FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

The Effect of Ascorbic Acid and Nicotinamide on Panton-Valentine Leukocidin Cytotoxicity: An Ex Vivo Study.

Background: Panton−Valentine Leukocidin sustains a strong cytotoxic activity, targeting immune cells and, consequently, perforating the plasma membrane and inducing cell death. The present study is aimed to examine the individual effect of ascorbic acid and nicotinamide on PVL cytotoxicity ex vivo, as well as their effect on granulocytes viability when treated with PVL. Materials and Methods: The PVL cytotoxicity assay was performed in triplicates using the commercial Cytotoxicity Detection Kit PLUS (LDH). LDH release was measured to determine cell damage and cell viability was measured via flow cytometry. Results and discussion: A clear reduction in PVL cytotoxicity was demonstrated (p < 0.001). Treatment with ascorbic acid at 5 mg/mL has shown a 3-fold reduction in PVL cytotoxicity; likewise, nicotinamide illustrated a 4-fold reduction in PVL cytotoxicity. Moreover, granulocytes' viability after PVL treatment was maintained when incubated with 5 mg/mL of ascorbic acid and nicotinamide. Conclusions: our findings illustrated that ascorbic acid and nicotinamide exhibit an inhibitory effect on PVL cytotoxicity and promote cell viability, as the cytotoxic effect of the toxin is postulated to be neutralized by antioxidant incubation. Further investigations are needed to assess whether these antioxidants may be viable options in PVL cytotoxicity attenuation in PVL-associated diseases.

Panton-Valentine Leukocidin Induces Cytokine Release and Cytotoxicity Mediated by the C5a Receptor on Rabbit Alveolar Macrophages.

Necrotizing pneumonia caused by Panton-Valentine leukocidin (PVL)-positive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has high mortality rates and is currently a serious clinical issue. PVL is a two-component toxin (LukS-PV and LukF-PV). It can cause necrosis in target cells by forming pores consisting of an octamer comprised of LukS-PV and LukF-PV. However, considering the specificity of PVL towards several target cells and species, the specific effect of PVL remains controversial. Therefore, we focused on necrotizing pneumonia caused by PVL-positive S. aureus and clarified the effect of PVL on alveolar macrophages, which play a central role in innate immunity in the alveolar space. We constructed recombinant PVL (rPVL) components and stimulated alveolar macrophages isolated from rabbits to evaluate cytotoxicity and pro-inflammatory cytokine release. Recombinant LukS-PV (rLukS-PV), but not recombinant LukF-PV (rLukF-PV), induced pro-inflammatory cytokine release. Specifically, tumor necrosis factor (TNF)-α release was mediated by the C5a receptor (C5aR) expressed on rabbit alveolar macrophages, and the toxicity of rPVL, consisting of rLukS-PV and rLukF-PV, towards rabbit alveolar macrophages was mediated by the same receptor. Overall, our findings shed light on the C5aR-mediated cytotoxic effect of PVL on alveolar macrophages, which may be useful for understanding the mechanism of necrotizing pneumonia caused by PVL.

Staphylococcal Panton-Valentine Leucocidin and Gamma Haemolysin Target and Lyse Mature Bone Marrow Leucocytes.

Staphylococcus aureus is a major human pathogen, inducing several infections ranging from the benign to the life-threatening, such as necrotising pneumonia. S. aureus is capable of producing a great variety of virulence factors, such as bicomponent pore-forming leucocidin, which take part in the physiopathology of staphylococcal infection. In necrotising pneumonia, Panton-Valentine leucocidin (PVL) induces not only lung injury and necrosis, but also leukopenia, regarded as a major factor of a poor prognosis. The aim of the present study was to evaluate the effect of bicomponent pore-forming leucocidin, PVL and gamma haemolysin on bone marrow leucocytes, to better understand the origin of leukopenia. Using multi-parameter cytometry, the expression of leucocidin receptors (C5aR, CXCR1, CXCR2, and CCR2) was assessed and toxin-induced lysis was measured for each bone marrow leucocyte population. We observed that PVL resulted in myeloid-derived cells lysis according to their maturation and their C5aR expression; it also induced monocytes lysis according to host susceptibility. Haemolysin gamma A, B, and C (HlgABC) displayed cytotoxicity to monocytes and natural killer cells, hypothetically through CXCR2 and CXCR1 receptors, respectively. Taken together, the data suggest that PVL and HlgABC can lyse bone marrow leucocytes. Nevertheless, the origin of leukopenia in severe staphylococcal infection is predominantly peripheral, since immature cells stay insensitive to leucocidins.

