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.

Tissue factor-induced fibrinogenesis mediates cancer cell clustering and multiclonal peritoneal metastasis.

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.

Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs.

BACKGROUND: Lysophosphatidic acid acyltransferase (LPAAT) is a phospholipid biosynthesis enzyme that introduces a particular set of fatty acids at the sn-2 position of phospholipids. Many bacteria have multiple LPAAT paralogs, and these enzymes are considered to have different fatty acid selectivities and to produce diverse phospholipids with distinct fatty acid compositions. This feature is advantageous for controlling the physicochemical properties of lipid membranes to maintain membrane integrity in response to the environment. However, it remains unclear how LPAAT paralogs are functionally differentiated and biologically significant. RESULTS: To better understand the division of roles of the LPAAT paralogs, we analyzed the functions of two LPAAT paralogs, PlsC4 and PlsC5, from the psychrotrophic bacterium Shewanella livingstonensis Ac10. As for their enzymatic function, lipid analysis of plsC4- and plsC5-inactivated mutants revealed that PlsC4 prefers iso-tridecanoic acid (C12-chain length, methyl-branched), whereas PlsC5 prefers palmitoleic acid (C16-chain length, monounsaturated). Regarding the physiological role, we found that plsC4, not plsC5, contributes to tolerance to cold stress. Using bioinformatics analysis, we demonstrated that orthologs of PlsC4/PlsC5 and their close relatives, constituting a new clade of LPAATs, are present in many γ-proteobacteria. We also found that LPAATs of this clade are phylogenetically distant from principal LPAATs, such as PlsC1 of S. livingstonensis Ac10, which are universally conserved among bacteria, suggesting the presence of functionally differentiated LPAATs in these bacteria. CONCLUSIONS: PlsC4 and PlsC5, which are LPAAT paralogs of S. livingstonensis Ac10, play different roles in phospholipid production and bacterial physiology. An enzyme belonging to PlsC4/PlsC5 subfamilies and their close relatives are present, in addition to principal LPAATs, in many γ-proteobacteria, suggesting that the division of roles is more common than previously thought. Thus, both principal LPAATs and PlsC4/PlsC5-related enzymes should be considered to decipher the metabolism and physiology of bacterial cell membranes.

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.

Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis.

Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

Capsular polysaccharide from a fish-gut bacterium induces/promotes apoptosis of colon cancer cells in vitro through Caspases' pathway activation.

Among the widespread malignancies colorectal cancer is the most lethal. Treatments of this malignant tumor include surgery for lesions and metastases, radiotherapy, immunotherapy, and chemotherapy. Nevertheless, novel therapies to reduce morbidity and mortality are demanding. Natural products, such as polysaccharides, can be a valuable alternative to sometimes very toxic chemotherapeutical agents, also because they are biocompatible and biodegradable biomaterials. Microbial polysaccharides have been demonstrated to fulfill this requirement. In this paper, the results about the structure and the activity of a capsular polysaccharide isolated from the psychrotroph Pseudoalteromonas nigrifaciens Sq02-Rifr, newly isolated from the fish intestine, have been described. The characterization has been obtained by spectroscopic and chemical methods, and it is supported by the bioinformatic analysis. The polymer activates Caspases 3 and 9 on colon cancer cells CaCo-2 and HCT-116, indicating a promising antitumor effect, and suggesting a potential capacity of CPS to induce apoptosis.

Membrane Vesicles Produced by Shewanella vesiculosa HM13 as a Prospective Platform for Secretory Production of Heterologous Proteins at Low Temperatures.

Extracellular membrane vesicles (EMVs) produced by Gram-negative bacteria are useful as a vaccine platform. During growth in broth at 18 °C, Shewanella vesiculosa HM13 produces a large number of EMVs that contain a 49-kDa major cargo protein, named P49. Enhanced green fluorescent protein fused to the C-terminus of P49 is delivered to EMVs, suggesting that P49 is useful as a carrier to target foreign proteins to EMVs for production of artificial EMVs with desired functions. This method is potentially useful for the preparation of designed vaccines and is described in detail in this chapter.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

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.

[A Case of Central Venous Catheter Fracture after Its Implantation for Liver Metastasis of Rectal and Appendiceal Cancers].

This is the case of a 72-year-old man in whom multiple colorectal cancers including rectal and appendiceal cancers and synchronous S3 liver metastases were observed in 2014, and resection was performed in 2 stages. In 2017, a single recurrence was found in the liver S8, and he underwent a liver S8 sub-segmental resection. Implantation of a CV port for postoperative chemotherapy was planned. At the time of insertion, the catheter was punctured from the exterior portion of the left subclavian vein to avoid the pinch-off syndrome wherein the catheter is crushed between the clavicle and the first rib. Subsequently, FOLFOX therapy was started, but it was discontinued because of allergic symptoms, which appeared during the third course. Two years after the CV port was implanted, a catheter fracture was found on a chest X-ray performed during a regular visit. Since the detached catheter did not fall into the vein, it was possible to remove the port under fluoroscopy. When a catheter is implanted, even under ultrasound guidance, it is considered important to always keep in mind the possibility of a catheter fracture and to detect and respond to it early.

