CD4+ T cell-mediated recognition of a conserved cholesterol-dependent cytolysin epitope generates broad antibacterial immunity.

CD4+ T cell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and invasive pneumococcal diseases (IPDs). Although such immune responses are common, the pertinent antigens have remained elusive. We identified an immunodominant CD4+ T cell epitope derived from pneumolysin (Ply), a member of the bacterial cholesterol-dependent cytolysins (CDCs). This epitope was broadly immunogenic as a consequence of presentation by the pervasive human leukocyte antigen (HLA) allotypes DPB1∗02 and DPB1∗04 and recognition via architecturally diverse T cell receptors (TCRs). Moreover, the immunogenicity of Ply427-444 was underpinned by core residues in the conserved undecapeptide region (ECTGLAWEWWR), enabling cross-recognition of heterologous bacterial pathogens expressing CDCs. Molecular studies further showed that HLA-DP4-Ply427-441 was engaged similarly by private and public TCRs. Collectively, these findings reveal the mechanistic determinants of near-global immune focusing on a trans-phyla bacterial epitope, which could inform ancillary strategies to combat various life-threatening infectious diseases, including IPDs.

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins.

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L. major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L. major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding.

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.

Repurposing rabeprazole sodium as an anti-Clostridium perfringens drug by inhibiting perfringolysin O.

AIMS: Clostridium perfringens infections affect food safety, human health, and the development of the poultry feed industry. Anti-virulence is an alternative strategy to develop new drug. Perfringolysin O (PFO) is an exotoxin of C. perfringens that has been demonstrated to play critical roles in the pathogenesis of this organism, promising it an attractive target to explore drugs to combat C. perfringens infection. METHODS AND RESULTS: Based on an activity-based screening, we identified six PFO inhibitors from the Food and Drug Administration (FDA)-approved drug library, among which rabeprazole sodium (RS) showed an optimal inhibitory effect with an IC50 of 1.82 ± 0.746 µg ml-1. The GLY57, ASP58, SER190, SER193-194, ASN199, GLU204, ASN377, THR379, and ALA200 in PFO interacted with RS during binding based on an energy analysis and H-bond analysis. This interaction blocked the oligomer formation of PFO, thereby inhibiting its cytotoxicity. RS treatment significantly increased the survival rate and alleviated pathological damage in C. perfringens or PFO-treated Galleria mellonella. CONCLUSIONS: RS could potentially be used as a candidate drug for treating C. perfringens infection.

Cholesterol-dependent cytolysins: The outstanding questions.

The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.

Evaluation of the available cholesterol concentration in the inner leaflet of the plasma membrane of mammalian cells.

Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that, although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.

The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria.

The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant ß-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their ß-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS.

Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is "accessible" to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with 15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-ß-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.

The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes.

Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membrane-insertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in Pseudomonas aeruginosa assemble heterodimers in membranes, leading to stably inserted hetero-complexes. Using site-directed fluorescence labeling with an environment-sensitive probe, we found that hydrophobic segments in PopD anchor the translocator to the membrane, but without adopting a typical transmembrane orientation. A fluorescence dual-quenching assay revealed that the presence of PopB changes the conformation adopted by PopD segments in membranes. Furthermore, analysis of PopD's interaction with human cell membranes revealed that PopD adopts a distinctive conformation when PopB is present. An N-terminal region of PopD is only exposed to the host cytosol when PopB is present. We conclude that PopB assists with the proper insertion of PopD in cell membranes, required for the formation of a functional translocon and host infection.

Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

Detectors for evaluating the cellular landscape of sphingomyelin- and cholesterol-rich membrane domains.

Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis.

BACKGROUND: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. RESULTS: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. CONCLUSION: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

Antibody-mediated neutralization of perfringolysin o for intracellular protein delivery.

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming proteins, which are highly efficient in delivering exogenous proteins to the cytoplasm. However, the indiscriminate and potent cytotoxicity of PFO limits its practical use as an intracellular delivery system. In this study, we describe the design and engineering of a bispecific, neutralizing antibody against PFO, which targets reversibly attenuated PFO to endocytic compartments via receptor-mediated internalization. This PFO-based system efficiently mediated the endosomal release of a co-targeted gelonin construct with high specificity and minimal toxicity in vitro. Consequently, the therapeutic window of PFO was improved by more than 5 orders of magnitude. Our results demonstrating that the activity of pore-forming proteins can be controlled by antibody-mediated neutralization present a novel strategy for utilizing these potent membrane-lytic agents as a safe and effective intracellular delivery vehicle.

Immunogenic and neutralization efficacy of recombinant perfringolysin O of Clostridium perfringens and its C-terminal receptor-binding domain in a murine model.

