The Yin and Yang of Pneumolysin During Pneumococcal Infection

Abstract

Pneumolysin (PLY) is a pore-forming toxin produced by the human pathobiont Streptococcus pneumoniae, the major cause of pneumonia worldwide. PLY, a key pneumococcal virulence factor, can form transmembrane pores in host cells, disrupting plasma membrane integrity and deregulating cellular homeostasis. At lytic concentrations, PLY causes cell death. At sub-lytic concentrations, PLY triggers host cell survival pathways that cooperate to reseal the damaged plasma membrane and restore cell homeostasis. While PLY is generally considered a pivotal factor promoting S. pneumoniae colonization and survival, it is also a powerful trigger of the innate and adaptive host immune response against bacterial infection. The dichotomy of PLY as both a key bacterial virulence factor and a trigger for host immune modulation allows the toxin to display both “Yin” and “Yang” properties during infection, promoting disease by membrane perforation and activating inflammatory pathways, while also mitigating damage by triggering host cell repair and initiating anti-inflammatory responses. Due to its cytolytic activity and diverse immunomodulatory properties, PLY is integral to every stage of S. pneumoniae pathogenesis and may tip the balance towards either the pathogen or the host depending on the context of infection.

1 Introduction

Streptococcus pneumoniae (Sp or pneumococcus) is an extracellular Gram-positive bacterium that asymptomatically colonizes the upper respiratory tract of 5–10% healthy adults and 20–40% of children (1). Sp was considered the major cause of lower respiratory infections (LRI) with a global incidence of 2.7% worldwide, resulting in more than a million deaths per year and thus ranking among the deadliest bacteria (2). In the United States alone, pneumococcal pneumonia leads to over 150,000 annual hospitalizations. Other forms of Sp infectious complications such as otitis media, bacteremia, and meningitis are also significant healthcare burdens, leading to an average of 5 000 000, 4 000, and 2 000 cases/year respectively (source CDC: https://www.cdc.gov/pneumococcal/clinicians/clinical-features.html). Elderly and children under 5 are particularly susceptible to severe disease, with an invasive pulmonary disease (IPD) incidence of 7%. A major health concern and economic burden, Sp infection results in over $17 billion in direct medical costs annually in the US (3, 4).

Sp is naturally transformable and displays high genome plasticity, contributing to the rapid emergence of antibiotic resistance and evasion of vaccine-mediated immunity (1, 5). Effective Sp prevention and treatment is complicated by the appearance multidrug-resistant infections, non-vaccine serotypes and aging of the world’s population (4, 6). Approximately 30% of Sp cases involved isolates resistant to one or more antibiotics (https://www.cdc.gov/drugresistance/pdf/threats-report/strep-pneumoniae-508.pdf). Worldwide, pneumococci resistant to penicillin, erythromycin, and trimethoprim-sulfamethoxazole are on the rise (7). To a lower extent, resistance to tetracycline, chloramphenicol and fluoroquinolone have also been identified (8). While in the United States, multi-drug resistant pneumococci prevalence has been reduced since introduction of pneumococcal vaccines, the risk remains high in susceptible populations, especially individuals aged 65 and over (9). Vaccination has been the cornerstone of pneumococcal disease prevention. However, currently, the two classes of pneumococcal vaccines, the 23-valent pneumococcal polysaccharide vaccine (PPSV23) and the pneumococcal vaccines based on protein-conjugated polysaccharides (PCV13/PCV23), only protect against a subset of over 90 different pneumococcal capsular variants (capsular serotypes) (10). Through both the expansion of pre-existing non-vaccine pneumococcal serotypes and serotype ‘switching’, an exchange of capsular polysaccharide genes through transformation, infectious strains not covered by standard vaccination are on the rise (11). As the efficacy of traditional antibiotics and vaccines become compromised, understanding the mechanisms of action of pneumococcal virulence determinants is of critical importance for the development of new therapeutics.