Hematogenous osteomyelitis in childhood can relapse many years later into adulthood: A retrospective multicentric cohort study in France.

To describe the epidemiological, clinical, laboratory, and radiological features and the management of adult patients who experienced a relapse between 2003 and 2015 of an acute hematogenous osteomyelitis acquired in childhood.A retrospective multicentric cohort study was conducted in 5 centers in France.Thirty-seven patients were included. The median age was 40 years (28-56), and 26 (70%) were male. The first site of infection was the distal femur (n = 23, 62%). The median time between the osteomyelitis in childhood and the relapse in adulthood was 26 years (13-45). Thirty-four (92%) patients reported inflammatory local clinical manifestations, 17 (46%) draining fistula, 10 (27%) fever. Most patients had intramedullary gadolinium deposition (with or without abscess) on magnetic resonance imaging. Most relapses were monomicrobial infections (82%). Staphylococcus aureus was the most commonly found microorganism (82%), expressing a small colony variant phenotype in 3 cases. Most patients (97%) had a surgical treatment, and the median duration of antibiotics for the relapse was 12 weeks. All patients had a favorable outcome, no patient died and no further relapse occurred. We count 2 femoral fractures on osteotomy site.Osteomyelitis in childhood can relapse later in adulthood, especially in patients with lack of care during the initial episode. Osteotomy and prolonged antimicrobial therapy are required for clinical remission.

DNA damages induced by both endotoxin and exotoxin produced by cyanobacteria.

Cyanobacteria are prokaryotes involved in the contamination of aquatic environments since they release toxins that are highly potent and dangerous for living organisms. Prokaryotes produce endo and exotoxins, among others. Exotoxins are highly toxic, while endotoxins have milder toxic effects. The present study evaluated the cytotoxicogenetic potency of both toxins studying them in different concentrations of cyanobacterial biomasses (1 µg/L, 1.5 µg/L, 2 µg/L), to assess the amount of exotoxin present in the cultured medium in which the cyanobacteria were grown. For this evaluation, we used an extract taken from the medium in a concentration of 10%. Our results showed that genotoxic and mutagenic changes in Allium cepa could be observed in all of the varying concentrations of biomass (endotoxin action) and also in the medium induced with exotoxin. Even at low concentrations, these toxins were highly effective at triggering changes in the DNA molecules of organisms exposed to them. This information is highly significant when considering environmental contamination caused by cyanobacteria blooms, since the results of this study show that these toxins may not only kill organisms when found in high concentrations, but also induce mutations when found in low concentrations. Since these mutations are expressed later on in the organisms, it is impossible to associate the observed effect with the event that induced the damage.

[Mechanisms of action of Vibrio parahaemoltyicus cytotoxins].

Vibrio parahaemolyticus, one of the Gram-negative common enteric pathogens, was first isolated in Japan in 1950. Since its discovery, this bacterium has been a major cause of food-poisoning in Japan, and its infection has recently undergone a global expansion. V. parahaemolyticus possesses a classical exotoxin, thermostable direct hemolysin, and two sets of type III secretion systems (T3SSs) that are able to inject effectors directly into host cells, which are its key virulence factors. Exotoxin/effector is exploited by many Gram-negative pathogens, and plays critical roles in pathogenesis by damaging host cells or by modulating host cell functions, through its activity on/in host cells. In recent years, functional activities of T3SS effectors produced by V. parahaemolyticus have been extensively studied, which has substantially increased our understanding of the pathogenic mechanisms of the bacterium. In paricular, some T3SS effectors of V. parahaemolyticus act as cytotoxins and thereby damage host cells. Here, I focus on these cytotoxic effectors of V. parahaemolyticus and describe recent advances in our understanding of their mechanisms of action.

RTX Toxins Ambush Immunity's First Cellular Responders.

