ABSTRACT
An important element of antibody-guided vaccine design is the use of neutralizing or opsonic monoclonal antibodies to define protective epitopes in their native three-dimensional conformation. Here, we demonstrate a multimodal mass spectrometry-based strategy for in-depth characterization of antigen-antibody complexes to enable the identification of protective epitopes using the cytolytic exotoxin Streptolysin O (SLO) from Streptococcus pyogenes as a showcase. We first discovered a monoclonal antibody with an undisclosed sequence capable of neutralizing SLO-mediated cytolysis. The amino acid sequence of both the antibody light and the heavy chain was determined using mass-spectrometry-based de novo sequencing, followed by chemical cross-linking mass spectrometry to generate distance constraints between the antibody fragment antigen-binding region and SLO. Subsequent integrative computational modeling revealed a discontinuous epitope located in domain 3 of SLO that was experimentally validated by hydrogen-deuterium exchange mass spectrometry and reverse engineering of the targeted epitope. The results show that the antibody inhibits SLO-mediated cytolysis by binding to a discontinuous epitope in domain 3, likely preventing oligomerization and subsequent secondary structure transitions critical for pore-formation. The epitope is highly conserved across >98% of the characterized S. pyogenes isolates, making it an attractive target for antibody-based therapy and vaccine design against severe streptococcal infections.
Subject(s)
Bacterial Proteins , Epitopes , Mass Spectrometry , Streptococcus pyogenes , Streptolysins , Streptococcus pyogenes/immunology , Streptococcus pyogenes/chemistry , Streptolysins/chemistry , Streptolysins/immunology , Streptolysins/metabolism , Bacterial Proteins/immunology , Bacterial Proteins/chemistry , Epitopes/immunology , Epitopes/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Amino Acid Sequence , Models, MolecularABSTRACT
Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.
Subject(s)
Bacterial Proteins , Bacterial Toxins , Hemolysin Proteins , Hemolysis , Streptococcus pneumoniae , Streptolysins , Streptolysins/metabolism , Streptolysins/chemistry , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/antagonists & inhibitors , Streptococcus pneumoniae/drug effects , Bacterial Toxins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/antagonists & inhibitors , Hemolysis/drug effects , Hemolysin Proteins/metabolism , Hemolysin Proteins/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , A549 Cells , Cholesterol/metabolism , Cryoelectron Microscopy , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Virulence Factors/metabolismABSTRACT
Pore-forming proteins (PFPs) are produced by various organisms, including pathogenic bacteria, and form pores within the target cell membrane. Streptolysin O (SLO) is a PFP produced by Streptococcus pyogenes and forms high-order oligomers on the membrane surface. In this prepore state, multiple α-helices in domain 3 of each subunit exist as unfolded structures and transiently interact with each other. They subsequently transition into transmembrane ß-hairpins (TMHs) and form pores with diameters of 20-30 nm. However, in this pore formation process, the trigger of the transition in a subunit and collaboration between subunits remains elusive. Here, I observed the dynamic pore formation process using high-speed atomic force microscopy. During the oligomer transition process, each subunit was sequentially inserted into the membrane, propagating along the oligomer in a domino-like fashion (chain reaction). This process also occurred on hybrid oligomers containing wildtype and mutant subunits, which cannot insert into the membrane because of an introduced disulfide bond. Furthermore, propagation still occurred when an excessive force was added to hybrid oligomers in the prepore state. Based on the observed chain reactions, I estimate the free energies and forces that trigger the transition in a subunit. Furthermore, I hypothesize that the collaboration between subunits is related to the structure of their TMH regions and interactions between TMH-TMH and TMH-lipid molecules.
Subject(s)
Bacterial Proteins , Streptolysins , Streptolysins/analysis , Streptolysins/chemistry , Streptolysins/metabolism , Cell Membrane/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistryABSTRACT
The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.
