Proteomics-based diagnostic peptide discovery for severe fever with thrombocytopenia syndrome virus in patients.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging infectious virus which causes severe hemorrhage, thrombocytopenia, and leukopenia, with a high fatality rate. Since there is no approved therapeutics or vaccines for SFTS, early diagnosis is essential to manage this infectious disease. METHODS: Here, we tried to detect SFTS virus in serum samples from SFTS patients by proteomic analysis. Firstly, in order to obtain the reference MS/MS spectral data of SFTS virus, medium from infected Vero cell culture was used for shotgun proteomic analysis. Then, tryptic peptides in sera from SFTS patients were confirmed by comparative analysis with the reference MS/MS spectral data of SFTS virus. RESULTS: Proteomic analysis of culture medium successfully discovered tryptic peptides from all the five antigen proteins of SFTS virus. The comparative spectral analysis of sera of SFTS patients revealed that the N-terminal tryptic peptide of the nucleocapsid (N) protein is the major epitope of SFTS virus detected in the patient samples. The prevalence of the peptides was strongly correlated with the viral load in the clinical samples. CONCLUSIONS: Proteomic analysis of SFTS patient samples revealed that nucleocapsid (N) protein is the major antigen proteins in sera of SFTS patients and N-terminal tryptic peptide of the N protein might be a useful proteomic target for direct detection of SFTS virus. These findings suggest that proteomic analysis could be an alternative tool for detection of pathogens in clinical samples and diagnosis of infectious diseases.

Serum proteomics of severe fever with thrombocytopenia syndrome patients.

BACKGROUND: Dabie bandavirus, also termed as severe fever with thrombocytopenia syndrome virus (SFTSV), was first isolated in China in 2010. At this time, the virus was found to have spread to South Korea, Japan, and other countries. A high case fatality rate is reported for SFTS, ranging from 12-50% within various sources. Several omics for clinical studies among SFTS patients as well as studies of cultured SFTSV have attempted to characterize the relevant molecular biology and epidemiology of the disease. However, a global serum proteomics analysis among SFTS patients has not yet been reported to date. METHODS: In the current study, we evaluated comparative serum proteomics among SFTS patients (eight recovered patients and three deceased patients) with the goal of identifying the protein expression patterns associated with the clinical manifestations of SFTS. RESULTS: The proteomic results in the current study showed that the coagulation factor proteins, protein S and protein C, were statistically significantly downregulated among the deceased patients. Downregulation of the complement system as well as prolonged neutrophil activation were also observed. Additionally, the downstream proteins of tumour necrosis factor alpha, neutrophil-activating cytokine, and interleukin-1ß, an inflammatory cytokine, were overexpressed. CONCLUSIONS: Thrombocytopenia and multiple organ failure are the major immediate causes of death among SFTS patients. In this study, serum proteomic changes related to thrombocytopenia, abnormal immune response, and inflammatory activation were documented in SFTS patients. These findings provide useful information for understanding the clinical manifestations of SFTS.

Transmission Electron Microscopy Confirmation of Orientia tsutsugamushi in Human Bile.

Scrub typhus, the third most frequently reported infectious disease in South Korea, causes serious public health problems. In 2019, we collected a bile specimen from a patient with scrub typhus through percutaneous transhepatic gallbladder drainage and performed transmission electron microscopy to confirm the ultrastructure of Orientia tsutsugamushi.

Proteomic Analysis of Urothelium of Rats with Detrusor Overactivity Induced by Bladder Outlet Obstruction.

