Cholesterol-dependent Cytolysins Research Articles

Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Bacterial cholesterol-dependent cytolysins and their interaction with the human immune response.

Many cholesterol-dependent cytolysin (CDC)-producing pathogens pose a significant threat to human health. Herein, we review the pore-dependent and -independent properties CDCs possess to assist pathogens in evading the host immune response. Within the last 5 years, exciting new research suggests CDCs can act to inhibit important immune functions, disrupt critical cell signaling pathways, and have tissue-specific effects. Additionally, recent studies have identified a key region of CDCs that generates robust immunity, providing resources for the development of CDC-based vaccines. This review provides new information on how CDCs alter host immune responses to aid bacteria in pathogenesis. These studies can assist in the design of more efficient vaccines and therapeutics against CDCs that will enhance the immune response to CDC-producing pathogens while mitigating the dampening effects CDCs have on the host immune response.

Populational genomic insights of Paraclostridium bifermentans as an emerging human pathogen.

Paraclostridium bifermentans (P.b) is an emerging human pathogen that is phylogenomically close to Paeniclostridium sordellii (P.s), while their populational genomic features and virulence capacity remain understudied. Here, we performed comparative genomic analyses of P.b and compared their pan-genomic features and virulence coding profiles to those of P.s. Our results revealed that P.b has a more plastic pangenome, a larger genome size, and a higher GC content than P.s. Interestingly, the P.b and P.s share similar core-genomic functions, but P.b encodes more functions in nutrient metabolism and energy conversion and fewer functions in host defense in their accessory-genomes. The P.b may initiate extracellular infection processes similar to those of P.s and Clostridium perfringens by encoding three toxin homologs (i.e., microbial collagenase, thiol-activated cytolysin, phospholipase C, which are involved in extracellular matrices degradation and membrane damaging) in their core-genomes. However, P.b is less toxic than the P.s by encoding fewer secretion toxins in the core-genome and fewer lethal toxins in the accessory-genome. Notably, P.b carries more toxins genes in their accessory-genomes, particularly those of plasmid origin. Moreover, three within-species and highly conserved plasmid groups, encoding virulence, gene acquisition, and adaptation, were carried by 25-33% of P.b strains and clustered by isolation source rather than geography. This study characterized the pan-genomic virulence features of P.b for the first time, and revealed that P. bifermentans is an emerging pathogen that can threaten human health in many aspects, emphasizing the importance of phenotypic and genomic characterizations of in situ clinical isolates.

Impact of Endogenous Pneumococcal Hydrogen Peroxide on the Activity and Release of Pneumolysin.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

Deciphering the early stages of pore formation in the cholesterol-dependent cytolysins

Deciphering the early stages of pore formation in the cholesterol-dependent cytolysins

LXR Signaling Mediates Protection from Bacterial Toxins

Abstract Necrotizing fasciitis (NF) is an aggressive skin and soft tissue infection characterized by massive tissue destruction. Current treatment strategies are limited to aggressive and repeated surgical debridement, coupled with systemic antibiotics. Thus, new therapeutic approaches that preserve tissue integrity would be beneficial. Cholesterol-dependent cytolysins (CDCs) are toxins produced by select gram-positive microbes implicated in NF. CDCs recognize cholesterol in membranes of cells for pore formation and subsequent cellular and tissue damage. Recently, we have shown that interferon signaling is able to protect cells from CDCs via reprogramming cholesterol homeostasis. This observation led to us to ask if pharmacological targeting of cholesterol metabolism provide similar protection. Liver X Receptor (LXRs) are key transcriptional regulators of cholesterol metabolism. We find that pharmacological LXR activation protects mice from Streptolysin O (SLO)-induced tissue damage. LXR stimulation in various cell types, such as macrophages, neutrophils, and endothelial cells induced resistance to CDCs by abrogating the ability of CDCs to directly bind to cholesterol in the plasma membrane. Ongoing mechanistic studies are investigating which metabolic pathways induced by LXRs (e.g., cholesterol efflux, biosynthesis, import, or trafficking) are responsible for the protective effects. Translationally, LXR stimulation may serve as an adjunctive therapeutics for necrotizing fasciitis and other CDC-mediated pathologies (e.g., pneumonia, pharyngitis, and septic cardiomyopathy). P01 HL146358

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

CD4+ Tcell-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+ Tcell 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 Tcell 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.

Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include: toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but Ca2+ flux triggered by aerolysin is controversial. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

The root canal microbiome diversity and function. A whole-metagenome shotgun analysis.

To evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. Twenty-two samples from patients with primary root canal infections, and 18 samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analysed with whole-metagenome shotgun sequencing at a depth of 20 M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. Microbial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity p = .001). Community composition was significantly different in primary versus secondary infection (R = .11, p = .005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum, Prevotella oris, Eubacterium infirmum, Tannerella forsythia, Atopobium rimae, Peptostreptococcus stomatis, Bacteroidetes bacterium oral taxon 272, Parvimonas micra, Olsenella profusa, Streptococcus anginosus, Lactobacillus rhamnosus, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, Fusobacterium nucleatum, Eubacterium brachy and Solobacterium moorei. The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signalling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, haemolysins, thiol-activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. Despite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.

A simple method to differentiate three classes of cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs) are proteinaceous toxins widely distributed in gram-positive pathogenic bacteria. CDCs can be classified into three groups (I–III) based on the mode of receptor recognition. Group I CDCs recognize cholesterol as their receptor. Group II CDC specifically recognizes human CD59 as the primary receptor on the cell membrane. Only intermedilysin from Streptococcus intermedius has been reported as a group II CDC. Group III CDCs recognize both human CD59 and cholesterol as receptors. CD59 contains five disulfide bridges in its tertiary structure. Therefore, we treated human erythrocytes with dithiothreitol (DTT) to inactivate CD59 on membranes. Our data showed that DTT treatment caused a complete loss of recognition of intermedilysin and an anti-human CD59 monoclonal antibody. In contrast, this treatment did not affect the recognition of group I CDCs, judging from the fact that DTT-treated erythrocytes were lysed with the same efficiency as mock-treated human erythrocytes. The recognition of group III CDCs toward DTT-treated erythrocytes was partially reduced, and these results are likely due to the loss of human CD59 recognition. Therefore, the degree of human CD59 and cholesterol requirements of uncharacterized group III CDCs frequently found in Mitis group streptococci can be easily estimated by comparing the amounts of hemolysis between DTT-treated and mock-treated erythrocytes.

Glucocorticoids increase tissue cell protection against pore-forming toxins from pathogenic bacteria

Many species of pathogenic bacteria damage tissue cells by secreting toxins that form pores in plasma membranes. Here we show that glucocorticoids increase the intrinsic protection of tissue cells against pore-forming toxins. Dexamethasone protected several cell types against the cholesterol-dependent cytolysin, pyolysin, from Trueperella pyogenes. Dexamethasone treatment reduced pyolysin-induced leakage of potassium and lactate dehydrogenase, limited actin cytoskeleton alterations, reduced plasma membrane blebbing, and prevented cytolysis. Hydrocortisone and fluticasone also protected against pyolysin-induced cell damage. Furthermore, dexamethasone protected HeLa and A549 cells against the pore-forming toxins streptolysin O from Streptococcus pyogenes, and alpha-hemolysin from Staphylococcus aureus. Dexamethasone cytoprotection was not associated with changes in cellular cholesterol or activating mitogen-activated protein kinase (MAPK) cell stress responses. However, cytoprotection was dependent on the glucocorticoid receptor and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR). Collectively, our findings imply that glucocorticoids could be exploited to limit tissue damage caused by pathogens secreting pore-forming toxins.

Cholesterol dependent cytolysins and the brain: Revealing a potential therapeutic avenue for bacterial meningitis.

Bacterial meningitis is a catastrophic nervous system disorder with high mortality and wide range of morbidities. Some of the meningitis-causing bacteria occupy cholesterol dependent cytolysins (CDCs) to increase their pathogenicity and arrange immune-evasion strategy. Studies have observed that the relationship between CDCs and pathogenicity in these meningitides is complex and involves interactions between CDC, blood-brain barrier (BBB), glial cells and neurons. In BBB, these CDCs acts on capillary endothelium, tight junction (TJ) proteins and neurovascular unit (NVU). CDCs also observed to elicit intriguing effects on brain inflammation which involves microglia and astrocyte activations, along with neuronal damage as the end-point of pathological pathways in bacterial meningitis. As some studies mentioned potential advantage of CDC-targeted therapeutic mechanisms to combat CNS infections, it might be a fruitful avenue to deepen our understanding of CDC as a candidate for adjuvant therapy to combat bacterial meningitis.

