Cholesterol-dependent Cytolysins Research Articles

Streptolysin O concentration and activity is central to in vivo phenotype and disease outcome in Group A Streptococcus infection

Group A Streptoccocus (GAS) is among the most diverse of all human pathogens, responsible for a range of clinical manifestations, from mild superficial infections such as pharyngitis to serious invasive infections such as necrotising fasciitis and sepsis. The drivers of these different disease phenotypes are not known. The GAS cholesterol-dependent cytolysin, Streptolysin O (SLO), has well established cell and tissue destructive activity. We investigated the role of SLO in determining disease outcome in vivo, by using two different clinical lineages; the recently emerged hypervirulent outbreak emm type 32.2 strains, which result in sepsis, and the emm type 1.0 strains which cause septic arthritis. Using clinically relevant in vivo mouse models of sepsis and a novel septic arthritis model, we found that the amount and activity of SLO was vital in determining the course of infection. The emm type 32.2 strain produced large quantities of highly haemolytic SLO that resulted in rapid development of sepsis. By contrast, the reduced concentration and lower haemolytic activity of emm type 1.0 SLO led to translocation of bacteria from blood to joints. Importantly, sepsis associated strains that were attenuated by deletion or inhibition of SLO, then also translocated to the joint, confirming the key role of SLO in determining infection niche. Our findings demonstrate that SLO is key to in vivo phenotype and disease outcome. Careful consideration should be given to novel therapy or vaccination strategies that target SLO. Whilst neutralising SLO activity may reduce severe invasive disease, it has the potential to promote chronic inflammatory conditions such as septic arthritis.

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.

Molecular Mechanisms of Mast Cell Activation by Cholesterol-Dependent Cytolysins.

Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity.

Electrochemical properties of tethered lipid bilayers on thin film silver substrates

In this work, the assembly of functional tethered bilayer membranes (tBLMs) on silver surface was demonstrated for the first time. To avoid degradation of thin silver films the non-corrosive mixture of thiols composed of WC14 molecular anchor and 3-mercapto-1-propanol (3M1P) serving as a backfiller was used to self-assemble monolayers which allowed fusion of multilamellar vesicles and tBLM formation. tBLMs on silver films exhibited high stability and low residual defect densities as measured by the electrochemical impedance spectroscopy (EIS). Silver supported tBLMs demonstrated all features typical for interactions of pore-forming proteins and bilayer membranes. Specifically, the EIS spectral response was typical for the formation of water filled defects when tBLMs were exposed to the solution of vaginolysin (VLY), the cholesterol dependent cytolysin. Increase of defect density was observed only in case tBLMs contained significant amount, e.g., 50 %, of cholesterol, thus confirming functional reconstitution of vaginolysin into tBLMs. This observation is consistent with the biological nature of interaction between the bilayer and the cholesterol dependent cytolysins. The reconstitution of VLY was confirmed by the direct imaging of the protein entities in tBLMs by the atomic force microscopy (AFM). Notably, the quantitative estimate of the functional protein-pores in tBLMs measured by EIS, was found to be close to the number of VLY entities in tBLMs measured by the AFM. Our work demonstrates that silver can serve as less expensive alternative to gold in designing tBLM based devices, for example phospholipid biosensors, as well as the experimental platform for studying membrane protein interactions by electrochemical, and microscopy techniques.

Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol-dependent cytolysins.

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.

Macrophages activate a MLK3/MEK signaling axis to trigger Annexin mediated membrane repair after cholesterol-dependent cytolysin damage