Overactive bladder (OAB) syndrome is a condition that has four symptoms: urgency, urinary frequency, nocturia, and urge incontinence and negatively affects a patient's life. Recently, it is considered that the urinary bladder urothelium is closely linked to pathogenesis of OAB. However, the mechanisms of pathogenesis of OAB at the molecular level remain poorly understood, mainly because of lack of modern molecular analysis. The goal of this study is to identify a potential target protein that could act as a predictive factor for effective diagnosis and aid in the development of therapeutic strategies for the treatment of OAB syndrome. We produced OAB in a rat model and performed the first proteomic analysis on the mucosal layer (urothelium) of the bladders of sham control and OAB rats. The resulting data revealed the differential expression of 355 proteins in the bladder urothelium of OAB rats compared with sham subjects. Signaling pathway analysis revealed that the differentially expressed proteins were mainly involved in the inflammatory response and apoptosis. Our findings suggest a new target for accurate diagnosis of OAB that can provide essential information for the development of drug treatment strategies as well as establish criteria for screening patients in the clinical environment.

Clinical proteomic analysis of scrub typhus infection.

BACKGROUND: Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. METHODS: Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. RESULTS: Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi, and normal expression was largely rescued by antibiotic treatment. CONCLUSIONS: This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202.

BACKGROUND: Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis. METHODS: Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed. RESULTS: OMV secretion was increased > twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which ß-lactamase OXA-23, various proteases, outer membrane proteins, ß-barrel assembly machine proteins, peptidyl-prolyl cis-trans isomerases and inherent prophage head subunit proteins were significantly upregulated. CONCLUSION: In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

Role of Tbx2 in defining the territory of the pronephric nephron.

Despite extensive study of the development of the nephron, which is the functional unit of the kidney, the molecular mechanisms underlying the determination of nephron size remain largely unknown. Using the Xenopus pronephros, we demonstrate here that Tbx2, a T-box transcriptional repressor, functions to demarcate the territory of the pronephric nephron. Tbx2 is specifically expressed around three distinct components of the pronephric nephron: the tubule, duct and glomus. Gain of function of Tbx2 inhibits nephric mesoderm formation. Conversely, Tbx2 loss of function expands the boundary of each component of the pronephric nephron, resulting in an enlarged pronephros. BMP signals induce Tbx2 in the non-nephric mesoderm, which inhibits the expression of the nephric markers Hey1 and Gremlin. Importantly, these pronephric molecules repress Tbx2 expression by antagonizing BMP signals in the nephric mesoderm. These results suggest that the negative regulatory loops between BMP/Tbx2 and Gremlin or Hey1 are responsible for defining the territory of the pronephric nephron.

Proteogenomic characterization of antimicrobial resistance in extensively drug-resistant Acinetobacter baumannii DU202.

OBJECTIVES: To determine the genomic sequence of extensively drug-resistant Acinetobacter baumannii DU202 and to perform proteomic characterization of antibiotic resistance in this strain using genome data. METHODS: The genome sequence of A. baumannii DU202 was determined using the Hi-Seq 2000 system and comparative analysis was performed to determine the unique characteristics of A. baumannii DU202. Previous proteomic results from the cell wall membrane fraction by one-dimensional electrophoresis and liquid chromatography combined with mass spectrometry analysis (1DE-LC-MS/MS), using the A. baumannii ATCC 17978 genome as a reference, were reanalysed to elucidate the resistance mechanisms of A. baumannii DU202 using strain-specific genome data. Additional proteomic data from the cytosolic fraction were also analysed. RESULTS: The genome of A. baumannii DU202 consists of 3660 genes and is most closely related to the Korean A. baumannii 1656-2 strain. More than 144 resistance genes were annotated in the A. baumannii DU202 genome, of which 72 that encoded proteins associated with antibiotic resistance were identified in the proteomic analysis of A. baumannii DU202 cultured in tetracycline, imipenem and Luria-Bertani broth (control) medium. Strong induction of ß-lactamases, a multidrug resistance efflux pump and resistance-nodulation-cell division (RND) multidrug efflux proteins was found to be important in the antibiotic resistance responses of A. baumannii DU202. CONCLUSIONS: Combining genomic and proteomic methods provided comprehensive information about the unique antibiotic resistance responses of A. baumannii DU202.

Platycodi Radix Extract Prevents Hepatic Steatosis by Enhancing Bile Acid Synthesis in a High-Fat Diet-Induced Fatty Liver Mouse Model.