Clostridium perfringens is a Gram-positive anaerobe ubiquitously present in different environments, including the gut of humans and animals. C. perfringens have been classified in the seven toxinotypes based on the secreted toxins that cause different diseases in humans and animals. Perfringolysin O (PFO), a cholesterol-dependent pore-forming cytolysin, is one of the potent toxins secreted by almost all C. perfringens isolates. The PFO acts in synergy with α-toxin in the progression of gas gangrene in humans and necrohemorrhagic enteritis in the calves.C. perfringens infections spread very fast, and the animals die within a few hours of the onset of infection. This necessitates the use of vaccines to control clostridial infections. Though the vaccine potential of other toxins has been reported, PFO has remained unexplored. The present study describes the immunogenic and protective potential of native recombinant PFO (WTrPFO). Since the PFO is toxic to the host cells, the non-toxic C-terminal domain of PFO (rPFOC-ter) was also assessed for its immunogenicity and protective efficacy. Immunization of mice with the purified soluble recombinant histidine-tagged WTrPFO and rPFOC-ter, expressed in E. coli, generated robust mixed immune response and T cell memory. Pre-incubation of the WTrPFO with anti-WTrPFO and rPFOC-ter antisera negated its hemolytic activity in mice RBCs, as well as its cytotoxic effect in mice peritoneal macrophages in vitro. Thus, immunization with the WTrPFO and its non-toxic C-terminal domain generated neutralizing antibodies, suggesting their vaccine potential against the PFO. Thus, the non-toxic C-terminal domain of PFO could serve as an alternative to PFO as a vaccine candidate.

Imaging of the Ciliary Cholesterol Underlying the Sonic Hedgehog Signal Transduction.

Primary cilia are antenna-like structures that develop on the surface of quiescent G0-phase cells and receive extracellular signals including sonic hedgehog (Shh) for embryogenesis and adult tissue homeostasis. In mammalian cells, cholesterol activates the seven-transmembrane protein Smoothened to transduce the Shh signal. Germline mutations of the DHCR7 gene encoding the cholesterol biogenesis enzyme 7-dehydrocholesterol reductase cause Smith-Lemli-Opitz syndrome with ciliopathy-related symptoms such as polycystic kidney and polydactyly, implying that cholesterol is indeed involved in ciliary functions. Notably, it has been reported that the cholesterol in ciliary membranes is significantly more abundant than that in the rest of the plasma membrane. However, several studies have failed to image the enriched ciliary cholesterol. Here, we propose a set of protocols for the sensitive imaging of ciliary cholesterol using the fluorescent small compound Filipin III and the green fluorescent protein tagged Domain 4 of the exotoxin Perfringolysin O derived from the anaerobic bacterium Clostridium perfringens. These cholesterol probes should be powerful tools for understanding the physiological and pathological roles of ciliary cholesterol in the context of Shh signaling in mammalian cells.

Massive intravascular hemolysis is an important factor in Clostridium perfringens-induced bacteremia.

Clostridium perfringens bacteremia is rare but often fatal. In particular, once bacteremia with massive intravascular hemolysis (MIH) occurs, the mortality rate is extremely high. However, because of its rarity, the detailed pathophysiology of this fulminant form of bacteremia is unclear. To elucidate the detailed pathogenesis of MIH, we retrospectively reviewed the data of all patients with C. perfringens bacteremia from two university hospitals from 2000 to 2014. The medical records and laboratory data of 60 patients with bacteremia, including 6 patients with MIH and 54 patients without MIH, were analyzed. Patients with MIH had higher rates of intense pain at onset, impaired consciousness, shock at presentation, hematuria, metabolic acidosis, and gas formation than patients without MIH. The antibiotic susceptibility of the clinical isolates was not significantly different between the two groups. All patients with MIH, although treated with appropriate antimicrobial agents, died within 26 h of admission due to rapidly progressive acute lung injury or acute respiratory distress syndrome, and the median time from arrival at the hospital to death was only 4 h and 20 min. When clinicians observe intravascular hemolysis in blood samples from patients with characteristic symptoms of MIH, they should prepare for a severe disease outcome. The underlying pathophysiology of fulminant cases must be investigated.

Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling.

Cholesterol is an abundant lipid in mammalian plasma membranes that regulates the reception of the Hedgehog (Hh) signal in target cells. In vertebrates, cell-surface organelles called primary cilia function as compartments for the propagation of Hh signals. Recent structural, biochemical, and cell-biological studies have led to the model that Patched-1 (PTCH1), the receptor for Hh ligands, uses its transporter-like activity to lower cholesterol accessibility in the membrane surrounding primary cilia. Cholesterol restriction at cilia may represent the long-sought-after mechanism by which PTCH1 inhibits Smoothened (SMO), a cholesterol-responsive transmembrane protein of the G protein-coupled receptor superfamily that transmits the Hh signal across the membrane.Protein probes based on microbial cholesterol-binding proteins revealed that PTCH1 controls only a subset of the total cholesterol molecules, a biochemically defined fraction called accessible cholesterol. The accessible cholesterol pool coexists (and exchanges) with a pool of sequestered cholesterol, which is bound to phospholipids like sphingomyelin. In this chapter, we describe how to measure the accessible and sequestered cholesterol pools in live cells with protein-based probes. We discuss how to purify and fluorescently label these probes for use in flow cytometry and microscopy-based measurements of the cholesterol pools. Additionally, we describe how to modulate accessible cholesterol levels to determine if this pool regulates Hh signaling (or any other cellular process of interest).

Pathogenic Characterization of Clostridium perfringens Strains Isolated From Patients With Massive Intravascular Hemolysis.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.