Surface-associated Proteins Research Articles

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of Gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Formation of multinucleated osteoclasts depends on an oxidized species of cell surface-associated La protein.

The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells - generated by an increased number of cell fusion events - have higher resorptive activity. We find that osteoclast fusion and bone resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La's unique regulatory role in osteoclast multinucleation and function is controlled by an ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.

Formation of multinucleated osteoclasts depends on an oxidized species of cell surface-associated La protein

The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells – generated by an increased number of cell fusion events – have higher resorptive activity. We find that osteoclast fusion and bone resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La’s unique regulatory role in osteoclast multinucleation and function is controlled by an ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

A Flagellin-Adjuvanted Trivalent Mucosal Vaccine Targeting Key Periodontopathic Bacteria.

Periodontal disease (PD) is caused by microbial dysbiosis and accompanying adverse inflammatory responses. Due to its high incidence and association with various systemic diseases, disease-modifying treatments that modulate dysbiosis serve as promising therapeutic approaches. In this study, to simulate the pathophysiological situation, we established a "temporary ligature plus oral infection model" that incorporates a temporary silk ligature and oral infection with a cocktail of live Tannerella forsythia (Tf), Pophyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn) in mice and tested the efficacy of a new trivalent mucosal vaccine. It has been reported that Tf, a red complex pathogen, amplifies periodontitis severity by interacting with periodontopathic bacteria such as Pg and Fn. Here, we developed a recombinant mucosal vaccine targeting a surface-associated protein, BspA, of Tf by genetically combining truncated BspA with built-in adjuvant flagellin (FlaB). To simultaneously induce Tf-, Pg-, and Fn-specific immune responses, it was formulated as a trivalent mucosal vaccine containing Tf-FlaB-tBspA (BtB), Pg-Hgp44-FlaB (HB), and Fn-FlaB-tFomA (BtA). Intranasal immunization with the trivalent mucosal vaccine (BtB + HB + BtA) prevented alveolar bone loss and gingival proinflammatory cytokine production. Vaccinated mice exhibited significant induction of Tf-tBspA-, Pg-Hgp44-, and Fn-tFomA-specific IgG and IgA responses in the serum and saliva, respectively. The anti-sera and anti-saliva efficiently inhibited epithelial cell invasion by Tf and Pg and interfered with biofilm formation by Fn. The flagellin-adjuvanted trivalent mucosal vaccine offers a novel method for modulating dysbiotic bacteria associated with periodontitis. This approach leverages the adjuvant properties of flagellin to enhance the immune response, aiming to restore a balanced microbial environment and improve periodontal health.

Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Immunogenicity and Protective Capacity of Sugar ABC Transporter Substrate-Binding Protein against Streptococcus suis Serotype 2, 7 and 9 Infection in Mice.

Background:Streptococcus suis (S. suis) is a Gram-positive bacterium that causes substantial disease in pigs. S. suis is also an emerging zoonoses in humans, primarily in Asia, through the consumption of undercooked pork and the handling of infected pig meat as well as carcasses. The complexity of S. suis epidemiology, characterized by the presence of multiple bacterial serotypes and strains with diverse sequence types, identifies a critical need for a universal vaccine with the ability to confer cross-protective immunity. Highly conserved immunogenic proteins are generally considered good candidate antigens for subunit universal vaccines. Methods: In this study, the cross-protection of the sugar ABC transporter substrate-binding protein (S-ABC), a surface-associated immunogenic protein of S. suis, was examined in mice for evaluation as a universal vaccine candidate. Results: S-ABC was shown to be highly conserved, with 97% amino acid sequence identity across 31 S. suis strains deposited in GenBank. Recombinantly expressed S-ABC (rS-ABC) was recognized via rabbit sera specific to S. suis serotype 2. The immunization of mice with rS-ABC induced antigen-specific antibody responses, as well as IFN-γ and IL-4, in multiple organs, including the lungs. rS-ABC immunization conferred high (87.5% and 100%) protection against challenges with S. suis serotypes 2 and 9, demonstrating high cross-protection against these serotypes. Protection, albeit lower (50%), was also observed in mice challenged with S. suis serotype 7. Conclusions: These data identify S-ABC as a promising antigenic target within a universal subunit vaccine against S. suis.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Colloidal gold based immunochromatographic detection of Mycoplasmopsis synoviae infection and its prevalence in avian species of Karachi, Pakistan