Subject(s)
Cell Membrane Permeability/drug effects , Drug Delivery Systems/methods , Molecular Biology/methods , Streptolysins/pharmacokinetics , T-Lymphocytes/drug effects , Adenosine Triphosphate/administration & dosage , Adenosine Triphosphate/pharmacokinetics , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacokinetics , Cell Separation , Fluorescent Antibody Technique , Immunoblotting , Lymphocyte Activation , Mice , Molecular Biology/instrumentation , Receptors, Antigen, T-Cell/metabolism , Spleen/cytology , Streptolysins/chemistry , T-Lymphocytes/metabolismABSTRACT
Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in â¼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors.
Subject(s)
Streptolysins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA-Directed RNA Polymerases/metabolism , Humans , Liposomes/chemistry , Liposomes/metabolism , RNA/biosynthesis , RNA/chemistry , Streptolysins/chemistry , Viral Proteins/metabolismABSTRACT
The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo. Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity "orchestra." We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.
Subject(s)
Erythrocytes/immunology , Hemolysis/immunology , Immunity, Innate , Serum Albumin, Human/immunology , Streptococcus pyogenes/immunology , Streptolysins/immunology , A549 Cells , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Humans , Serum Albumin, Human/chemistry , Streptococcus pyogenes/chemistry , Streptolysins/chemistryABSTRACT
The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.
Subject(s)
Antibodies, Monoclonal/pharmacology , Epitope Mapping/methods , Epitopes/immunology , Streptococcus pneumoniae/immunology , Streptolysins/chemistry , Streptolysins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Binding Sites , Cell Line, Tumor , Cholesterol/metabolism , Humans , Lung/immunology , Lung/microbiology , Mice , Models, Molecular , Protein Binding/drug effects , Protein Conformation , Protein Domains , Streptolysins/immunologyABSTRACT
Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.
Subject(s)
Inflammation/drug therapy , Mutant Proteins/pharmacology , Mutant Proteins/therapeutic use , Streptococcus pneumoniae/metabolism , Streptolysins/pharmacology , Toll-Like Receptor 4/antagonists & inhibitors , Amino Acid Sequence , Animals , Apoptosis/drug effects , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacology , Binding Sites , Caspase 3/metabolism , Cell Survival/drug effects , Diet, High-Fat , E-Selectin/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intercellular Adhesion Molecule-1/metabolism , Lipopolysaccharides , Mice , Molecular Docking Simulation , Mutant Proteins/chemistry , NF-kappa B/metabolism , Neutrophils/cytology , Neutrophils/drug effects , Phosphorylation/drug effects , Solubility , Streptolysins/chemistry , Streptozocin , Toll-Like Receptor 4/metabolism , Transendothelial and Transepithelial Migration/drug effects , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Oligomers of pneumolysin form transmembrane channels in cholesterol-containing lipid bilayers. The mechanism of pore formation involves a multistage process in which the protein, at first, assembles into a ring-shaped complex on the outer-bilayer leaflet. In a subsequent step, the complex inserts into the membrane. Contrary to most investigations of pore formation that have focussed on protein changes, we have deduced how the lipid-packing order is altered in different stages of the pore-forming mechanism. An optical tweezing apparatus was used, in combination with microfluidics, to isolate large-unilamellar vesicles and control exposure of the bilayer to pneumolysin. By monitoring Raman-scattered light from a single-trapped liposome, the effect of the protein on short-range order and rotational diffusion of lipids could be inferred from changes in the envelope of the C-H stretch. A significant change in the lipid-packing order takes place during assembly of pre-pore oligomers. We were not able to detect a change in the lipid-packing order during the initial stage of protein binding, or any further change during the insertion of oligomers. Pre-pore complexes induce a transformation in which a bilayer, resembling a liquid-ordered phase is changed into a bilayer resembling a fluid-liquid-disordered phase surrounding ordered microdomains enriched in cholesterol and protein complexes.
