Membrane Vesicles Research Articles

Fatty Acid Uptake in Klebsiella pneumoniae and the Landscape of Its Infectious Niches.

Klebsiella pneumoniae is consistently ranked among the most problematic multidrug-resistant bacterial pathogens in healthcare systems. Developing novel treatments requires a better understanding of its interaction with the host environment. Although bacteria can synthesize fatty acids, emerging findings suggest a potential preference for their acquisition from the host. Fatty acid profiling of mice revealed a dramatic increase in the level of hepatic lipids during K. pneumoniae infection. The K. pneumoniae fatty acid composition and uptake capabilities were found to be largely clonally conserved. Correlations between fatty acid uptake, outer membrane vesicle production, and cell permeability were observed, but this did not translate to alterations in cell morphology, capsule production, or antimicrobial susceptibility. Importantly, hyper-capsulation did not prevent the uptake of hydrophobic fatty acids. The uptake of a saturated fatty acid by hypervirulent K. pneumoniae isolate may provide insights into the clinical association of K. pneumoniae infections with hyperlipidemic and/or obese individuals.

The aryl hydrocarbon receptor and FOS mediate cytotoxicity induced by Acinetobacter baumannii

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

Brain and Serum Membrane Vesicle (Exosome) Profiles in Experimental Alcohol-Related Brain Degeneration: Forging the Path to Non-Invasive Liquid Biopsy Diagnostics

Background: Alcohol-related brain degeneration (ARBD) is associated with cognitive–motor impairments that can progress to disability and dementia. White matter (WM) is prominently targeted in ARBD due to chronic neurotoxic and degenerative effects on oligodendrocytes and myelin. Early detection and monitoring of WM pathology in ARBD could lead to therapeutic interventions. Objective: This study examines the potential utility of a non-invasive strategy for detecting WM ARBD using exosomes isolated from serum. Comparative analyses were made with paired tissue (Tx) and membrane vesicles (MVs) from the temporal lobe (TL). Methods: Long Evans rats were fed for 8 weeks with isocaloric liquid diets containing 37% or 0% caloric ethanol (n = 8/group). TL-Tx, TL-MVs, and serum exosomes (S-EVs) were used to examine ethanol’s effects on oligodendrocyte glycoprotein, astrocyte, and oxidative stress markers. Results: Ethanol significantly decreased the TL-Tx expression of platelet-derived growth factor receptor alpha (PDGFRA), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), glial fibrillary acidic protein (GFAP), and 8-OHdG, whereas in the TL-MVs, ethanol increased CNPase, PDGFRA, and 8-OHdG, but decreased MOG and GFAP concordantly with TL-Tx. Ethanol modulated the S-EV expression by reducing PLP, nestin, GFAP, and 4-hydroxynonenal (HNE). Conclusion: Chronic ethanol exposures differentially alter the expression of oligodendrocyte/myelin, astrocyte, and oxidative stress markers in the brain, brain MVs, and S-EVs. However, directionally concordant effects across all three compartments were limited. Future studies should advance these efforts by characterizing the relationship between ABRD and molecular pathological changes in brain WM-specific exosomes in serum.

Bacterial Membrane Vesicles: The Missing Link Between Bacterial Infection and Alzheimer Disease.

Periodontitis is a common chronic inflammatory disease, affecting approximately 19% of the global adult population. A relationship between periodontal disease and Alzheimer disease has long been recognized, and recent evidence has been uncovered to link these 2 diseases mechanistically. Periodontitis is caused by dysbiosis in the subgingival plaque microbiome, with a pronounced shift in the oral microbiota from one consisting primarily of Gram-positive aerobic bacteria to one predominated by Gram-negative anaerobes, such as Porphyromonas gingivalis. A common phenomenon shared by all bacteria is the release of membrane vesicles to facilitate biomolecule delivery across long distances. In particular, the vesicles released by P gingivalis and other oral pathogens have been found to transport bacterial components across the blood-brain barrier, initiating the physiologic changes involved in Alzheimer disease. In this review, we summarize recent data that support the relationship between vesicles secreted by periodontal pathogens to Alzheimer disease pathology.