Sp transmits predominantly via aerosol as the bacteria is harbored in the nasopharynx. Asymptomatic Sp carriage can lead to localized infection of tissues contiguous with the nasopharynx, causing sinusitis, otitis media, and pneumonia (1). The bacterium is also capable of subsequent systemic spread to the heart and brain upon access to the bloodstream, causing serious diseases such as cardiac dysfunction and meningitis (1) (Figure 1). The versatility of Sp is reflected by the diversity of interactions with its host depending on the site of infection and degree of disease. To colonize the host nasopharynx, Sp forms biofilms on the mucosa of the upper respiratory tract (12). This causes mucosal inflammation which promotes bacterial shedding in secretions leading to transmission (13). Long-term asymptomatic nasopharyngeal carriage supports inflammation-induced transmission and predisposes the host to develop disease (Figure 1A). Sp can then spread from the nasopharynx to neighboring tissues, such as the sinusoidal cavities to cause sinusitis, the middle ear to cause otitis media, or the eye to cause keratitis (Figures 1B, C), all of which can be recurrent infections especially in immunocompromised patients (14). Sp aspiration into the lower respiratory tract causes pneumonia, where infection damages alveolar epithelial and endothelial cells promoting tissue permeability and induces thrombotic events (Figure 1D). If the immune response mounted in the lungs is not sufficient to eliminate the bacteria, Sp may gain access to the bloodstream and disseminate to cause IPD (Figures 1E–G). During IPD, Sp may invade the spleen (15), the heart (16–18), and/or may cross the blood-brain barrier (19). In the heart, Sp forms biofilms and damages both cardiomyocytes and immune cells, leading to myocardial dysfunction and long-term pneumonia-associated adverse cardiac events (PACE; Figure 1F). Sp-induced remodeling of brain tissue during meningitis likely accounts for permanent neurological damage reported in about 50% of the survivors (Figure 1G).

Figure 1

Integral to all stages of Sp pathogenesis is the host-pathogen interaction mediated by pneumolysin (PLY), a key virulence factor produced by almost all clinical isolates and currently known Sp serotypes (20). PLY can promote asymptomatic carriage, colonization, transmission, immune evasion and dissemination, highlighting the multifaceted functions of this bacterial toxin. As a member of the cholesterol-dependent cytolysin (CDC) family, PLY is a pore-forming toxin (PFT) that at lytic concentrations disrupts host cell plasma membrane (PM) and promotes the uncontrolled influx and efflux of ions, small molecules, and proteins (21). PLY-induced pore formation interferes with a plethora of cellular signal transduction pathways. Depending on PLY concentration, which dictates the extent of PM damage and intracellular Ca²⁺ overload, host cells respond by activation of either cell death or cell survival and damage repair pathways. At lytic levels, PLY-induced pores can overwhelm cellular homeostatic mechanisms, triggering both cellular proinflammatory signaling pathways and irreversible cellular injury that results in the release of molecules heightening inflammation. The ensuing tissue damage is integral to pneumococcal disease (22). For example, in the lung, exacerbated inflammation promotes tissue invasion and systemic spread (21, 23). In contrast, at the sub-lytic concentrations likely present during the early stages of infection, PLY stimulates cell-autonomous repair mechanisms that overcome PM damage, restore cell integrity, and promote cell survival (24, 25), curtailing inflammation. In addition, depending on infection dose, site, disease stage, target cell, and the immune state of the host, PLY may display either “Yin” or “Yang” components, each with potentially opposing effects on Sp pathogenesis. The Yin of PLY promotes disease by promoting cell damage either directly or by excessive inflammation. The Yang of PLY mitigates the damage by triggering host repair mechanisms and modulating inflammation, allowing the carriage of Sp without symptoms. The remarkable ability of PLY to trigger both “Yin” and “Yang” responses depending on infection context is showcased in several in vitro and in vivo infection models (26, 27). Interestingly, heterogeneous expression of PLY within individual bacteria of an isogenic Sp population favors the appearance of subpopulations expressing different levels of PLY. Recent studies showed that genetically modified PLY expressing Sp strains producing different levels of PLY triggered different outcomes during infection. Specifically, high-PLY producing bacteria induced extensive autophagosome damage allowing efficient clearance of Sp from the host cells, yet, low-PLY producing bacteria facilitated evasion of host defense mechanism and promoted the cross of the blood-brain barrier (28, 29). Furthermore, rapid and high levels of PLY release drives hypervirulence in infection by serotype 1 pneumococci (30, 31), showcasing that the plasticity of PLY expression during Sp pathogenesis is fundamental and can influence the outcome of the infection.