Subject(s)
Cholesterol/metabolism , Streptococcus pneumoniae/metabolism , Streptolysins/chemistry , Streptolysins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cholesterol/chemistry , Hemolysis , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Microfluidic Analytical Techniques , Models, Molecular , Mutation , Optical Tweezers , Protein Binding , Spectrum Analysis, Raman , Streptolysins/genetics , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolismABSTRACT
BACKGROUND: Cell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells. SCOPE OF REVIEW: We review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (~30â¯nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment. MAJOR CONCLUSIONS: As confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique. GENERAL SIGNIFICANCE: SLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity.
Subject(s)
Bacterial Toxins/chemistry , Cell Membrane Permeability/drug effects , Cytological Techniques , Heat-Shock Proteins/chemistry , Hemolysin Proteins/chemistry , Streptolysins/chemistry , Animals , Bacterial Proteins/chemistry , Biological Products , Cell Membrane/metabolism , Cytosol/metabolism , Diabetes Mellitus, Experimental , Fluorescein/chemistry , Gene Editing , Genotype , HEK293 Cells , Humans , Membranes/metabolism , Mice , PhenotypeABSTRACT
BACKGROUND: Native American populations experience a substantial burden of pneumococcal disease despite use of highly effective pneumococcal conjugate vaccines (PCVs). Protein-based pneumococcal vaccines may extend protection beyond the serotype-specific protection elicited by PCVs. METHODS: In this phase IIb, double-blind, controlled trial, 6-12â¯weeks-old Native American infants randomized 1:1, received either a protein-based pneumococcal vaccine (dPly/PhtD) containing pneumolysin toxoid (dPly, 10⯵g) and pneumococcal histidine triad protein D (PhtD, 10⯵g) or placebo, administered along with 13-valent PCV (PCV13) at ages 2, 4, 6 and 12-15â¯months. Other pediatric vaccines were given per the routine immunization schedule. We assessed vaccine efficacy (VE) against acute otitis media (AOM) and acute lower respiratory tract infection (ALRI) endpoints. Immunogenicity, reactogenicity and unsolicited adverse events were assessed in a sub-cohort and serious adverse events were assessed in all children. RESULTS: 1803 infants were randomized (900 dPly/PhtD; 903 Control). VE against all episodes of American Academy of Pediatrics (AAP)-defined AOM was 3.8% (95% confidence interval: -11.4, 16.9). Point estimates of VE against other AOM outcomes ranged between 2.9% (-9.5, 14.0) and 5.2% (-8.0, 16.8). Point estimates of VE against ALRI outcomes ranged between -4.4% (-39.2, 21.8) and 2.0% (-18.3, 18.8). Point estimates of VE tended to be higher against first than all episodes but the confidence intervals included zero. dPly/PhtD vaccine was immunogenic and had an acceptable reactogenicity and safety profile after primary and booster vaccination in Native American infants. CONCLUSIONS: The dPly/PhtD vaccine was immunogenic and well tolerated, however, incremental efficacy in preventing AAP-AOM over PCV13 was not demonstrated. CLINICAL TRIALS REGISTRATION: NCT01545375 (www.clinicaltrials.gov).
Subject(s)
Immunization, Secondary/methods , Otitis Media/prevention & control , Pneumococcal Vaccines/administration & dosage , Pneumonia, Pneumococcal/prevention & control , Respiratory Tract Infections/prevention & control , Streptococcus pneumoniae/immunology , Acute Disease , Antibodies, Bacterial/blood , Bacterial Proteins/administration & dosage , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Female , Humans , Hydrolases/administration & dosage , Hydrolases/chemistry , Hydrolases/immunology , Immunization Schedule , Immunogenicity, Vaccine , Infant , Infant, Newborn , Male , Otitis Media/immunology , Otitis Media/microbiology , Otitis Media/pathology , Patient Safety , Pneumococcal Vaccines/chemistry , Pneumococcal Vaccines/immunology , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/microbiology , Pneumonia, Pneumococcal/pathology , Respiratory Tract Infections/immunology , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/pathology , Streptococcus pneumoniae/pathogenicity , Streptolysins/administration & dosage , Streptolysins/chemistry , Streptolysins/immunology , Vaccines, Conjugate , Vaccines, SubunitABSTRACT
The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.