Microdroplet-enhanced chip platform for high-throughput immunotherapy marker screening from extracellular vesicle RNAs and membrane proteins

Extracellular vesicles (EVs) are considered as promising candidates for predicting patients who respond to immunotherapy. Nevertheless, simultaneous detection of multiple EVs markers still presents significant technical challenges. In this work, we developed a high-throughput microdroplet-enhanced chip (MEC) platform, which utilizes thousands of individual microchambers (∼pL) as reactors, accelerating the detection efficiency of the CRISPR/Cas systems and increasing the sensitivity by up to 100-fold (aM level). Ten biomarkers (including 5 RNAs and 5 proteins) from patients’ EVs are successfully detected on one chip, and the comprehensive markers show increased accuracy (AUC 0.911) than the individual marker for the efficacy prediction of immunotherapy. This platform provides a high-throughput yet sensitive strategy for screening immunotherapy markers in clinical.

Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Membrane vesicles can contribute to cellulose degradation by Teredinibacter turnerae, a cultivable intracellular endosymbiont of shipworms.

Teredinibacter turnerae is a cultivable cellulolytic Gammaproteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose, and pectin and contribute to wood (lignocellulose) digestion in the shipworm gut. However, the mechanisms by which T. turnerae secretes lignocellulolytic enzymes are incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce membrane vesicles (MVs) that include a variety of proteins identified by LC-MS/MS as carbohydrate-active enzymes (CAZymes) with predicted activities against cellulose, hemicellulose, and pectin. Reducing sugar assays and zymography confirm that these MVs exhibit cellulolytic activity, as evidenced by the hydrolysis of CMC. Additionally, these MVs were enriched with TonB-dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations indicate a potential role for MVs in lignocellulose utilization by T. turnerae in the free-living state, suggest possible mechanisms for host-symbiont interaction, and may be informative for commercial applications such as enzyme production and lignocellulosic biomass conversion.

In-host intra- and inter-species transfer of blaKPC-2 and blaNDM-1 in Serratia marcescens and its local and global epidemiology

ObjectivesThe aim of this study was to investigate interspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of resistance gene blaKPC-2 in Serratia marcescens, and explore the epidemical and evolutionary characteristics of carbapenemase-producing S. marcescens (CPSM) regionally and globally. MethodsInterspecies and intraspecies transfer of blaKPC-2- or blaNDM-1 were identified by antimicrobial susceptibility testing, plasmid conjugation and curing, discovery of transposable units (TUs), outer membrane vesicles (OMVs), qPCR, whole-genome sequencing (WGS) and bioinformatic analysis. The genomic evolution of CPSM strains was explored by cgSNP and maximum-likelihood phylogenetic tree. ResultsCPSM S50079 strain, co-carrying blaKPC-2 and blaNDM-1 on one plasmid, was isolated from the blood of a patient with acute pancreatitis and could generate TUs carrying either blaKPC-2 or blaNDM-1. The interspecies transfer of blaNDM-1-carrying plasmid from Providencia rettgeri P50213, producing the identical blaNDM-1-carrying TUs, to S. marcescens S50079K, an S50079 variant via plasmid curing, was identified through blaNDM-1-harbouring plasmid conjugation and OMVs transfer. Moreover, the intraspecies transfer of blaKPC-2, mediated by IS26 from plasmid to chromosome in S50079, was also identified. In another patient, who underwent lung transplantation, interspecies transfer of blaNDM-1 carried by IncX3 plasmid was identified among S. marcescens and Citrobacter freundii as well as Enterobacter hormaechei via plasmid transfer. Furthermore, 11 CPSM from 349 non-repetitive S. marcescens strains were identified in the same hospital, and clonal dissemination, with carbapenemase evolution from blaKPC-2 to both blaKPC-2 and blaNDM-1, was found in the 8 CPSM across 4 years. Finally, the analysis of 236 global CPSM from 835 non-repetitive S. marcescens genomes, retrieved from the NCBI database, revealed long-term spread and evolution worldwide, and would cause the convergence of more carbapenemase genes. ConclusionsInterspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of resistance gene blaKPC-2 in CPSM were identified. Nosocomial and global dissemination of CPSM were revealed and more urgent surveillance was acquired.