We aimed to identify the mechanism underlying the preventive effects of non-alcoholic fatty liver disease (NAFLD) through Platycodi Radix consumption using liver proteomic and bioinformatic analysis. C57BL/6J mice were categorized into three groups: those receiving a standard chow diet (NCD), those on a high-fat diet (HFD), and those on an HFD supplemented with 5% Platycodi Radix extract (PRE). After a 12-week period, PRE-fed mice exhibited a noteworthy prevention of hepatic steatosis. Protein identification and quantification in liver samples were conducted using LC-MS/MS. The identified proteins were analyzed through Ingenuity Pathway Analysis software, revealing a decrease in proteins associated with FXR/RXR activation and a concurrent increase in cholesterol biosynthesis proteins in the PRE-treated mouse liver. Subsequent network analysis predicted enhanced bile acid synthesis from these proteins. Indeed, the quantity of bile acids, which was reduced in HFD conditions, increased in the PRE group, accompanied by an elevation in the expression of synthesis-related proteins. Our findings suggest that the beneficial effects of PRE in preventing hepatic steatosis may be mediated, at least in part, through the modulation of FXR/RXR activation, cholesterol biosynthesis, and bile acid synthesis pathways.

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver.

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress-induced, nine proteins with inflammatory stress-induced, and 14 proteins with endoplasmic reticulum stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c-Jun amino-terminal kinase-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

ß-Arrestin 1 mediates non-canonical Wnt pathway to regulate convergent extension movements.

ß-Arrestins are multifaceted proteins that play critical roles in termination of G protein-coupled receptor (GPCR) signaling by inducing its desensitization and internalization as well as in facilitation of many intracellular signaling pathways. Here, we examine using Xenopus embryos whether ß-arrestin 1 might act as a mediator of ß-catenin-independent Wnt (non-canonical) signaling. Xenopus ß-arrestin 1 (xßarr1) is expressed in the tissues undergoing extensive cell rearrangements in early development. Gain- and loss-of-function analyses of xßarr1 revealed that it regulates convergent extension (CE) movements of mesodermal tissue with no effect on cell fate specification. In addition, rescue experiments showed that xßarr1 controls CE movements downstream of Wnt11/Fz7 signal and via activation of RhoA and JNK. In line with this, xßarr1 associated with key Wnt components including Ryk, Fz, and Dishevelled. Furthermore, we found that xßarr1 could recover CE movements inhibited by xßarr2 knockdown or its endocytosis defective mutant. Overall, these results suggest that ß-arrestin 1 and 2 share interchangeable endocytic activity to regulate CE movements downstream of the non-canonical Wnt pathway.

Ultrasensitive biosensing platform for Mycobacterium tuberculosis detection based on functionalized graphene devices.

Tuberculosis (TB) has high morbidity as a chronic infectious disease transmitted mainly through the respiratory tract. However, the conventional diagnosis methods for TB are time-consuming and require specialists, making the diagnosis of TB with point-of-care (POC) detection difficult. Here, we developed a graphene-based field-effect transistor (GFET) biosensor for detecting the MPT64 protein of Mycobacterium tuberculosis with high sensitivity as a POC detection platform for TB. For effective conjugation of antibodies, the graphene channels of the GFET were functionalized by immobilizing 1,5-diaminonaphthalene (1,5-DAN) and glutaraldehyde linker molecules onto the graphene surface. The successful immobilization of linker molecules with spatial uniformity on the graphene surface and subsequent antibody conjugation were confirmed by Raman spectroscopy and X-ray photoelectron spectroscopy. The GFET functionalized with MPT64 antibodies showed MPT64 detection with a detection limit of 1 fg/mL in real-time, indicating that the GFET biosensor is highly sensitive. Compared to rapid detection tests (RDT) and enzyme-linked immunosorbent assays, the GFET biosensor platform developed in this study showed much higher sensitivity but much smaller dynamic range. Due to its high sensitivity, the GFET biosensor platform can bridge the gap between time-consuming molecular diagnostics and low-sensitivity RDT, potentially aiding in early detection or management of relapses in infectious diseases.