Subject(s)
Hemolysis , Streptolysins/chemistry , Streptolysins/metabolism , Toll-Like Receptor 4/metabolism , Animals , Apoptosis/drug effects , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Female , Humans , Mice, Inbred ICR , Protein Domains , Recombinant Proteins/pharmacology , Streptococcus pneumoniae/pathogenicity , Streptolysins/pharmacology , Structure-Activity Relationship , Virulence/drug effectsABSTRACT
BACKGROUND: Pneumolysin (Ply), as a major virulence factor of Streptococcus pneumoniae, has attracted increased attention for its potential value in the development of next-generation protein-based pneumococcal vaccines. This study aimed to analyze the genetic and antigenic diversity that can influence the immunogenicity of vaccines. METHODS: A total of 96 pneumococcal isolate samples were obtained from children of 1-35 months old with invasive pneumococcal diseases in Shanghai Children's Medical Center (Shanghai, China). After DNA amplification by PCR and Sanger sequencing, Ply DNA sequences were analyzed by bioinformatics tools, including ClustalX, BioEdit and MEGA7. RESULTS: Two alleles, allele 1 and 2, and 10 subtypes, of which were 6 novel subtypes, were identified. Nucleotide and amino acid sequence identity among these pneumococcal isolates were >99%. Subtypes with the same amino acid sequence were more closely evolutionarily related in the phylogenetic tree. Only minor differences in the B-cell epitopes were identified in the antigenicity plots of alleles 1 and 2. The most common serotype was serotype 19A. CONCLUSIONS: The sequence diversity of Ply is limited although some allelic variations are detected. Different alleles exhibit similar antigenic patterns. Development of Ply-based vaccines may be a promising method to combat pneumococcal infection in the future.
Subject(s)
Pneumococcal Infections/microbiology , Streptolysins/genetics , Streptolysins/immunology , Alleles , Amino Acid Sequence , Antigenic Variation , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Child, Preschool , Genetic Variation , Humans , Infant , Phylogeny , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines , Serogroup , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/isolation & purification , Streptolysins/chemistry , Virulence Factors/genetics , Virulence Factors/immunologyABSTRACT
Protein-membrane interactions that modify the shape of membranes are important for generating curvature, membrane deformation by protein-protein crowding or trafficking of vesicles. Giant vesicles represent a simplified but versatile model for biological membranes and are commonly employed for the study of lipid domains and permeation across compartments. In this study, we investigated the interaction of pneumolysin (PLY), a pore-forming toxin secreted by Streptococcus pneumoniae, with multilamellar and unilamellar membranes. It reveals an enlargement of membrane area due to the insertion of pores into the bilayer and protein-membrane aggregations that induce membrane deformation and wrinkling. Moreover, we demonstrate that PLY peel-off layers from multilamellar giant vesicles in a hitherto unknown layer-by-layer peeling mechanism, which reveals the structure and number of membrane lamellae. We employed microfluidic methods to capture giant vesicles and confocal laser scanning microscopy, transmission microscopy, dynamic light scattering and cryo-electron microscopy to disclose the structure of multilamellar vesicles. Based on our findings we suggest how back-to-back pore arrangements stabilize large PLY-membrane entities and that pore-displaced lipids possibly remain in the membrane.