Outer Membrane Vesicle Production by Escherichia coli Enhances Its Defense against Phage Infection.

Several studies have investigated the multifunctional characteristics of outer membrane vesicles (OMVs), but research on their role in mediating phage-bacteria interactions is limited. Employing Escherichia coli as a model, we engineered a mutant strain overproducing OMVs for protective experiments against phage infections. The addition of exogenous OMVs proved highly effective in safeguarding the bacterial host against various phages, mitigating predatory threats. Screening for phage-resistant strains and adsorption experiments revealed that inhibiting phage adsorption is a crucial pathway through which OMVs protect against phage predation. Although OMVs conferred tolerance to the phage-sensitive strains (those easily infected by phages), they could not restore the phage-resistant strains (those that effectively resist phage infection) to a sensitive phenotype. This study provides valuable insights for the future development of novel biotechnological approaches aimed at utilizing OMVs to protect fermentative strains and reduce the risk of phage contamination.

Apoptotic vesicles (apoVs) derived from fibroblast-converted hepatocyte-like cells effectively ameliorate liver fibrosis

Liver fibrosis is a serious global health issue for which effective treatment remains elusive. Chemical-induced hepatocyte-like cells (ciHeps) have emerged as an appealing source for cell transplantation therapy, although they present several challenges such as the risk of lung thromboembolism or hemorrhage. Apoptotic vesicles (apoVs), small membrane vesicles generated during the apoptosis process, have gained attention for their role in regulating various physiological and pathological processes. In this study, we generated ciHep-derived apoVs (ciHep-apoVs) and investigated their therapeutic potential in alleviating liver fibrosis. Our findings revealed that ciHep-apoVs induced the transformation of macrophages into an anti-inflammatory phenotype, effectively suppressed the activity of activated hepatic stellate cells (aHSCs), and enhanced the survival of hepatocytes. When intravenously administered to mice with liver fibrosis, ciHep-apoVs were primarily engulfed by macrophages and myofibroblasts, leading to a reduction in liver inflammation and fibrosis. Proteomic and miRNA analyses showed that ciHep-apoVs were enriched in various functional molecules that modulate crucial cellular processes, including metabolism, signaling transduction, and ECM-receptor interactions. ciHep-apoVs effectively suppressed aHSCs activity through the synergistic inhibition of glycolysis, the PI3K/AKT/mTOR pathway, and epithelial-to-mesenchymal transition (EMT) cascades. These findings highlight the potential of ciHep-apoVs as multifunctional nanotherapeutics for liver fibrosis and provide insights into the treatment of other liver diseases and fibrosis in other organs.Graphical abstract

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Mitochondrial Extracellular Vesicles: A Promising Avenue for Diagnosing and Treating Lung Diseases.

Mitochondria, pivotal organelles governing cellular biosynthesis, energy metabolism, and signal transduction, maintain dynamic equilibrium through processes such as biogenesis, fusion, fission, and mitophagy. Growing evidence implicates mitochondrial dysfunction in a spectrum of respiratory diseases including acute lung injury/acute respiratory distress syndrome, bronchial asthma, pulmonary fibrosis, chronic obstructive pulmonary disease, and lung cancer. Consequently, identifying methods capable of ameliorating damaged mitochondrial function is crucial for the treatment of pulmonary diseases. Extracellular vesicles (EVs), nanosized membrane vesicles released by cells into the extracellular space, facilitate intercellular communication by transferring bioactive substances or signals between cells or organs. Recent studies have identified abundant mitochondrial components within specific subsets of EVs, termed mitochondrial extracellular vesicles (mitoEVs), whose contents and compositions vary with disease progression. Moreover, mitoEVs have demonstrated reparative mitochondrial functions in injured recipient cells. However, a comprehensive understanding of mitoEVs is currently lacking, limiting their clinical translation prospects. This Review explores the biogenesis, classification, functional mitochondrial cargo, and biological effects of mitoEVs, with a focus on their role in pulmonary diseases. Emphasis is placed on their potential as biological markers and innovative therapeutic strategies in pulmonary diseases, offering fresh insights for mechanistic studies and drug development in various pulmonary disorders.