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

Analysis of the endoplasmic reticulum subproteome in the livers of type 2 diabetic mice.

Type 2 diabetes is a chronic metabolic disease that results from insulin resistance in the liver, muscle, and adipose tissue and relative insulin deficiency. The endoplasmic reticulum (ER) plays a crucial role in the regulation of the cellular response to insulin. Recently, ER stress has been known to reduce the insulin sensitivity of the liver and lead to type 2 diabetes. However, detailed mechanisms of ER stress response that leads to type 2 diabetes remains unknown. To obtain a global view of ER function in type 2 diabetic liver and identify proteins that may be responsible for hepatic ER stress and insulin resistance, we performed proteomics analysis of mouse liver ER using nano UPLC-MSE. A total of 1584 proteins were identified in control C57 and type 2 diabetic db/db mice livers. Comparison of the rER and sER proteomes from normal mice showed that proteins involved in protein synthesis and metabolic process were enriched in the rER, while those associated with transport and cellular homeostasis were localized to the sER. In addition, proteins involved in protein folding and ER stress were found only in the rER. In the livers of db/db mice, however, the functions of the rER and sER were severely disrupted, including the capacity to resolve ER stress. These results provide new insight into the research on hepatic insulin resistance and type 2 diabetes and are suggestive of the potential use of the differentially expressed hepatic ER proteins as biomarkers for hepatic insulin resistance and type 2 diabetes.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Computational Method-Based Optimization of Carbon Nanotube Thin-Film Immunosensor for Rapid Detection of SARS-CoV-2 Virus.

The recent global spread of COVID-19 stresses the importance of developing diagnostic testing that is rapid and does not require specialized laboratories. In this regard, nanomaterial thin-film-based immunosensors fabricated via solution processing are promising, potentially due to their mass manufacturability, on-site detection, and high sensitivity that enable direct detection of virus without the need for molecular amplification. However, thus far, thin-film-based biosensors have been fabricated without properly analyzing how the thin-film properties are correlated with the biosensor performance, limiting the understanding of property-performance relationships and the optimization process. Herein, the correlations between various thin-film properties and the sensitivity of carbon nanotube thin-film-based immunosensors are systematically analyzed, through which optimal sensitivity is attained. Sensitivities toward SARS-CoV-2 nucleocapsid protein in buffer solution and in the lysed virus are 0.024 [fg/mL]-1 and 0.048 [copies/mL]-1, respectively, which are sufficient for diagnosing patients in the early stages of COVID-19. The technique, therefore, can potentially elucidate complex relationships between properties and performance of biosensors, thereby enabling systematic optimization to further advance the applicability of biosensors for accurate and rapid point-of-care (POC) diagnosis.

Identification of a Novel Antigen for Serological Diagnosis of Scrub Typhus.

Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection.

Streptococcus equi subspecies equi is a pathogenic bacterium that causes strangles, a highly contagious respiratory infection in horses and other equines. The limitations of current vaccines against S. equi infection warrants the development of an affordable, safe, and effective vaccine. Because gram-positive extracellular vesicles (EVs) transport various immunogenic antigens, they are attractive vaccine candidates. Here, we purified the EVs of S. equi ATCC 39506 and evaluated them as a vaccine candidate against S. equi infection in mice. As an initial step, comparative proteomic analysis was performed to characterize the functional features of the EVs. Reverse vaccinology and knowledge-based annotations were then used to screen potential vaccine candidates (PVCs) for S. equi ATCC 39506. Finally, 32 PVCs were found to be enriched in the EV fraction, suggesting the usefulness of this fraction as a vaccine. Importantly, a significantly higher survival rate after S. equi infection was detected in mice immunized with S. equi-derived EVs via the intraperitoneal route than in mice immunized with heat-killed bacteria. Of note, immunoprecipitation-mass spectrometry results validated various immunogenic antigens within the EV proteome. In conclusion, our results suggest that S. equi-derived EVs can serve as a vaccine candidate against S. equi infection.