Here we provide a comprehensive review of PLY functions and interactions with host cells by describing the mechanism of pore formation, its multiple targets and effects on host cells, and its antipodal interactions with the host immune system. We summarize the available data and recent discoveries that highlight the “Yin” and “Yang” perspectives of PLY activity contributing to Sp disease progression, unveiling the myriad and sometimes conflicting properties of this fascinating bacterial toxin.

2 Structural and Functional Insights of Pneumolysin

2.1 PLY Export

Secreted PLY is key for infection; however, the localization and export mechanism of PLY is still debated (32). PLY can be detected in the bacterial cytosol (32, 33), non-covalently attached to the cell wall (32), in culture supernatants (32), and in circulation during Sp infection (34). Given the lack of a canonical N-terminal signal peptide that promotes protein secretion, PLY was thought to be released solely by autolysis, an idea supported by experimental data showing reduced PLY release in the presence of non-bacteriolytic antibiotics (35). Nevertheless, autolysis may not be the only mechanism for PLY secretion (32, 36). In fact, although attenuated for virulence, Sp strains lacking autolysin release wild-type levels of PLY when cultured in vitro (37, 38). In addition, a functional SecA2 system is required for the non-covalent association of PLY to cell wall peptidoglycan (39), a structure that can hinder PLY delivery to the bacterial surface (40). Indeed, enzymatic digestion of Sp peptidoglycan promotes PLY release and is detrimental for virulence (40). Thus, PLY secretion is likely determined by several mechanisms that may cooperate to foster disease (31).

2.2 Mechanism of PLY-Mediated Membrane Permeabilization

PLY shares structural and functional properties with other bacterial PFTs belonging to the CDC family, such as listeriolysin (LLO), perfringolysin (PFO), and streptolysin (SLO) (41). It binds to cholesterol residues at the PM of host cells, oligomerizes, and undergoes conformational changes to form stable pores with well-defined sizes (Figure 2).

Figure 2

To generate pores, soluble PLY interacts with the PM, multimerizes into two successive “prepore” complexes, and then inserts into the PM to generate a large pore (Figure 2A). Soluble PLY, in its monomeric state, has been shown by crystallographic methods to be an asymmetric molecule composed of 4 major domains (D1 to D4), including α-helices and β-sheets (Figure 2B). The N-terminal D1 domain, although not essential for cell binding, stabilizes overall protein structure and enables hemolytic activity (42, 43). The non-contiguous D1 and D3 are adjacent in the 3D crystal structure and linked to the C-terminal of D4 via D2 (44) (Figure 2B). Blocking monoclonal antibodies and the analysis of binding-defective point mutants indicate that D4 promotes membrane binding (21, 45–49). In particular, two residues (T459 and L460) in a conserved D4 undecapeptide that comprises a Trp-rich loop are essential for cholesterol recognition (21, 41, 50, 51) (Figure 2B, arrow). In addition, the recombinant PLY toxoid B “PdB” harboring a mutation in Trp 433 in D4 is unable to undergo the conformational changes required for membrane insertion and strains expressing this mutant show reduced virulence, further reinforcing the role of D4 in membrane binding and pore formation (13, 34).