Exosomes of immune cell origin and their therapeutic potential for tumors

AbstractExosomes are nanoscale membrane vesicles identified by electron microscopy in 1946. They are approximately 30–150 nm in size. Originally, exosomes were thought to be used to eliminate excess components from cells to maintain their normal physiology. Later, it was found that the function and targeting of specific cellular components in exosomes have important implications for the regulation of cellular communication. In the last few years, exosomes have been linked to tumors, infections, and other diseases. New advances in cancer immunotherapy have occurred as a result of the identification of exosomes of immune cell origin, expanding the existing anticancer immune response. This article details exosomes derived from dendritic cells, T lymphocytes (CD4+ T cells, CD8+ T cells, and CAR‐T cells), natural killer cells, and their promising applications in tumor therapy.

Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Thermophilic fungus uses anthraquinones to modulate ferrous excretion, sterol-mediated endocytosis, and iron storage in response to cold stress.

To date, there are no real physiological mechanisms for iron excretion in eukaryote, and no physiological "actuator" that can control all the three fundamental biologic processes of absorption, storage, and excretion. Here, we observed that the accumulation of anthraquinones by Thermomyces dupontii under cold stress can achieve this process. Through mutation analysis, we found that mutant ΔAn deficiency in anthraquinones accumulated ferrous and total free iron due to adopting a rare lifestyle with no endocytosis but accumulation of membrane-derived vesicles. Anthraquinone complement indicated that the vesicles in ΔAn could coat the extrinsic anthraquinone-induced granules to prevent contact with the fungal interiors. Detailed chemical investigation on ΔAn led to characterization of a rare oxygen-free ergosterene with unstable nature in air as the major membrane steroid in ΔAn, suggesting hypoxia inner in ΔAn cells, consistent with dramatically low oxygen-consuming rates in ΔAn. A series of physiological and metabolic analyses indicated anthraquinones were involved in exporting ferrous and promoting formation of oxygen-containing metabolites, including ergosterols for endocytosis and iron chelators for iron storage. Moreover, we found that both the anticancer agent mitoxantrone with well-known-cardiotoxicity side effect and the major terpenoid-derived polycyclic aromatics from Danshen for treating cardiovascular disease showed potent ferrous transporting capabilities in human cancer cells. Our findings provide a novel insight into the underlying mechanisms of polycyclic aromatics in nature and pharmacology, and offer a new strategy for developing potential therapeutics and agents for membrane transport, iron homestasis, and anticold.

Enhancing protective immunity against bacterial infection via coating nano-Rehmannia glutinosa polysaccharide with outer membrane vesicles.

With the coming of the post-antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non-replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live-attenuated vaccines. Here, we developed a novel nano-vaccine by coating OMVs onto PEGylated nano-Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL-OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL-OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial-specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T-cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV-coated nano-carriers in antimicrobial immunotherapy.

Tumor-associated exosomes in cancer progression and therapeutic targets.

Exosomes are small membrane vesicles that are released by cells into the extracellular environment. Tumor-associated exosomes (TAEs) are extracellular vesicles that play a significant role in cancer progression by mediating intercellular communication and contributing to various hallmarks of cancer. These vesicles carry a cargo of proteins, lipids, nucleic acids, and other biomolecules that can be transferred to recipient cells, modifying their behavior and promoting tumor growth, angiogenesis, immune modulation, and drug resistance. Several potential therapeutic targets within the TAEs cargo have been identified, including oncogenic proteins, miRNAs, tumor-associated antigens, immune checkpoint proteins, drug resistance proteins, and tissue factor. In this review, we will systematically summarize the biogenesis, composition, and function of TAEs in cancer progression and highlight potential therapeutic targets. Considering the complexity of exosome-mediated signaling and the pleiotropic effects of exosome cargoes has challenge in developing effective therapeutic strategies. Further research is needed to fully understand the role of TAEs in cancer and to develop effective therapies that target them. In particular, the development of strategies to block TAEs release, target TAEs cargo, inhibit TAEs uptake, and modulate TAEs content could provide novel approaches to cancer treatment.