Abstract The innate immune system is critical to promoting host survival during bacterial necrotizing soft tissue infections (NSTIs). NSTI causing pathogens like Streptococcus pyogenes and Clostridium perfringens use pore-forming toxins to kill innate immune cells like macrophages. Toxins cause rapid influx of Ca2+, which triggers repair responses. Ca2+ sensitive proteins like Annexins repair via clogging the injured site and are shed with toxin pores on microvesicles during repair. However, the molecular mechanism orchestrating this repair is unknown. Since Mitogen Activated Protein Kinase (MAPK) pathways initiate long-term inflammatory responses, short-term signaling could drive membrane repair. We hypothesized that Mixed Lineage Kinase 3 (MLK3) activates a prosurvival kinase cascade (MEK/ERK) to activate membrane repair by microvesicle shedding in macrophages. We tested cell viability and repair responses to toxins after blocking proteins in the MAPK pathways using inhibitors or RNAi in primary murine macrophages and cell lines. We found MLK3 or MEK inhibition significantly reduced toxin resistance. Protective MLK3/MEK activity was downstream of Ca2+ and accounted for ~ 60% of all Ca2+ dependent repair. MEK inhibition led to increased cytosolic Ca2+ flux after toxin damage and reduced ANXA2, but not ANXA1 or ANXA6, translocation to damaged site. MEK blockade reduced shedding of toxin pores. Overall, these findings suggest that a Ca2+ dependent MLK3/MEK axis protects macrophages and other cells from pore-forming toxins by recruiting ANXA2 to the cell membrane to promote repair. Modulation of this axis could promote resistance to NSTIs.

Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition.

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.

Oligomeric State Transitions of Pore Forming Toxins Induce Non-Monotonic Lipid Dynamics in Biomembranes

Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane remodelling and related repair processes during attack by PFTs remain largely unexplored. in our recent experiments [1] with GUVs exposed to prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO), we were able to correlate changes in lipid dynamics to membrane bound conformational states of LLO oligomers. We now report non-monotonic variations in lipid diffusion due to interplay of lipid ejection and crowding by membrane bound oligomers of LLO. Our experiments monitor lipid dynamics and membrane micro-rheology over a broad range of protein-to-lipid ratios encompassing the dilute, intermediate as well as the crowded regime. This systematic variation of protein concentration reveals for the first time a unique non-monotonic behaviour of lipid diffusivity as a function of PFT concentration which falls outside the purview of existing membrane dynamics models. by monitoring the fluorescent intensity of lipids using confocal microscopy and number of lipids in FCS we provide direct evidence of lipid loss during pore formation. Not only do we observe the anticipated crowding induced decreasing lipid diffusivities at high protein concentration, but our results reveal a counter-intuitive increase in lipid diffusivity at intermediate LLO concentrations. We quantitatively capture these trends using an established model based on the dependence of membrane lipidxdiffusion on the available free area. We correlate changes in observed lipid diffusivity to the free area contributions from different PFT oligomeric states, thus providing unique insights connecting lipid dynamics to bound protein distributions. 1. Ilanila IP, Ramesh Cheerla, K Ganapathy Ayappa and Jaydeep K Basu, “Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins”, Proc Nat Acad Sci. 116, 12839 (2019).

Alginate Nanoparticles Enhance Anti-Clostridium perfringens Activity of the Leaderless Two-Peptide Enterocin DD14 and Affect Expression of Some Virulence Factors.

Here, we report a novel approach to improve the anti-Clostridium perfringens activity of the leaderless two-peptide enterocin 14 (EntDD14), produced by Enterococcus faecalis 14. This strategy consists of loading EntDD14 onto alginate nanoparticles (Alg NPs), which are made of a safe polymer. The resulting formulation (EntDD14/Alg NPs) was able to reduce up to four times the minimum inhibitory concentration (MIC) of EntDD14 against C. perfringens pathogenic strains isolated from a chicken affected by necrotic enteritis (NE). Interestingly, this formulation remained active under conditions mimicking the human and chicken gastric tract. Assays conducted to establish the impact of this formulation on the intestinal epithelial cell line Caco-2 and the human colorectal adenocarcinoma cell line HT29 revealed the absence of cytotoxicity of both free-EntDD14 and EntDD14 loaded onto the alginate nanoparticles (EntDD14/Alg NPs) against the aforementioned eukaryotic cells, after 24h of contact. Notably, EntDD14 and EntDD14/Alg NPs, both at a sub-inhibitory concentration, affected the expression of genes coding for clostridialtoxins such as toxin α, enteritis B-like toxin, collagen adhesion protein and thiol-activated cytolysin. Further, expression of these genes was significantly down-regulated following the addition of EntDD14/Alg NPs, but not affected upon addition of EntDD14 alone. This study revealed that adsorption of EntDD14 onto Alg NPs leads to a safe and active formulation (EntDD14/Alg NPs) capable of affecting the pathogenicity of C. perfringens. This formulation could therefore be used in the poultry industry as a novel approach to tackle NE.