Avian mycoplasmosis is an infection that commonly prevails in birds, particularly in poultry chickens. Among mycoplasmosis causing organisms, Mycoplasmopsis synoviae is a predominant and lethal pathogen to the aves. Considering the increased incidence of infections by M. synoviae, the prevalence of M. synoviae was deduced in poultry chickens and fancy birds of Karachi region. The lungs and tracheal samples from chicken and dead fancy birds and swab samples from live fancy birds were collected and investigated by amplifying 16 s rRNA gene of M. synoviae. Biochemical characteristics of M. synoviae was also evaluated. Furthermore, surface-associated membrane proteins, that represent key antigens for diagnosis of M. synoviae infection was extracted by Triton X- 114 method. Results showed that M. synoviae was detected more frequently in lungs than in trachea, that could be due to its invasion capacity and tissue affinity. SDS PAGE analysis of extracted membrane proteins showed two prominent hydrophobic proteins of different molecular mass including proteins of 150 and 50 kDa. Protein of 150 kDa was purified by size exclusion chromatography and it exhibited agglutinogen activity. Purified protein was used in the development of one-step immunochromatographic (ICT) assay for the detection of antibodies against M. synoviae using gold nanoparticles coated with polyclonal antibodies. Low levels of antibodies were detected by the developed ICT kit, which has 88% sensitivity with 92% specificity.

Temporal Tracking of Insulin Action on the Cell Surface of Proteins at a Resolution of Ten Seconds.

The ligand-receptor signaling occurring on the cell surface governs cell growth, proliferation, and survival via rapidly triggering a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling at a temporal resolution of ten seconds. By this innovation, about 1022 cell surface-associated proteins were reproducibly identified and quantified. It is noteworthy that, upon a model ligand insulin stimulus, a few rapid-responding proteins at 10 s to 2 min were identified, e.g., CNNM3. Moreover, temporal response patterns were established for the members of GLUT4 storage vesicles (GSVs; responsible for glucose transportation) and confirmed with five known GSV proteins. This pattern was then exploited to uncover seven new regulatory proteins (LDLR, HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a powerful surface proteomic tool to decipher rapid signaling of cell-surface proteins and to uncover new subunits involved in rapidly trafficking vesicles.

Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.

Unusual carriage of virulence genes sasX/sesI/shsA by nosocomial Staphylococcus haemolyticus from Brazil.

Background: Staphylococcus haemolyticus is an emerging threat in the nosocomial environmentbut only some virulence factors are known. Materials & methods: The frequency of the sasX gene (or orthologues sesI/shsA), encoding an invasiveness-related surface-associated protein,in S. haemolyticus was detectedin different hospitals in Rio de Janeiro. Results: 9.4% of strains were sasX/sesI/shsA-positive, some were in the context of the ΦSPβ-like prophage and devoid of CRISPR systems, indicating potential transferability of their virulence genes. Gene sequencing evidenced that Brazilian S. haemolyticus harbored sesI, instead of the usual sasX, while S. epidermidis had sasX instead of sesI, suggesting horizontal acquisition. Conclusion: The contexts of Brazilian sasX/sesI/shsA favor transfer, which is alarming given the difficulty in treating infections caused by S. haemolyticus.

The Pseudomonas aeruginosa Biofilm Matrix Protein CdrA Has Similarities to Other Fibrillar Adhesin Proteins.

The ability of bacteria to adhere to each other and both biotic and abiotic surfaces is key to biofilm formation, and one way that bacteria adhere is using fibrillar adhesins. Fibrillar adhesins share several key characteristics, including (i) they are extracellular, surface-associated proteins, (ii) they contain an adhesive domain as well as a repetitive stalk domain, and (iii) they are either a monomer or homotrimer (i.e., identical, coiled-coil) of a high molecular weight protein. Pseudomonas aeruginosa uses the fibrillar adhesin called CdrA to promote bacterial aggregation and biofilm formation. Here, the current literature on CdrA is reviewed, including its transcriptional and posttranslational regulation by the second messenger c-di-GMP as well as what is known about its structure and ability to interact with other molecules. I highlight its similarities to other fibrillar adhesins and discuss open questions that remain to be answered toward a better understanding of CdrA.

Lipid Droplets from Plants and Microalgae: Characteristics, Extractions, and Applications.

Plant and algal LDs are gaining popularity as a promising non-chemical technology for the production of lipids and oils. In general, these organelles are composed of a neutral lipid core surrounded by a phospholipid monolayer and various surface-associated proteins. Many studies have shown that LDs are involved in numerous biological processes such as lipid trafficking and signaling, membrane remodeling, and intercellular organelle communications. To fully exploit the potential of LDs for scientific research and commercial applications, it is important to develop suitable extraction processes that preserve their properties and functions. However, research on LD extraction strategies is limited. This review first describes recent progress in understanding the characteristics of LDs, and then systematically introduces LD extraction strategies. Finally, the potential functions and applications of LDs in various fields are discussed. Overall, this review provides valuable insights into the properties and functions of LDs, as well as potential approaches for their extraction and utilization. It is hoped that these findings will inspire further research and innovation in the field of LD-based technology.

