, whereas stapled peptides showed substantially decreased binding affinities for bacterial EVs covered with capsular polysaccharides. This is probably because curvature-sensing peptides must pass through the layer of hydrophilic polysaccharide chains prior to binding to the hydrophobic membrane surface. While stapled peptides with restricted structures cannot easily pass through the layer of polysaccharide chains, unstapled peptides with flexible structures can easily approach the membrane surface. Therefore, we concluded that the structural flexibility of curvature-sensing peptides is a key factor for governing the highly sensitive detection of bacterial EVs.

Polysaccharide corona: The acetyl-rich envelope wraps the extracellular membrane vesicles and the cells of Shewanella vesiculosa providing adhesiveness.

Bacterial extracellular membrane vesicles (EMVs) play an active role in many physiological and pathogenic processes. Here, we report the identification and the detailed structural characterization of the capsular polysaccharide from both cells and EMVs from Shewanella vesiculosa by NMR and chemical analysis. The polysaccharide consists of a pentasaccharide repeating unit containing neutral monosaccharides together with amino sugars, of which one has never been isolated from a natural source. The adhesion ability of the polymer both on synthetic surfaces, such as polystyrene nanoparticles and on vesicles with a bilayer mimicking the bacterial membrane in the presence and absence of lipopolysaccharide was investigated. In both cases, a "CPS-corona" that could be the first stage of biofilm formation was observed. The polymer also activates Caspases on colon cancer cells, making S. vesiculosa EMVs as natural nanocarriers for drug delivery.

Parallel study of transient dosing of antibiotics in a microfluidic device.

Microfluidic tools are well suited for studying bacteria as they enable the analysis of small colonies or single cells. However, current techniques for studying bacterial response to antibiotics are largely limited to static dosing. Here, we describe a microfluidic device and a method for entrapping and cultivating bacteria in hydrogel plugs. Ring-shaped isolation valves are used to define the shape of the plugs and also to control exposure of the plugs to the surrounding medium. We demonstrate bacterial cultivation, determination of the minimum inhibitory concentration of an antibiotic, and transient dosing of an antibiotic at sub-1-h doses. The transient dosing experiments reveal that at dose durations on the order of minutes, ampicillin's bactericidal effect has both a time and concentration dependency.

Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium, Shewanella vesiculosa HM13.

Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production at high lysine concentration in a hypervesiculating Gram-negative bacterium, Shewanella vesiculosa HM13. This protein was predicted to possess typical sensing and signaling domains of sensor proteins, such as methyl-accepting chemotaxis proteins. Comparison of vesicle production between the hm1275-disrupted mutant and the parent strain revealed that HM1275 is involved in lysine-induced hypervesiculation. Moreover, HM1275 has sequence similarity to a biofilm dispersion protein, BdlA, of Pseudomonas aeruginosa PAO1, and hm1275 disruption increased the amount of biofilm. Thus, this study showed that the induction of vesicle production and suppression of biofilm formation in response to lysine concentration are under the control of the same putative sensor protein.

Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media.

Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.

Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of Shewanella vesiculosa HM13.

Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria. Shewanella vesiculosa HM13, a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel, is able to produce a large amount of EMVs. S. vesiculosa HM13 has been found to include a cargo protein, P49, in the EMVs, but the entire mechanism in which P49 is preferentially included in the vesicles has still not been completely deciphered. Given these premises, and since the structural study of the components of the EMVs is crucial for deciphering the P49 transport mechanism, in this study the complete characterization of the lipooligosaccharide (LOS) isolated from the cells and from the EMVs of S. vesiculosa HM13 grown at 18 °C is reported. Both lipid A and core oligosaccharide have been characterized by chemical and spectroscopic methods.

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation.

Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into the sn-2 position of membrane phospholipids (PLs). Various bacteria produce multiple LPAATs, whereas it is believed that Escherichia coli produces only one essential LPAAT homolog, PlsC-the deletion of which is lethal. However, we found that E. coli possesses another LPAAT homolog named YihG. Here, we show that overexpression of YihG in E. coli carrying a temperature-sensitive mutation in plsC allowed its growth at non-permissive temperatures. Analysis of the fatty acyl composition of PLs from the yihG-deletion mutant (∆yihG) revealed that endogenous YihG introduces the cis-vaccenoyl group into the sn-2 position of PLs. Loss of YihG did not affect cell growth or morphology, but ∆yihG cells swam well in liquid medium in contrast to wild-type cells. Immunoblot analysis showed that FliC was highly expressed in ∆yihG cells, and this phenotype was suppressed by expression of recombinant YihG in ∆yihG cells. Transmission electron microscopy confirmed that the flagellar structure was observed only in ∆yihG cells. These results suggest that YihG has specific functions related to flagellar formation through modulation of the fatty acyl composition of membrane PLs.

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13.

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.

Characterization of extracellular membrane vesicles of an Antarctic bacterium, Shewanella livingstonensis Ac10, and their enhanced production by alteration of phospholipid composition.

A cold-adapted bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid (EPA) as a component of its membrane phospholipids, is useful as a model to study the function of EPA and as a host for heterologous production of thermolabile proteins at low temperatures. In this study, we characterized extracellular membrane vesicles (EMVs) of this bacterium to examine the involvement of EPA in the biogenesis of EMVs and for the future application of EMVs to extracellular protein production. We found that this strain produced EMVs from the cell surface. Cryo-electron microscopic observation showed that the majority of the EMVs had a single-bilayer structure with an average diameter of 110 nm, though EMVs with double-bilayer membranes and other diverse structures were also observed. Quantitative analysis demonstrated that the EMV production was significantly increased (3-5 fold) by the depletion of EPA-containing phospholipids. The lack of EPA also altered the protein composition of EMVs. In particular, incorporation of one of the cold-inducible outer membrane proteins, OmpC176, was significantly increased in EMVs after the depletion of EPA. These results provide a basis for the construction of an EMV-based, low-temperature protein production system and show the involvement of EPA in the regulation of EMV biogenesis.

A case of lymphomatoid gastropathy: a self-limited pseudomalignant natural killer (NK)-cell proliferative disease mimicking NK/T-cell lymphomas.

Natural killer (NK)/T-cell lymphomas exhibit aggressive tumor behavior and have a poor prognosis. Recently, self-limited pseudomalignant NK-cell proliferative disorders of the stomach mimicking NK/T-cell lymphomas have been recognized. We report a rare case of lymphomatoid gastropathy in a 71-year-old female. The patient underwent esophagogastroduodenoscopy (EGD) because of slight epigastric discomfort which revealed a 10-mm, reddish, flat elevation with erosion on the posterior wall of the lower gastric body. Histological examination of biopsy specimens showed atypical NK/T cell infiltration with cytoplasmic CD3+, CD4-, CD5-, CD7+, CD8-, CD16-, CD20-, CD56+, CD68-, CD117-, MPO-, TIA1+, and granzyme B+. Epstein-Barr virus-encoded RNA in situ hybridization was negative. Three months later, repeated endoscopic examination surprisingly revealed spontaneous regression of the lesion without any treatment. It is important that endoscopists consider this rare entity in the differential diagnosis, and excessive treatment should be avoided.

[Black esophagus due to acute necrotizing esophagitis: four case reports].

We describe four patients with acute esophageal necrosis who were admitted to hospital due to upper gastrointestinal bleeding. "Black esophagus" is endoscopically defined as diffuse dark pigmentation of the esophageal wall. The underlying conditions were ketoacidosis in three of the patients and diabetes mellitus in two. Three patients responded well to empirical supportive therapy and one patient died of coexisting illness rather than the esophageal status. Acute esophageal necrosis is a rare entity that should be considered in the differential diagnosis of upper gastrointestinal bleeding.