How protein engineering has revealed the molecular mechanisms of pore-forming toxins.

Pore-forming proteins are found in prokaryotes, vertebrates, and invertebrates, and when involved in pathogenic processes they are classified as pore-forming toxins (PFTs). The use of gene engineering methods in combination with the information provided by the high-resolution crystal structures of the PFTs have allowed investigators to gain a deep understanding of their pore-forming mechanisms. In this chapter, we discuss how protein engineering has helped us and others to reveal the molecular mechanisms of pore formation by prokaryotic PFTs with an emphasis on our experiences with the cholesterol-dependent cytolysins (CDCs).

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Structural basis for tuning activity and membrane specificity of bacterial cytolysins

Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.

Molecular Dynamics Study of Lipid and Cholesterol Reorganization Due to Membrane Binding and Pore Formation by Listeriolysin O.

Pore-forming toxins are proteinsexpressed by bacteria to primarily cause infections in the host cell. Cholesterol-dependent cytolysins (CDCs) are a class of proteins whose pore-forming ability requires the presence of cholesterol in the membrane. Upon binding to the target cell, cholesterol-recognizing residues in the membrane binding D4 subdomain assist in stabilizing both the pre-pore and pore states which occur during protein oligomerization on the cell membrane. Super resolution-stimulated emission depletion (STED) microscopy experiments (Sarangi et al. in Langmuir, 32:9649-9657, 2016) on supported lipid bilayers have shown that listeriolysin (LLO), a CDC expressed by Listeria monocytogenes, a food-borne pathogen, induces both spatial and dynamic heterogeneity in bilayer membranes. Here, we use all-atom molecular dynamics simulations to explore molecular details of the induced membrane reorganization by considering two distinct states of the oligomerized LLO protein in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol membrane. In the membrane bound (MB) state, four D4 subunits are placed at the bilayer interface in a pre-pore configuration and the membrane-inserted (MI) state consists of a tetrameric arc-like pore configuration. By analyzing lipid-order parameters, mobilities, and diffusion coefficients, we examine the induced spatial heterogeneity that occurs in both the MB and MI states. This heterogeneity is primarily driven by the local density enhancement of cholesterol in the vicinity of the MB, D4 subunits leading to distinct differences in lipid and cholesterol mobility across the two leaflets as well as enhanced lipid mobilities in regions where cholesterol is depleted. The leaflet-induced heterogeneity is greater for the MB state when compared with the MI state and the dynamic variations are more pronounced in the extracellular leaflet when compared with the cytosolic leaflet. Our study provides molecular-level insights into the inhomogeneity and perturbation induced in bilayer membranes upon LLO binding and pore formation and is expected to represent trends across PFTs in the broad CDC subclass of proteins.

Assessing the Genomic Variability of Gardnerella vaginalis through Comparative Genomic Analyses: Evolutionary and Ecological Implications.

Gardnerella vaginalis is described as a common anaerobic vaginal bacterium whose presence may correlate with vaginal dysbiotic conditions. In the current study, we performed phylogenomic analyses of 72 G. vaginalis genome sequences, revealing noteworthy genome differences underlying a polyphyletic organization of this taxon. Particularly, the genomic survey revealed that this species may actually include nine distinct genotypes (GGtype1 to GGtype9). Furthermore, the observed link between sialidase and phylogenomic grouping provided clues of a connection between virulence potential and the evolutionary history of this microbial taxon. Specifically, based on the outcomes of these in silico analyses, GGtype3, GGtype7, GGtype8, and GGtype9 appear to have virulence potential since they exhibited the sialidase gene in their genomes. Notably, the analysis of 34 publicly available vaginal metagenomic samples allowed us to trace the distribution of the nine G. vaginalis genotypes identified in this study among the human population, highlighting how differences in genetic makeup could be related to specific ecological properties. Furthermore, comparative genomic analyses provided details about the G. vaginalis pan- and core genome contents, including putative genetic elements involved in the adaptation to the ecological niche as well as many putative virulence factors. Among these putative virulence factors, particularly noteworthy genes identified were the gene encoding cholesterol-dependent cytolysin (CDC) toxin vaginolysin and genes related to microbial biofilm formation, iron uptake, adhesion to the vaginal epithelium, as well as macrolide antibiotic resistance.IMPORTANCE The identification of nine different genotypes among members of G. vaginalis allowed us to distinguish an uneven distribution of virulence-associated genetic traits within this taxon and thus suggest the potential occurrence of putative pathogen and commensal G. vaginalis strains. These findings, coupled with metagenomics microbial profiling of human vaginal microbiota, permitted us to get insights into the distribution of the genotypes among the human population, highlighting the presence of different structural communities in terms of G. vaginalis genotypes.