Vaccinomics to design a multi-epitope-based vaccine against monkeypox virus using surface-associated proteins

In 2022, the ongoing multi-country outbreak of monkeypox virus—now occurring outside Africa, too is a global health concern. Monkeypox is a zoonotic virus, which causes disease mainly in animals, and then it is transferred to humans. Recently, in the monkeypox epidemic, a large number of human cases emerged while the global health community worked to tackle the outbreak and save lives. Herein, a multi-epitope-based vaccine is designed against monkeypox virus using two surface-associated proteins: MPXVgp002 accession number > YP_010377003.1 and MPXVgp008 accession number > YP_010377007.1 proteins. These proteins were utilized for B- and T-cell epitopes prediction. The epitopes were further screened, and the screen filtered KCKDNEYRSR, RSCNTTHNR, and RTRRETGAS with the antigenicity scores of 0.5279, 0.5604, and 0.7628, respectively. Overall, the epitopes can induce immunity in 99.74% population of the world. Further, GPGPG linkers were used for joining the epitopes and EAAAK linker was used for adjuvant attachment. It has a three-dimensional structure modelled for retaining the structural stability. Three pairs of amino acid residues that were able to make disulfide bonds were chosen: Gly1-Ser82, Cys7-Tyr10, and Phe51-Ile55. Molecular docking of vaccine was done with toll-like receptors, viz., 2, 3, 4, and 8 immune cell receptors. The docking results revealed that the vaccine as potential molecule due to its better binding affinity with toll-like receptors 2, 3, 4 and 8. Top complex in docking in with each receptor was selected based on lowest energy scores— −888.7 kcal/mol (TLR-2), −976.3 kcal/mol (TLR-3), −801.9 kcal/mol (TLR-4), and −955.4 kcal/mol (TLR-4)—were subjected to simulation. The docked complexes were evaluated in 500 ns of MD simulation. Throughout the simulation time, no significant deviation occurred. This confirmed that the vaccine as potential vaccine candidate to interact with immune cell receptors. This interaction is important for the immune system activation. In conclusion, the proposed vaccine construct against monkeypox could induce an effective immune response and speed up the vaccine development process. However, the study is completely based on the computational approach, hence, the experimental validation is required. Communicated by Ramaswamy H. Sarma

Cell surface-associated protein elongation factor Tu interacts with fibronectin mediating the adhesion of Lactobacillus plantarum HC-2 to Penaeus vannamei intestinal epithelium and inhibiting the apoptosis induced by LPS and pathogen in Caco-2 cells

The adherent colonization of lactic acid bacteria in the animal intestine is the basis for their probiotic effect, and their bacteria surface proteins play an important role in this process. Previous work has demonstrated that Lactobacillus plantarum HC-2 can adhere and colonize the intestine of Penaeus vannamei, modulate the intestinal immune response and microbial diversity, protect the intestinal tissues from pathogenic damage, and improve the protection rate of shrimp. The aim of this work was to identify adhesion molecules on the surface of HC-2 and its adhesion receptors in the intestinal epithelium of shrimp. The elongation factor Tu (EF-Tu) on the surface of HC-2 was found to interact with Fibronectin (Fib) in the shrimp intestine by immunoblotting and yeast two-hybrid assays, and this interaction relationship was verified by immunoprecipitation (Co-IP). The adhesion of HC-2 to Caco-2 cells could be blocked via EF-Tu antibody confinement, and the adhesion of Fib to HC-2 could be blocked by Fib antibody confinement. Expression of Fib on the surface of HEK293T cells revealed a significant increase in the adhesion rate of HC-2 to HEK293T cells. Using immunofluorescence, a significant reduction in HC-2 adhesion to the intestine of shrimp was observed after blocking the Fib site in the shrimp intestine, particularly in Vibrio parahaemolyticus E1-infected intestines. In addition, the recombinant protein rEF-Tu was found to promote the growth of Caco-2 cells in a certain concentration range and significantly inhibit the apoptosis induced by LPS, Staphylococcus aureus and V. parahaemolyticus E1. Our results indicate that EF-Tu might participate in gut immunity and homeostasis, through its binding to the shrimp intestinal cells and inhibiting apoptosis.