The repeats-in-toxin (RTX) family represents a unique class of bacterial exoproteins. The first family members described were toxins from Gram-negative bacterial pathogens; however, additional members included exoproteins with diverse functions. Our review focuses on well-characterized RTX family toxins from Aggregatibacteractinomycetemcomitans (LtxA), Mannheimiahaemolytica (LktA), Bordetella pertussis (CyaA), uropathogenic Escherichia coli (HlyA), and Actinobacillus pleuropneumoniae (ApxIIIA), as well as the studies that have honed in on a single host cell receptor for RTX toxin interactions, the ß2 integrins. The ß2 integrin family is composed of heterodimeric members with four unique alpha subunits and a single beta subunit. ß2 integrins are only found on leukocytes, including neutrophils and monocytes, the first responders to inflammation following bacterial infection. The LtxA, LktA, HlyA, and ApxIIIA toxins target the shared beta subunit, thereby targeting all types of leukocytes. Specific ß2 integrin family domains are required for the RTX toxin's cytotoxic activity and are summarized here. Research examining the domains of the RTX toxins required for cytotoxic and hemolytic activity is also summarized. RTX toxins attack and kill phagocytic immune cells expressing a single integrin family, providing an obvious advantage to the pathogen. The critical question that remains, can the specificity of the RTX-ß2 integrin interaction be therapeutically targeted?

Advanced Proteomics as a Powerful Tool for Studying Toxins of Human Bacterial Pathogens.

Exotoxins contribute to the infectious processes of many bacterial pathogens, mainly by causing host tissue damages. The production of exotoxins varies according to the bacterial species. Recent advances in proteomics revealed that pathogenic bacteria are capable of simultaneously producing more than a dozen exotoxins. Interestingly, these toxins may be subject to post-transcriptional modifications in response to environmental conditions. In this review, we give an outline of different bacterial exotoxins and their mechanism of action. We also report how proteomics contributed to immense progress in the study of toxinogenic potential of pathogenic bacteria over the last two decades.

Identification, cloning and characterization of SpEX exotoxin produced by Staphylococcus pseudintermedius.

Staphylococci have evolved numerous strategies to evade their hosts' immune systems. Some staphylococcal toxins target essential components of host innate immunity, one of the two main branches of the immune system. Analysis of the Staphylococcus pseudintermedius secretome using liquid chromatography mass spectrometry guided by genomic data, was used to identify an S. pseudintermedius exotoxin provisionally named SpEX. This exoprotein has low overall amino acid identity with the Staphylococcus aureus group of proteins named staphylococcal superantigen like proteins (SSLs) and staphylococcal enterotoxin- like toxin X (SEIX), but predictive modeling showed that it shares similar folds and domain architecture to these important virulence factors. In this study, we found SpEX binds to complement component C5, prevents complement mediated lysis of sensitized bovine red blood cells, kills polymorphonuclear leukocytes and monocytes and inhibits neutrophil migration at sub-lethal concentrations. A mutant version of SpEX, produced through amino acid substitution at selected positions, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine neutrophil migration and protected immune cells from the toxic effects of the native recombinant protein. These results suggest that SpEX likely plays an important role in S. pseudintermedius virulence and that attenuated SpEX may be an important candidate for inclusion in a vaccine against S. pseudintermedius infections.

Global Proteomic Response of Caenorhabditis elegans Against PemKSa Toxin.

Bacterial exotoxins are major causative agents that infect by promoting cell and tissue damages through disabling the invading host immune system. However, the mode of action by which toxins modulate host immune system and lead cell death is still not completely understood. The nematode, Caenorhabditis elegans has been used as an attractive model host for toxicological studies. In this regard, the present study was undertaken to assess the impact of Staphylococcus aureus toxin (PemK) on the host C. elegans through global proteomics approach. Our proteomic data obtained through LC-MS/MS, subsequent bioinformatics and biochemical analyses revealed that in response to PemKSa a total of 601 proteins of C. elegans were differentially regulated in response to PemKSa. The identified proteins were found to mainly participate in ATP generation, protein synthesis, lipid synthesis, cytoskeleton, heat shock proteins, innate immune defense, stress response, neuron degeneration, and muscle assembly. Current findings suggested that involvement of several regulatory proteins that appear to play a role in various molecular functions in combating PemKSa toxin-mediated microbial pathogenicity and/or host C. elegans immunity modulation. The results provided a preliminary view of the physiological and molecular response of a host toward a toxin and provided insight into highly complex host-toxin interactions.