Vibrio vulnificus Hemolysin: Biological Activity, Regulation of vvhA Expression, and Role in Pathogenesis.

The Vibrio vulnificus (V. vulnificus) hemolysin (VVH) is a pore-forming cholesterol-dependent cytolysin (CDC). Although there has been some debate surrounding the in vivo virulence effects of the VVH, it is becoming increasingly clear that it drives different cellular outcomes and is involved in the pathogenesis of V. vulnificus. This minireview outlines recent advances in our understanding of the regulation of vvhA gene expression, the biological activity of the VVH and its role in pathogenesis. An in-depth examination of the role of the VVH in V. vulnificus pathogenesis will help reveal the potential targets for therapeutic and preventive interventions to treat fatal V. vulnificus septicemia in humans. Future directions in VVH research will also be discussed.

Ancient but Not Forgotten: New Insights Into MPEG1, a Macrophage Perforin-Like Immune Effector.

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data.

A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins.

The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.

Mannose receptor‐derived peptides neutralize pore‐forming toxins and reduce inflammation and development of pneumococcal disease

Cholesterol‐dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC‐1) on specific immune cells thereby evoking an anti‐inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC‐1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC‐1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC‐induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY‐induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre‐treatment of human DCs with peptides blocked bacterial uptake via MRC‐1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co‐administration of peptide‐loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC‐1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs.

Interaction of Macrophages and Cholesterol-Dependent Cytolysins: The Impact on Immune Response and Cellular Survival

Cholesterol-dependent cytolysins (CDCs) are key virulence factors involved in many lethal bacterial infections, including pneumonia, necrotizing soft tissue infections, bacterial meningitis, and miscarriage. Host responses to these diseases involve myeloid cells, especially macrophages. Macrophages use several systems to detect and respond to cholesterol-dependent cytolysins, including membrane repair, mitogen-activated protein (MAP) kinase signaling, phagocytosis, cytokine production, and activation of the adaptive immune system. However, CDCs also promote immune evasion by silencing and/or destroying myeloid cells. While there are many common themes between the various CDCs, each CDC also possesses specific features to optimally benefit the pathogen producing it. This review highlights host responses to CDC pathogenesis with a focus on macrophages. Due to their robust plasticity, macrophages play key roles in the outcome of bacterial infections. Understanding the unique features and differences within the common theme of CDCs bolsters new tools for research and therapy.

Cytotoxic property of Streptococcus mitis strain producing two different types of cholesterol-dependent cytolysins

Streptococcus mitis strain Nm-65 secretes an atypical 5-domain-type cholesterol-dependent cytolysin (CDC) called S. mitis-derived human platelet aggregation factor (Sm-hPAF) originally described as a platelet aggregation factor. Sm-hPAF belongs to Group III CDC that recognize both membrane cholesterol and human CD59 as the receptors, and shows preferential activity towards human cells. Draft genome analyses have shown that the Nm-65 strain also harbors a gene encoding another CDC called mitilysin (MLY). This CDC belongs to Group I CDC that recognizes only membrane cholesterol as a receptor, and it is a homolog of the pneumococcal CDC, pneumolysin. The genes encoding each CDC are located about 20 kb apart on the Nm-65 genome. Analysis of the genomic locus of these CDC-encoding genes in silico showed that the gene encoding Sm-hPAF and the region including the gene encoding MLY were both inserted into a specific locus of the S. mitis genome. The results obtained using deletion mutants of the gene(s) encoding CDC in Nm-65 indicated that each CDC contributes to both hemolysis and cytotoxicity, and that MLY is the major hemolysin/cytolysin in Nm-65. The present study aimed to determine the potential pathogenicity of an S. mitis strain that produces two CDC with different receptor recognition properties and secretion modes.