Cytolytic Pore-forming Toxin Research Articles

The natural product, echinatin, protects mice from methicillin-resistant Staphylococcus aureus pneumonia by inhibition of alpha-hemolysin expression.

Antimicrobial resistance (AMR) is a global, multifaceted crisis that poses significant challenges to the successful eradication of devastating pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), a persistent superbug that causes devastating infections. The scarcity of new antibacterial drugs is obvious, and antivirulence strategies that reduce the pathogenicity of bacteria by weakening their virulence have become the subject of intense investigation. Alpha-hemolysin (Hla), a cytolytic pore-forming toxin, has a pivotal role in S. aureus pathogenesis. Here, we demonstrated that echinatin, a natural compound isolated from licorice, effectively inhibited the hemolytic activity of MRSA at 32 μg/mL. In addition, echinatin did not interfere with bacterial growth and had no significant cytotoxicity at the inhibitory concentration of S. aureus hemolysis. Heptamer formation tightly correlated with Hla-mediated cell invasion, whereas echinatin did not affect deoxycholic acid-induced oligomerization of Hla. Echinatin affected hemolytic activity through indirect binding to Hla as confirmed by the neutralization assay and cellular thermal shift assay (CETSA). Furthermore, qRT-PCR and western blot analyses revealed that echinatin suppressed Hla expression at both the mRNA and protein levels as well as the transcript levels of Agr quorum-sensing system-related genes. Additionally, when echinatin was added to a coculture system of A549 cells and S. aureus, it significantly reduced cell damage. Importantly, echinatin exhibited a significant therapeutic effect in an MRSA-induced mouse pneumonia model. In conclusion, the present findings demonstrated that echinatin significantly inhibits the hemolysin effect and may be a potential candidate compound for combating drug-resistant MRSA infections.

Alanine Scanning Mutagenesis of the MEDI4893 (Suvratoxumab) Epitope Reduces Alpha Toxin Lytic Activity In Vitro and Staphylococcus aureus Fitness in Infection Models.

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.

A Critical Role for HlgA in Staphylococcus aureus Pathogenesis Revealed by A Switch in the SaeRS Two-Component Regulatory System.

Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans.

Rapid Microfluidic Double-Jump Mixing Device for Single-Molecule Spectroscopy.

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g., transiently populated intermediates that are inaccessible with existing single-molecule approaches. We demonstrate the potential and reliability of the method on time scales from milliseconds to minutes by investigating the coupled folding and binding reaction of two intrinsically disordered proteins and the conformational changes occurring in a large cytolytic pore-forming toxin.

Targeting Alpha Toxin and ClfA with a Multimechanistic Monoclonal-Antibody-Based Approach for Prophylaxis of Serious Staphylococcus aureus Disease

ABSTRACTStaphylococcus aureus produces numerous virulence factors, each contributing different mechanisms to bacterial pathogenesis in a spectrum of diseases. Alpha toxin (AT), a cytolytic pore-forming toxin, plays a key role in skin and soft tissue infections and pneumonia, and a human anti-AT monoclonal antibody (MAb), MEDI4893*, has been shown to reduce disease severity in dermonecrosis and pneumonia infection models. However, interstrain diversity and the complex pathogenesis of S. aureus bloodstream infections suggests that MEDI4893* alone may not provide adequate protection against S. aureus sepsis. Clumping factor A (ClfA), a fibrinogen binding protein, is an important virulence factor facilitating S. aureus bloodstream infections. Herein, we report on the identification of a high-affinity anti-ClfA MAb, 11H10, that inhibits ClfA binding to fibrinogen, prevents bacterial agglutination in human plasma, and promotes opsonophagocytic bacterial killing (OPK). 11H10 prophylaxis reduced disease severity in a mouse bacteremia model and was dependent on Fc effector function and OPK. Additionally, prophylaxis with 11H10 in combination with MEDI4893* provided enhanced strain coverage in this model and increased survival compared to that obtained with the individual MAbs. The MAb combination also reduced disease severity in murine dermonecrosis and pneumonia models, with activity similar to that of MEDI4893* alone. These results indicate that an MAb combination targeting multiple virulence factors provides benefit over a single MAb neutralizing one virulence mechanism by providing improved efficacy, broader strain coverage, and protection against multiple infection pathologies.

Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration.

Cell ablation is a powerful tool for studying cell lineage and/or function; however, current cell-ablation models have limitations. Intermedilysin (ILY), a cytolytic pore-forming toxin that is secreted by Streptococcus intermedius, lyses human cells exclusively by binding to the human complement regulator CD59 (hCD59), but does not react with CD59 from nonprimates. Here, we took advantage of this feature of ILY and developed a model of conditional and targeted cell ablation by generating floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination. The administration of ILY to ihCD59+ mice crossed with various Cre-driver lines resulted in the rapid and specific ablation of immune, epithelial, or neural cells without off-target effects. ILY had a large pharmacological window, which allowed us to perform dose-dependent studies. Finally, the ILY/ihCD59-mediated cell-ablation method was tested in several disease models to study immune cell functionalities, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. Together, the results of this study demonstrate the utility of the ihCD59 mouse model for studying the effects of cell ablation in specific organ systems in a variety of developmental and disease states.