PLY monomers multimerize, forming transient pre-pore asymmetric structures with variable diameters and composed of 40 to 44 monomers (Figure 2A) (44, 46, 52–56). D2 undergoes a 90° rotation, bringing D1 and D3 towards the PM, compacting the overall structure and establishing the late pre-pore (Figures 2A, C) (21, 53). PM insertion occurs when the D3 helix bundles (HB1 and 2) are refolded into 85A° β-hairpins (HP1 and HP2) that perforate the hydrophobic membrane and assemble into an approximately 260A° diameter transmembrane channel (Figure 2D) (52, 53). Mutations in D3 residues Y150 and T172 are found in a naturally occurring non-hemolytic variant of PLY (45, 57), pointing to their critical role in the conformational alterations required for transmembrane hairpin formation, PM insertion and subsequent pore-formation (45). Although it is unclear what advantage Sp derives from loss of PLY hemolytic activity, serotypes harboring naturally occurring non-hemolytic PLY are commonly associated with non-lethal respiratory tract infections (57) pointing to roles played by non-pore formation dependent mechanisms of PLY during Sp pathogenesis.

Additionally, it has been reported that PLY monomers may be trapped in arcs or slit-shaped oligomers (21) that have the ability to generate pores (58, 59), possibly with variable sizes, different permeabilities and distinct functional roles (55, 60). At low PLY concentrations, likely occurring at early stages of infection, complete prepore ring structures are expected to form less efficiently than arcs, and so pore formation may rely on the efficiency of the conversion of arc oligomers to pores. At high PLY concentrations, potentially reached later in infection, binding of soluble PLY to PM is efficiently followed by assembly of full rings, so cell lysis mainly depends on the PLY affinity for cholesterol (60). PM disruption following arc or full ring assembly possibly dictates different host responses according to the phase of infection and could influence the balance of Yin and Yang outcomes.

2.3 PLY as a Ligand for Host Molecules

In addition to binding cholesterol, PLY interacts with several other host molecules. Such interactions contribute to its functions as a both pro- and anti-inflammatory agent, further discussed in Section 4 below. First, through its D4 domain, PLY binds the mannose receptor C type 1 (MRC-1), expressed at the cell surface of multiple immune cell types in the airways (61). MRC-1 acts as an internalization receptor, allowing Sp to invade MRC-1-expressing dendritic cells (DCs) and alveolar macrophages. At low infectious doses when immune stimulation by bacterial PAMPs is limited, MRC-1-PLY interaction and the consequent Sp phagocytosis modulate inflammation (discussed in section 4.2.2) and promote intracellular bacterial survival in the lungs in a murine pneumococcal pneumonia model (61). Second, PLY also has the capacity to modulate the interaction of Sp with complement by functioning as a molecular decoy, binding the Fc portion of human IgG and triggering C1q recruitment and complement cascade activation (62, 63) (reviewed in section 4.2.4). This interaction is dependent on specific residues within short non-contiguous PLY sequences that are homologous to the human C-reactive protein (CRP), which also activates the complement (63). In vivo, diversion of complement proteins by PLY promotes both pulmonary and systemic infection, as PLY-deficient Sp become more virulent in complement-deficient mice (64). Lastly, PLY was suggested to bind host cell glycans such as sialylated fucosylated glycan divalent-LewisX (sLeX) (65), and computational docking studies suggested the D4 and the interface between D3 and D4 as putative binding sites for LeX and sLeX, respectively (46). Nevertheless, interactions detected in vitro utilized high sLeX : PLY ratios and thus appear to be of low affinity, and PLY:sLeX co-crystals have never been observed (46), indicating the need for further studies are needed to explore potentially biologically interactions interaction PLY and sLeX.

2.4 PLY-Mediated Pathogenesis and Targeted Cells

Due to its ability to bind to cholesterol commonly found in mammalian PMs, PLY is able to target and modulate the function of virtually all cell types and thus plays key roles in many modes of infection, such as asymptomatic carriage, local disease, or life-threatening systemic disease (66) (Figure 1).