Subject(s)
Cell Membrane/chemistry , Streptococcus pneumoniae/chemistry , Streptolysins/chemistry , Unilamellar Liposomes/chemistry , Bacterial Proteins/chemistryABSTRACT
Bacterial vaginosis (BV) is a vaginal anaerobic dysbiosis that affects women of reproductive age worldwide. BV is microbiologically characterized by the depletion of vaginal lactobacilli and the overgrowth of anaerobic bacterial species. Accumulated evidence suggests that Gardnerella spp. have a pivotal role among BV-associated bacteria in the initiation and development of BV. However, Gardnerella spp. often colonize healthy women. Lactobacillus iners is considered as a prevalent constituent of healthy vaginal microbiota, and is abundant in BV. Gardnerella spp. and L. iners secrete the toxins vaginolysin (VLY) and inerolysin (INY), which have structural and activity features attributed to cholesterol-dependent cytolysins (CDCs). CDCs are produced by many pathogenic bacteria as virulence factors that participate in various stages of disease progression by forming lytic and non-lytic pores in cell membranes or via pore-independent pathways. VLY is expressed in the majority of Gardnerella spp. isolates; less is known about the prevalence of the gene that encodes INY. INY is a classical CDC; membrane cholesterol acts a receptor for INY. VLY uses human CD59 as its receptor, although cholesterol remains indispensable for VLY pore-forming activity. INY-induced damage of artificial membranes is directly dependent on cholesterol concentration in the bilayer, whereas VLY-induced damage occurs with high levels of membrane cholesterol (>40 mol%). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is specific for a healthy vagina. Increased VLY levels in vaginal mucosa in vivo were associated with clinical indicators of BV. However, experimental evidence is lacking for the specific roles of VLY and INY in BV. The interplay between vaginal bacterial species affects the expression of the gene encoding VLY, thereby modulating the virulence of Gardnerella spp. This review discusses the current evidence for VLY and INY cytolysins, including their structures and activities, factors affecting their expression, and their potential impacts on the progression of anaerobic dysbiosis.
Subject(s)
Bacteria/metabolism , Cholesterol/metabolism , Cytotoxins/metabolism , Dysbiosis , Vagina/microbiology , Vaginosis, Bacterial/metabolism , Animals , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins , CD59 Antigens , Cell Membrane/metabolism , Cryoelectron Microscopy , Female , Gardnerella/isolation & purification , Humans , Hydrogen-Ion Concentration , Lactobacillus/genetics , Lactobacillus/physiology , Microbiota/genetics , Streptolysins/chemistry , Virulence FactorsABSTRACT
Methods to efficiently deliver fluorophores across the cell membrane are crucial for imaging the dynamics of intracellular proteins using fluorescence. Here we describe a simple protocol for permeabilizing living cells using streptolysin O, a bacterial toxin, which allows transient uptake of fluorescent probes for labeling specific intracellular proteins. The technique is applicable for delivering different classes of fluorescent probes with a molecular weight of <150 kDa, and it is also applicable to a variety of different cell lines. The technique enables the utilization of a broad range of fluorophores for live cell imaging of intracellular proteins. Extended observation of intracellular fluorescence bound to specific proteins is now possible through super-resolution microscopy by using fluorophores that are photostable in "cell-friendly" deoxygenating and reducing conditions. © 2018 by John Wiley & Sons, Inc.
Subject(s)
Fluorescent Dyes/chemistry , Staining and Labeling/methods , Streptolysins/chemistry , Bacterial Proteins/chemistry , Cell Line , Microscopy, Fluorescence/methodsABSTRACT
Neutralization of bacterial toxins has become a compelling approach to treating bacterial infections as it may pose less selective pressure for the development of bacterial resistance. Currently, the majority of toxin neutralization platforms act by targeting the molecular structure of the toxin, which requires toxin identification and customized design for different diseases. Therefore, their development has been challenged by the enormous number and complexity of bacterial toxins. Herein, biomimetic toxin nanosponges are formulated by coating membranes of human red blood cells (hRBCs) onto polymeric nanoparticles, which act as a toxin decoy to absorb and neutralize a broad-spectrum of hemolytic toxins regardless of their molecular structure. When tested with model pore-forming toxins, including melittin, α-hemolysin of methicillin-resistant Staphylococcus aureus, listeriolysin O of Listeria monocytogenes, and streptolysin O of Group A Streptococcus, the hRBC nanosponges are able to completely inhibit toxin-induced hemolysis in a concentration-dependent manner. In addition, the nanosponge-detained toxins show no cytotoxicity when tested on human umbilical vein endothelial cells and no lethality when injected into mice, which together indicate effective toxin neutralization. Overall, these results demonstrate the broad applicability and high effectiveness of the hRBC nanosponges as a novel antivirulence platform against hemolytic toxins from various strains of bacteria.