Oral Microto-Nano Genome-Editing System Enabling Targeted Delivery and Conditional Activation of CRISPR-Cas9 for Gene Therapy of Inflammatory Bowel Disease.

The potent CRISPR-Cas9 technology can correct genes in human mutated cells to achieve the treatment of multiple diseases, but it lacks safe and effective delivery systems. Herein, we proposed an oral microto-nano genome-editing system aiming at the enteric excessive level of TNF-α for specific gene therapy of inflammatory bowel disease (IBD). This editing system facilitated the assembly of Cas9/sgRNA ribonucleoprotein (RNP) into nanoclusters (NCs) through the bridging of disulfide bonds. RNP-NCs were subsequently encapsulated within inflammatory cell-targeted lipopolysaccharide-deleted outer membrane vesicles (dOMVs) sourced from Escherichia coli Nissle 1917, which were further shielded by an outer layer of calcium alginate microspheres (CAMs). By leveraging the protection effect of CAMs, the oral administration system withstood gastric acid degradation upon entry into the stomach, achieving targeted delivery to the intestines with high efficiency. As the pH gradually rose, the microscale CAMs swelled and disintegrated, releasing nanoscale RNP-NCs encapsulated in dOMVs into the intestines. These RNP-NCs@dOMVs could traverse the mucosal barrier and target inflammatory macrophages where conditionally activated Cas9/sgRNA RNPs effectively perform genomic editing of TNF-α within the nucleus. Such oral microto-nano genome-editing systems represent a promising translational platform for the treatment of IBD.

The small GTPase RAB-18 is involved in regulating development/diapause by recruiting the intestinal cholesterol transporter NCR-1 onto the apical side in Caenorhabditis elegans

RAB family proteins, which are small GTPases, are integral to the process of eukaryotic membrane trafficking. In the nematode, Caenorhabditis elegans, 31 RAB proteins have been identified through genome sequencing. Using an RNAi screen specifically targeting C. elegans rab genes, we identified multiple genes that are involved in the regulation of larval development, in particular, the rab-18 gene. Our molecular genetic studies resulted in several findings. First, RAB-18 predominantly functions in the intestine to regulate larval development by modulating steroid hormone signaling. Second, the C. elegans cholesterol transporter NCR-1 is a target of RAB-18 in the intestine. Third, the membrane trafficking of NCR-1 to the apical side in intestinal cells is particularly influenced by RAB-18. Finally, RAB-18 and NCR-1 possibly co-localize on membrane vesicles. Our study is the first to demonstrate the relationship between a RAB protein and a cholesterol transporter, in which the RAB protein probably drives the transporter to the apical membrane in the intestine to regulate cholesterol uptake. This study provides insight into the molecular mechanisms underlying human disease stemming from a transport defect of cholesterol and its derivative.

Epinecidin-1 and lactic acid synergistically inhibit Aeromonas hydrophila through membrane disruption

Epinecidin-1 (Epi-1) is an antimicrobial peptide originated from fish with various pharmacological activities but carries the risk of acquiring resistance with long-term use. In the present study, we use L-lactic acid to enhance the antibacterial activity of synthesized Epi-1 against the aquaculture and food pathogen Aeromonas hydrophila. The results showed that 5.5 mmol/L lactic acid increased the inhibitory and bactericidal activity of 25 μmol/L Epi-1 against two strains of A. hydrophila. The laser confocal images proved that lactic acid pre-treatment improved the attachment efficiency of Epi-1 in A.hydrophila cells. In addition, lactic acid enhanced the damaging effect of Epi-1 on the cell membrane of A. hydrophila, evidenced by releasing more nucleic acids, proteins, and transmembrane pH ingredients decrease and electromotive force dissipation. SEM images showed that compared with the single Epi-1 treatment, the co-treatment of Epi-1 and lactic acid caused more outer membrane vesicles (OMVs) and more severe cell deformation. These findings proved that lactic acid could enhance the efficiency of Epi-1 against A. hydrophila and shed light on new aspects to avoid resistance of pathogens against Epi-1.