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death and reduced immunoglobulin release.

Streptococcal pyrogenic exotoxin (Spe) A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage to Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leucocyte profile of tonsil is unique, the impact of SpeA production on human tonsil cell function was investigated. Human tonsil cells from routine tonsillectomy were co-incubated with purified streptococcal superantigens or culture supernatants from isogenic streptococcal isolates, differing only in superantigen production. Tonsil cell proliferation was quantified by tritiated thymidine incorporation, and cell surface characteristics assessed by flow cytometry. Soluble mediators including immunoglobulin were measured using enzyme-linked immunosorbent assay. Tonsil T cells proliferated in response to SpeA and demonstrated typical release of proinflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7 days. In contrast, marked B cell apoptosis and abrogation of total immunoglobulin (Ig)A, IgM, and IgG production occurred in the presence of SpeA and other superantigens. In SpeA-stimulated cultures, T follicular helper (Tfh) cells showed a reduction in C-X-C chemokine receptor (CXCR)5 (CD185) expression, but up-regulation of OX40 (CD134) and inducible T cell co-stimulator (ICOS) (CD278) expression. The phenotypical change in the Tfh population was associated with impaired chemotactic response to CXCL13. SpeA and other superantigens cause dysregulated tonsil immune function, driving T cells from Tfh to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a probable survival advantage.

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Leukotoxic activity of Fusobacterium necrophorum of cattle origin.

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.

[Isolated hemorrhagic pleurisy due to Panton-Valentine leukocidin-positive Staphylococcus aureus]. / Pleurésie hémorragique isolée à Staphylococcus aureus à leucocidine de Panton-Valentine.

Panton-Valentine leucocidin is a major virulence factor produced by some strains of Staphylococcus aureus (SA-PVL). The best-described invasive infection is a necrotizing haemorrhagic pneumonia. Pleural effusion is not uncommon but is always associated with a parenchymal lesion. Here, we report a case of haemorrhagic pleurisy attributable to isolated SA-PVL.

Panton-Valentine Leucocidin Proves Direct Neuronal Targeting and Its Early Neuronal and Glial Impacts a Rabbit Retinal Explant Model.

: Panton-Valentine leukocidin (PVL) retinal intoxication induces glial activation and inflammatory response via the interaction with retinal neurons. In this study, rabbit retinal explant was used as a model to study neuronal and glial consequences of PVL intoxication. Retinal explants were treated with different concentrations of PVL. PVL location and neuronal and glial changes were examined using immunohistochemistry. Some inflammatory factors were quantified using RT-qPCR at 4 and 8 h. These results were compared with those of control explants. PVL co-localized rapidly with retinal ganglion cells and with horizontal cells. PVL induced Müller and microglial cell activation. Retinal structure was altered and some amacrine and microglial cells underwent apoptosis. Glial activation and cell apoptosis increased in a PVL concentration- and time-dependent manner. IL-6 and IL-8 expression increased in PVL-treated explants but less than in control explants, which may indicate that other factors were responsible for glial activation and retinal apoptosis. On retinal explants, PVL co-localized with neuronal cells and induced glial activation together with microglial apoptosis, which confirms previous results observed in in vivo model. Rabbit retinal explant seems to be suitable model to further study the process of PVL leading to glial activation and retinal cells apoptosis.

Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

Cytotoxicity analysis of staphylococcal bi-component ß-pore forming toxins using the CHO cells expressing human lymphocyte receptor CCR5.

CCR5-mediated cytotoxicity of staphylococcal bi-component toxins was investigated using human CCR5-expressing CHO cells. Cytotoxicity of rim domain loop-exchange mutants between LukE and Hlg2 indicated that loop-4 of LukE is essential for cytotoxicity in combination with LukD. Interestingly, Hlg2 showed LukF-dependent CCR5-mediated cytotoxicity, suggesting that the F-components of toxins also play a role in the cell-specific cytotoxicity.

Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin.

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential ß-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.