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Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens.

ABSTRACT: Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

SUBMITTER: Zou JT

PROVIDER: S-EPMC8294524 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pore-forming toxin alpha-hemolysin efficiently improve the immunogenicity and protective efficacy of protein antigens by increasing antigen uptake and targeting ADAM10

Project description:Highly immunogenic exotoxins were proved to be efficient in improving the immunogenicity of polysaccharides, traditionally known as carrier proteins. However, the efficacy of exotoxins on protein antigens remains to be excluded. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus. After immunization with the fusion protein, increased titers of PA0833-specific antibodies and higher protective efficacy were observed, companied with decreased bacterial burden and pro-inflammatory cytokines secretion when compared with PA0833 immunization alone. Then, by using fluorescently labeled antigens to track antigen uptake and delivery, we found that fusion to HlaH35A significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that increased antigen uptake by fusion to HlaH35A were mainly mediated by monocytes and macrophages in a macropinocytosis dependent manner, probably due to targeting ADAM10, the host receptor of Hla. Further, transcriptome analysis was performed and the result showed that several immune signaling pathways were activated by HPF, which also shed light on the mechanism of improved immunogenicity. Finally, improvement of immunogenicity by fusion to HlaH35A was also confirmed in two other anigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, which indicated that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

2021-02-20 | GSE167077 | GEO

Pore-forming toxin alpha-hemolysin efficiently improve the immunogenicity and protective efficacy of protein antigens by increasing antigen uptake and targeting ADAM10

Project description:Pore-forming toxin alpha-hemolysin efficiently improve the immunogenicity and protective efficacy of protein antigens by increasing antigen uptake and targeting ADAM10

| PRJNA702922 | ENA

Membrane-Pore Forming Characteristics of the Bordetella pertussis CyaA-Hemolysin Domain.

Project description:Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.

| S-EPMC4448159 | biostudies-literature

The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways.

Project description:Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell functions, but also disable macrophages and suppress inflammatory responses.

| S-EPMC3266558 | biostudies-literature

Redirecting Pore Assembly of Staphylococcal α-Hemolysin by Protein Engineering.

Project description:α-Hemolysin (αHL), a β-barrel pore-forming toxin (βPFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, αHL assembles to form heptameric membrane-spanning pores. We have previously engineered αHL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned αHL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of αHL with galectin-1 (αHLG1, αHL-Galectin-1 chimera). αHLG1 was cytolytic toward cells that lack a receptor for wild-type αHL. We then constructed protease-activatable mutants of αHLG1 (PAMαHLG1s). PAMαHLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications.

| S-EPMC6487460 | biostudies-literature

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems.

Project description:The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin's mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl's ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

| S-EPMC8623112 | biostudies-literature

Electrostatic Control of Polymer Translocation Speed through <i>α</i>‑Hemolysin Protein Pore.

Project description:The electrostatic origins behind the speed of translocation of a uniformly charged flexible macromolecule through α-hemolysin (αHL) protein pores under a voltage are investigated using variations in pH and electrolyte concentration. We have measured durations of successful threading of poly(styrenesulfonate) through αHL at two different pH conditions, pH 4.5 and pH 7.5, under various salt concentration conditions. Salt concentrations in the donor (cis) and the recipient (trans) compartments influence the polymer translocation dynamics differently, depending on pH. At both pH 4.5 and pH 7.5, decreasing the cis salt concentration, cs,cis , results in faster polymer translocations. On the other hand, a decrease in trans salt concentration, cs,trans , retards the polymer transport process at pH 4.5, while at pH 7.5 the translocation time is observed to be independent of cs,trans . We present a theoretical model to calculate the translocation times from the free energy of the polymer along the translocation process to describe our experimental results. We show that the charge density of the polymer inside the nanopore is significantly affected by cs,cis , explaining the cis salt effect on the speed of polymer translocation. The trans salt effects are attributed to the electrostatic interaction between the polymer and the exit portion of the αHL pore, which is determined by the pH of the trans compartment. At low pH where the net charge of the end of the αHL is positive, the attractive electrostatic interaction in trans becomes stronger, as cs,trans decreases, resulting in delays in translocation process.

| S-EPMC5919254 | biostudies-literature

Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore.

Project description:Nanopores are under investigation for single-molecule DNA sequencing. The alpha-hemolysin (alphaHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R(1) by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g., bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points.

| S-EPMC2935931 | biostudies-literature

Current Perspective on the Membrane-Damaging Action of Thermostable Direct Hemolysin, an Atypical Bacterial Pore-forming Toxin.

Project description:Thermostable direct hemolysin (TDH) is the major virulence determinant of the gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin (PFT). TDH shares remarkable structural similarity with the actinoporin family of eukaryotic PFTs produced by the sea anemones. Unlike most of the PFTs, it exists as tetramer in solution, and such assembly state is crucial for its functionality. Although the structure of the tetrameric assembly of TDH in solution is known, membrane pore structure is not available yet. Also, the specific membrane-interaction mechanisms of TDH, and the exact role of any receptor(s) in such process, still remain unclear. In this mini review, we discuss some of the unique structural and physicochemical properties of TDH, and their implications for the membrane-damaging action of the toxin. We also present our current understanding regarding the membrane pore-formation mechanism of this atypical bacterial PFT.

| S-EPMC8343067 | biostudies-literature

Polymer capture by α-hemolysin pore upon salt concentration gradient.

Project description:We have measured the rate of capture of single molecules of sodium poly(styrene sulfonate) by α-hemolysin protein pore by varying applied voltage, pH, and the salt concentration asymmetry across the pore. We show that electrostatic interaction between the polyelectrolyte and the protein pore significantly affects the polymer capture rate in addition to the enhancement of drift arising from electrolyte concentration gradient. At higher pH values where the electrostatic interaction between the polymer and the α-hemolysin pore is repulsive, an antagonistic coupling with the drift induced by salt concentration gradient emerges. This antagonistic coupling results in a nonmonotonic dependence of the polymer capture rate on the salt concentration in the donor compartment. The coupling between the pore-polymer interaction and drift can be weakened by increasing the strength of the electric field that drives the polymer translocation. In contrast, at lower pH values where the polymer-pore interaction is attractive, a synergy with the additional drift from salt concentration asymmetry arises and the capture rate depends monotonically on the donor salt concentration. For higher pH, we identify two regimes for the enhancement of capture rate by salt concentration gradient: (a) drift-dominated regime, where the capture rate is roughly quadratic in the ratio of salt concentration in the receiver compartment to that in the donor compartment, and (b) antagonistic coupling regime at higher values of this ratio with a linear relation for the polymer capture rate.

| S-EPMC3895088 | biostudies-other

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