Synthetic selenium nanoparticles as co-adjuvant improved immune responses against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-acquired infections worldwide, which is resistant to many antibiotics, resulting in significant mortality in societies. Vaccination is a well-known approach to preventing disease. Autolysin, a surface-associated protein in S. aureus with multiple functions, is a suitable candidate for vaccine development. As a co-adjuvant, selenium nanoparticles (SeNPs) can increase the immune system, presumably resulting in increased vaccine efficacy. The present study evaluated the immunogenicity and defense of recombinant autolysin formulated in SeNPs and Alum adjuvants against MRSA. r-Autolysin was expressed and purified by the Ni-NTA affinity chromatography. SeNPs were synthetically obtained from sodium dioxide, followed by an assessment of shape and size using SEM and DLS. Balb/c mice were injected subcutaneously with 20mg of r-autolysin formulated in Alum and SeNps adjuvants three times with the proper control group in 2 weeks intervals. Cytokine profile and isotyping ELISA were conducted to determine the type of induced immunity. Opsonophagocytosis tests assessed the functional activity of the vaccine, and the bacterial burden from the infected tissues was determined. Results showed that mice receiving SeNps and r-Autolysin had higher levels of total IgG and isotypes (IgG1 and IgG2a) and increased cytokine levels (IFN-γ, TNF-α, IL-12, and IL-4) as compared with those only receiving autolysin and PBS as a control. More importantly, mice immunized with SeNps and r-Autolysin exhibited a decrease in mortality and bacterial burden compared to the control group. We concluded that SeNps could stimulate immune responses and can be used as an adjuvant element in vaccine formulation.

Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test.

Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface-associated proteins on EVs using a multiparametric microarray-based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs. Previously, this analysis has been limited to measuring only one fluorescent signal per analysis. The analysis relies on antibodies printed onto a solid surface, for catching the EVs carrying the specific surface or surface-associated proteins, and on the subsequent fluorescent detection. For the optimization of detection, two antibodies with attached Cy3 or Cy5 were added to various combinations of the EV surface or surface-associated proteins: CD9, CD63, CD81, flotillin-1, and HSP90. In this study, the EV surface or surface-associated proteins were analyzed in human plasma from six healthy subjects. Changes observed in signal intensities from Cy3 and Cy5 related specifically to these combinations and allowed for a comparison of the two different fluorescent signals. When comparing the results, it was observed that it is possible to measure the EV surface or surface-associated proteins at both 532nm (Cy3) and 635nm (Cy5) simultaneously without a significant change in signals from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis, thereby more quickly finding complex biomarker patterns in a sample.

Membrane-Sensing Peptides for Extracellular Vesicle Analysis.

Analytical platforms for small extracellular vesicle (sEV) high-throughput analysis are highly desirable. These bionanoparticles present fairly distinctive lipid membrane features including high curvature, lipid-packing defects, and a relative abundance in lipids. sEV membrane could be considered as a "universal" marker, complementary or alternative to traditional surface-associated proteins. Here, we describe the use of membrane-sensing peptides as a new, highly efficient ligand to directly integrate sEV capturing and analysis on a microarray platform.

Evaluating complete surface-associated and secretory proteome of Leishmania donovani for discovering novel vaccines and diagnostic targets.

The protozoa Leishmania donovani causes visceral leishmaniasis (kala-azar), the third most common vector-borne disease. The visceral organs, particularly the spleen, liver, and bone marrow, are affected by the disease. The lack of effective treatment regimens makes curing and eradicating the disease difficult. The availability of complete L. donovani genome/proteome data allows for the development of specific and efficient vaccine candidates using the reverse vaccinology method, while utilizing the unique sequential and structural features of potential antigenic proteins to induce protective T cell and B cell responses. Such shortlisted candidates may then be tested quickly for their efficacy in the laboratory and later in clinical settings. These antigens will also be useful for designing antigen-based next-generation sero-diagnostic assays. L. donovani's cell surface-associated proteins and secretory proteins are among the first interacting entities to be exposed to the host immune machinery. As a result, potential antigenic epitope peptides derived from these proteins could serve as competent vaccine components. We used a stepwise filtering-based in silico approach to identify the entire surface-associated and secretory proteome of L. donovani, which may provide rationally selected most exposed antigenic proteins. Our study identified 12 glycosylphosphatidylinositol-anchored proteins, 45 transmembrane helix-containing proteins, and 73 secretory proteins as potent antigens unique to L. donovani. In addition, we used immunoinformatics to identify B and T cell epitopes in them. Out of the shortlisted surface-associated and secretory proteome, 66 protein targets were found to have the most potential overlapping B cell and T cell epitopes (linear and conformational; MHC class I and MHC class II).