Evaluation of the Protective Immunity of a Novel Subunit Fusion Vaccine in a Murine Model of Systemic MRSA Infection

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.

Evaluation of the protective immunity of a novel subunit fusion vaccine in a murine model of systemic MRSA infection (P4254)

Abstract Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on Hla and IsdB antigenic region. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). our results verified that the strategy of optimized chimeric bivalent antigens could be as an important approach for developing S. aureus mult-subunit vaccines against S. aureus infection.

Nrc of Streptococcus pneumoniae suppresses capsule expression and enhances anti-phagocytosis

Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Δ spd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).

Pneumolysin Localizes to the Cell Wall of Streptococcus pneumoniae

Streptococcus pneumoniae is the causative agent of multiple diseases, including otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolytic pore-forming toxins, is produced by virtually all clinical isolates of S. pneumoniae, and strains in which the Ply gene has been deleted are severely attenuated in mouse models of infection. In contrast to all other members of the cholesterol-dependent cytolysin family, Ply lacks a signal peptide for export. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains ill defined. We determined by use of cell fractionation and Western blotting that, during in vitro growth, exported Ply is localized primarily to the cell wall compartment in 18 different serotypes in the absence of detectable cell lysis. Hemolytic assays revealed that this cell wall-localized Ply is active. Additionally, cell wall-localized Ply is accessible to extracellular protease and is detergent releasable.

Abstract 3574: A Unique Model and Mechanisms of Hemolysis-Associated Pulmonary Hypertension

Pulmonary hypertension is a common complication of intravascular hemolysis and is suspected to be a strong mortality determinant of hemolytic disorders. However, the pathophysiologic mechanisms leading to hemolytic anemia-associated pulmonary hypertension remains unclear. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses only human cells due to its unique receptor specificity for human CD59 (hCD59). We recently generated a novel intravascular hemolysis mouse model in which ILY exclusively lyses erythrocytes transgenically expressing hCD59 ( ThCD59RBC mice) ( Nature Medicine, 2008, 14: 98 –103 ). Using this model, we investigated the pathogenesis of hemolytic anemia-associated pulmonary hypertension. In ThCD59RBC mice, but not wild type mice, intravenous injection of ILY with a 1.5-fold lethal dose induced rapid intravascular hemolysis with acute death (less than 1 minute) that was preceded by a sharp increase in the pulmonary pressure (10.40 ± 0. 89 mmHg increase, n = 5) with 0.65 ± 0.36 mmHg/second rate of rise of systolic pulmonary artery pressure as measured by right-heart catheterization. We also observed decreased NO levels indirectly measured by plasma nitrate + nitrite concentration [10.01 ± 2.47μM in ThCD59RBC (n=3) vs 22.16 ± 2.20 μM in WT (n=3); P <0.001] and increased plasma P-selectin levels [445.65 ± 54.85 ng/ml in ThCD59RBC (n=4) vs 186.95 ± 18.64 ng/ml in WT (n=4); P <0.001]. These results suggest that the lethality of acute intravascular hemolysis may result from a sudden increase in pulmonary pressure likely a consequence of the reduced bioavailability of NO and resulting pulmonary vasoconstriction and platelet activation. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59(RBC)) or on endothelia (strain ThCD59(END)). Intravenous injection of ILY in ThCD59(RBC) mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59(END) mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.

Alternate SlyA and H‐NS nucleoprotein complexes control hlyE expression in Escherichia coli K‐12

Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE–lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription.

Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins.

Cytolytic pore-forming toxins are important for the virulence of many disease-causing bacteria. How target cells molecularly respond to these toxins and whether or not they can mount a defense are poorly understood. By using microarrays, we demonstrate that the nematode Caenorhabditis elegans responds robustly to Cry5B, a member of the pore-forming Crystal toxin family made by Bacillus thuringiensis. This genomic response is distinct from that seen with a different stressor, the heavy metal cadmium. A p38 mitogen-activated protein kinase (MAPK) kinase and a c-Jun N-terminal-like MAPK are both transcriptionally up-regulated by Cry5B. Moreover, both MAPK pathways are functionally important because elimination of either leads to animals that are (i) hypersensitive to a low, chronic dose of toxin and (ii) hypersensitive to a high, brief dose of toxin such that the animal might naturally encounter in the wild. These results extend to mammalian cells because inhibition of p38 results in the hypersensitivity of baby hamster kidney cells to aerolysin, a pore-forming toxin that targets humans. Furthermore, we identify two downstream transcriptional targets of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Cry5B. Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.

A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins ( Escherichia coli hemolysin and Staphylococcus aureus α-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistence. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.