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.

[A Case of Laparoscopic Gastrojejunal Bypass Surgery for Malignant Stenosis after Chemoradiotherapy for Esophageal Cancer].

We report a case of malignant stenosis due to recurrence of lymph node metastasis treated with laparoscopic gastrojejunal bypass. A 83-year-old man who underwent chemoradiotherapy for esophageal cancer(cT3N2M0). About 3 and half years after chemoradiotherapy, he was referred to hospital for vomiting. As a result of the examination, we diagnosed malignant stenosis of descending part of duodenum due to retroperitoneum lymph node recurrence of esophageal cancer. We performed laparoscopic gastrojejunal bypass operation because we suggested self-expandable metallic stent make easy to migrate into anal side of the duodenum. The postoperative course was good. He was enrolled in oncology department on the 21 days after the operation. Gastroduodenal stenosis is common pathology by malignant tumor. Gastrojejunostomy and placement of self-expandable metallic stent is commonly performed for malignant gastroduodenal obstruction. Endoscopic metallic stent placement is minimally invasive treatment for malignant stenosis of the intestine, however sometime the stent placement will make easy to migrate by extra compression. Gastrojejunostomy mat be more safety than endoscopic stent placement for the malignant gastroduodenal obstruction.

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.

Role of acyl-CoA dehydrogenases from Shewanella livingstonensis Ac10 in docosahexaenoic acid conversion.

The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general ß-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the ß-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the ß-oxidation pathway that catalyzes DHA conversion.

Bioconversion From Docosahexaenoic Acid to Eicosapentaenoic Acid in the Marine Bacterium Shewanella livingstonensis Ac10.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which belong to the same class of long chain ω-3 polyunsaturated fatty acids (PUFAs), are present in marine γ-proteobacteria. In contrast to their de novo biosynthesis that has been intensively studied, their metabolic fates remain largely unknown. Detailed information regarding bacterial ω-3 PUFA metabolism would be beneficial for understanding the physiological roles of EPA/DHA as well as the industrial production of EPA, DHA, and other PUFAs. Our previous studies revealed that the EPA-producing marine bacterium Shewanella livingstonensis Ac10 produces EPA from exogenous DHA independently of de novo EPA biosynthesis, indicating the presence of an unidentified metabolic pathway that converts DHA into EPA. In this study, we attempted to reveal the molecular basis for the bioconversion through both in vivo and in vitro analyses. Mutagenesis experiments showed that the gene disruption of fadH, which encodes an auxiliary ß-oxidation enzyme 2,4-dienoyl-CoA reductase, impaired EPA production under DHA-supplemented conditions, and the estimated conversion rate decreased by 86% compared to that of the parent strain. We also found that the recombinant FadH had reductase activity toward the 2,4-dienoyl-CoA derivative of DHA, whereas the intermediate did not undergo ß-oxidation in the absence of the FadH protein. These results indicate that a typical ß-oxidation pathway is responsible for the conversion. Furthermore, we assessed whether DHA can act as a substitute for EPA by using an EPA-less and conversion-deficient mutant. The cold-sensitive phenotype of the mutant, which is caused by the lack of EPA, was suppressed by supplementation with EPA, whereas the DHA-supplementation suppressed it to a lesser extent. Therefore, DHA can partly substitute for, but is not biologically equivalent to, EPA in S. livingstonensis Ac10.

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.

[A Case of Synchronous Colorectal Cancer Successfully Treated by Laparoscopic Surgery with Two Segmental Anastomoses].

A 63-year-old man was admitted for the evaluation of Hb 4.8 g/dL anemia. He underwent colon fiberscopy and was subsequently diagnosed with synchronous cancers of the ascending colon and rectum. He underwent laparoscopic ileocecal resection and low anterior resection with 2 segmental anastomoses. The histopathological diagnosis of A/C and rectal cancer was Stage Ⅱ and Stage Ⅲa, respectively. His treatment was completed after 6months of adjuvant chemotherapy with oral TS-1, which was followed by a subsequent 2 year follow-up study, without disease recurrence. Operations of synchronous cancers with 2 segmental anastomoses usually require longer surgical time and are associated with more postoperative complications compared with a single segmental anastomosis. We report a case of synchronous colorectal cancer successfully treated by laparoscopic surgery with 2 segmental anastomoses.

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity.

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. ABBREVIATIONS: ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-ß-D-maltoside; DDM: n-dodecyl-ß-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

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.

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.