Early in infection, PLY has vital roles in colonization of the nasopharynx and in host-to-host transmission (13, 67, 68) (Figure 1A). In vitro, PLY-deficient mutants are impaired in adherence (69) and show lower bacterial burden in the nasopharynx of intranasally infected mice (67, 68). Sp grown in vitro as a biofilm show increased ply transcription and produced high levels of PLY (70, 71), suggesting that Sp biofilms developed in the respiratory mucosa might also produce high levels of PLY. The PLY-induced mucosal inflammation promotes increased bacterial shedding in secretions and promotes transmission in an infant mouse model of Sp infection (13).

PLY is also a key molecule in the progression of Sp infection from the nasopharynx to neighboring tissues. A PLY-deficient mutant causes mild histopathological changes and lower middle ear bacterial loads in chinchilla OM model (72, 73). In guinea pig infection models, PLY directly damages cochlear hair cells (74, 75). In addition, PLY intracochlear perfusion induces a strong cytotoxic effect possibly related to pore formation at the PM of inner hair cells (76, 77) (Figure 1B). PLY also plays a role in pneumococcal ocular infections causing endophthalmitis and keratitis (78, 79). Intravitreally injection of PLY causes inflammation and tissue damage (80, 81), likely related to PLY-induced corneal epithelial cell lysis (82) (Figure 1C).

In the lungs, PLY targets pivotal cell types during acute injury at the early phases of pneumonia and becomes a potent inducer of inflammation, facilitating damage of the respiratory epithelial barrier and host tissue penetration (Figure 1D). Purified PLY damages bronchial and alveolar epithelium and slows human ciliary beating in vitro (83); it damages alveolar epithelial and endothelial cells impairing barrier function (84, 85); and causes platelet destruction thus inhibiting platelet thrombus formation (86, 87). Anti-PLY polyvalent antibodies were reported to inhibit PLY-mediated platelet annihilation and were proposed to be of pharmacological interest to treat Sp community-acquired pneumonia (87). In addition, PLY can compromise effective immune response in the lungs through several mechanisms: it induces the necroptosis of alveolar macrophages (88–90), triggers caspase-dependent cell death of dendritic cells (91, 92), promotes neutrophil transepithelial cell migration and elastase release, thus impairing macrophage phagocytosis, and damages epithelial cells (93–95). The pro-inflammatory role of PLY is discussed in detail in section 4.

If not controlled in the lungs, the bacteria can reach the bloodstream and cause IPD (Figure 1E). In the heart, sub-lytic doses of PLY disrupt the PM of cardiomyocytes leading to the influx of Ca²⁺, membrane depolarization and induction of ER stress, which together cause alterations in cardiac rhythm and depression in contractile force. At higher concentrations, PLY causes cardiomyocyte cell death, inhibiting cell contractility and promoting extensive cardiac damage which may lead to microlesion formation and cardiac remodeling likely associated with long-term adverse cardiac events described in IPD patients (34, 96, 97) (Figure 1F). Sp biofilms, established in the heart of infected mice, release PLY causing rapid macrophage killing and impairing cytokine production (98).

During brain infection, PLY induces neuronal cell death (99, 100) and targets astrocytes, rearranging cytoskeleton and altering astrocyte cell shape, which leads to remodeling of brain tissue, astrocytic retraction, and cortical astroglial reorganization (101–103) (Figure 1G). PLY-dependent astrocyte cell death is mediated by connexin 43 (Cx43), a gap junction protein forming hemichannels, that amplifies ATP release and cytosolic Ca²⁺ influx, ultimately leading to astrocyte depletion and blood-brain barrier destabilization (100). PLY also targets other cells in the brain promoting endothelial cell disruption (104), limiting microglia motility (105), and decreasing cilia beating frequency in ependymal cells (106–108), which correlates with loss of cilia and damage of ependymal ultrastructure described in rat meningitis model (109).