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Species-specific gene duplication in Clostridia produces variations of cholesterol-dependent cytolysin with different cytotoxicity

Cholesterol-dependent cytolysin (CDC) is a bacterial toxin that binds to eukaryotic cholesterol-containing membranes, forms oligomeric complexes, and is inserted into the bilayer to create large aqueous pores. Recently, we reported a species-specific duplication of the hemolysin gene in group III Clostridium botulinum. The duplicated genes (bly1 and bly2) encoded two separate CDC proteins (botulinolysins; BLY1 and BLY2). Here, we aimed to investigate whether BLY1 and BLY2 exert differential cytotoxicity. We isolated two bly genes from C. botulinum and evaluated the cytotoxicity of two recombinant BLY proteins (rBLY1 and rBLY2) in HeLa, IEC-6, and NRK cells. rBLYs were cytotoxic to equine erythrocytes. rBLY1 showed higher hemolytic activity than rBLY2. rBLY2 showed no or very weak cytotoxicity to the HeLa, IEC-6, and NRK cells, whereas rBLY1 showed high cytotoxicity to these cells. The comparison of the amino acid sequence of BLYs with those of other CDCs revealed that the already-known amino acid residues involved in cholesterol-containing membrane recognition, oligomerization, and insertion into membranes are well conserved in both BLYs. However, several amino acid substitutions were observed in the conserved regions, particularly in L2 and L3 regions involved in cell binding. These findings suggest that gene duplication in group III C. botulinum evolved distinct functional specializations, and differential cytotoxicity of BLY1 and BLY2 could be due to the amino acid substitution in the conserved regions. However, the structural and functional comparisons of the two BLYs are essential to gain insights into the function of the CDCs.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins using Leishmania major.

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Single Point Mutation and Its Role in Specific Pathogenicity to Reveal the Mechanism of Related Protein Families.

ABSTRACTPyolysin (PLO) is secreted by Trueperella pyogenes as a water-soluble monomer after forming transmembrane β-barrel channels in the cell membrane by binding cholesterol. Two significantly conserved residues at domain 1 of PLO are mutated, which provides novel evidence of a relationship between conformational change and interaction with the cell membrane and uncovers the pore formation mechanism of the cholesterol-dependent cytolysin (CDC) family. Moreover, PLO is a special member of the CDCs, which the percentage of sequence identities between PLO and other CDC members is from 31% to 45%, while others are usually from 40% to 70%. It is important to understand that at very low sequence identities, models can be different in the pathogenic mechanisms of these CDC members, which are dedicated to a large number of Gram-positive bacterial pathogens. Our studies, for the first time, located and mutated two different highly conserved structural sites in the primary structure critical for PLO structure and function that proved the importance of these sites. Together, novel and repeatable observations into the pore formation mechanism of CDCs are provided by our findings.IMPORTANCE Postpartum disease of dairy cows caused by persistent bacterial infection is a global disease, which has a serious impact on the development of the dairy industry and brings huge economic losses. As one of the most relevant pathogenic bacteria for postpartum diseases in dairy cows, Trueperella pyogenes can secrete pyolysin (PLO), a member of the cholesterol-dependent cytolysin (CDC) family and recognized as the most important toxin of T. pyogenes. However, the current research work on PLO is still insufficient. The pathogenic mechanism of this toxin can be fully explored by changing the local structure and overall function of the toxin by a previously unidentified single point mutation. These studies lay the groundwork for future studies that will explore the contribution of this large family of CDC proteins to microbial survival and human disease.

V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy.

Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.

Single-molecule tracking of perfringolysin O assembly and membrane insertion uncoupling.

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol-dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.

Pneumolysin boosts the neuroinflammatory response to Streptococcus pneumoniae through enhanced endocytosis

In pneumococcal meningitis, bacterial growth in the cerebrospinal fluid results in lysis, the release of toxic factors, and subsequent neuroinflammation. Exposure of primary murine glia to Streptococcus pneumoniae lysates leads to strong proinflammatory cytokine and chemokine production, blocked by inhibition of the intracellular innate receptor Nod1. Lysates enhance dynamin-dependent endocytosis, and dynamin inhibition reduces neuroinflammation, blocking ligand internalization. Here we identify the cholesterol-dependent cytolysin pneumolysin as a pro-endocytotic factor in lysates, its elimination reduces their proinflammatory effect. Only pore-competent pneumolysin enhances endocytosis in a dynamin-, phosphatidylinositol-3-kinase- and potassium-dependent manner. Endocytic enhancement is limited to toxin-exposed parts of the membrane, the effect is rapid and pneumolysin permanently alters membrane dynamics. In a murine model of pneumococcal meningitis, mice treated with chlorpromazine, a neuroleptic with a complementary endocytosis inhibitory effect show reduced neuroinflammation. Thus, the dynamin-dependent endocytosis emerges as a factor in pneumococcal neuroinflammation, and its enhancement by a cytolysin represents a proinflammatory control mechanism.