Subject(s)
Bacterial Toxins/chemistry , Erythrocyte Membrane/chemistry , Nanostructures/chemistry , Animals , Bacterial Proteins/chemistry , Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , Biomimetic Materials , Hemolysin Proteins/chemistry , Hemolysis/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Melitten/chemistry , Mice, Inbred ICR , Streptolysins/chemistryABSTRACT
B-lymphocytes have the ability to repair their plasma membranes following injury, such as by bacterial cholesterol-dependent cytolysins. The repair process includes the removal of the pore from the inflicted region of the plasma membrane via lipid raft-mediated internalization. Lipid rafts are critical for B cell receptor (BCR) activation. Cholesterol-dependent pore forming bacterial toxins provide a useful tool for examining the role of lipid rafts in B cell activation and the underlying cellular mechanisms. This method serves as a great alternative of known cholesterol disruption reagents such as filipin, nystatin, and methyl-ß-cyclodextrin. Here, we describe a method of damaging primary murine B cell plasma membranes with the Streptococcus pyogenes cytolysin, Streptolysin O (SLO), and monitoring levels of damage, repair and BCR activation.
Subject(s)
B-Lymphocytes/chemistry , Cholesterol/chemistry , Lymphocyte Activation , Membrane Microdomains/chemistry , Receptors, Antigen, B-Cell/chemistry , Streptococcus pyogenes/chemistry , Streptolysins/chemistry , Animals , B-Lymphocytes/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Cholesterol/immunology , Humans , Membrane Microdomains/immunology , Mice , Receptors, Antigen, B-Cell/immunology , Streptococcus pyogenes/immunology , Streptolysins/immunologyABSTRACT
Vaccine design ushered in the era of nanotechnology, as the vaccine is being developed toward particulate formulation. We have previously shown that the attenuated pneumolysin mutant (ΔA146PLY) was a safe and effective pneumococcal vaccine candidate. Here, to further optimize the formulation, we fused calcium phosphate (CaP) binding domains with ΔA146PLY so that the biocompatible CaP can mineralize with the protein automatically, allowing simple production of nanoparticle antigen during preparation. We fabricated four different nanoparticles, and then we compared the characteristics of different CaP-ΔA146PLY nanoparticles and demonstrated the influence of CaP binding domains on the size, shape and surface calcium content of the nanoparticles. It was found that these self-biomineralized CaP-ΔA146PLY nanoparticles varied in their capacity to induce BMDCs and splenocytes production of cytokines. We further demonstrated that, compared to free proteins, nanoparticle antigens induced more efficient humoral and cellular immune responses which was strong enough to protect mice from both pneumonia and sepsis infection. Also, the integration of CaP to protein has no significant impairment on body weight of animals, and subcutaneous injection of ΔA146PLY-peptides@CaP nanoparticles did not lead to permanent formation of nodules in the skin relative to Alum adjuvant formulated antigens. Together, our data sufficiently suggest that soluble ΔA146PLY vaccine candidate could be processed into nanoparticles by self-biomineralization of CaP, the immunogenicity of which could be efficiently improved by the CaP binding domains and biomineralization.
Subject(s)
Calcium Phosphates/chemistry , Calcium Phosphates/metabolism , Nanoparticles/chemistry , Streptolysins/chemistry , Streptolysins/metabolism , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Mice , Pneumococcal Infections/metabolism , Streptococcus pneumoniae/metabolism , Streptococcus pneumoniae/pathogenicityABSTRACT
In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.