3 PLY Triggers Multiple Cellular Responses: Irreversible Damage or Repair

Upon interaction with cells, PLY interferes with a plethora of signal transduction pathways to induce multiple and antipodal cellular responses that define the pathogenesis in a whole organism (Figure 3). Such responses are cell-type specific and are dependent on PLY concentration, which correlates with the extent of the PM damage and of the intracellular Ca²⁺ overload. Activation of different cellular responses can tip the balance towards activation of cell death or cell survival pathways, thus promoting tissue damage or repair, respectively. At lytic concentrations, PLY triggers irreversible damage and cell death by causing massive mitochondrial damage, excessive inflammation, and tissue injury. In contrast, at sub-lytic amounts, PLY activates repair pathways triggering PM remodeling, cytoskeleton reorganization, and, by transiently activating MAPK, cell survival.

Figure 3

3.1 Mitochondrial Damage

The role of PLY in mitochondrial damage was first described in primary rat neurons infected with Sp or incubated with culture supernatants, which both induce the Ca²⁺-dependent mitochondrial release of apoptosis-inducing factor (AIF), a likely manifestation of massive mitochondrial damage (Figure 3, left) (110). In addition, PLY was shown to trigger mitochondrial swelling, loss of mitochondrial membrane potential, and impairment in mitochondrial metabolism (111, 112). Electron microscopy analysis of PLY-intoxicated neurons show direct binding of PLY to the mitochondrial membrane (111). In human alveolar epithelial cells, PLY also induces dramatic morphological alterations of the mitochondria, which are accompanied by cytosolic Ca²⁺ overload, reduction in ATP levels, membrane depolarization, increased mitochondria permeability and release of mitochondrial DNA (mtDNA) into the cytosol (Figure 3, left) (112). mtDNA can then be released extracellularly through microvesicle shedding and act as a danger signal, thus contributing to inflammation (Figure 3, right) (112). In macrophages, cytosolic mtDNA released by PLY-damaged mitochondria is recognized by STING and upregulates the expression and secretion of IFN-β, demonstrated both in vitro and in vivo in lungs of Sp infected mice (113).

3.2 Interactions With the Host Cytoskeleton

PLY was reported to bind and promote actin polymerization in vitro (114) and was shown to interact with actin filaments underneath the PM of astrocytes (115) or exposed at the surface of damaged neuronal cells (99) (Figure 3, left). In fact, PLY D4 domain interacting with cholesterol in the PM of neurons enables β-actin exposure to the outer surface of the cell, facilitating Sp adhesion and invasion and increasing cell death (99). In addition, in those cells, PLY induces cytoskeleton instability depolymerizing intracellular actin filaments likely due to increased Ca²⁺ levels (99). How these data relate to the PLY intrinsic ability to polymerize actin in vitro needs further investigation, and an understanding of the consequence of PLY-mediated Sp interactions with the actin cytoskeleton may require kinetic analysis of the infection in relevant models.

PLY also bundles and stabilizes host cell microtubules during intoxication of neuronal cells and during pneumococcal meningitis in rabbit infection models (Figure 3, left) (102). Consistent with increased microtubule stabilization, high levels of acetylated tubulin were found in both the cell culture and animal model (102). PLY-triggered microtubule stabilization is comparable to pharmacological microtubule-stabilizing agents (e.g., taxol) and has been suggested to perturb axonal transport, likely contributing to neuronal damage during pneumococcal meningitis (102). PLY-induced microtubule perturbations are dependent on cholesterol binding and require Src kinase activity but are independent of Ca²⁺ influx and actin remodeling (102).

Several reports, primarily in neuronal cells, suggest that at low concentrations, PLY interaction with host PM causes rapid cytoskeleton remodeling which translates into cell shape changes (Figure 3, right). Alterations in the brain structure detected in infected rats were associated with PLY-mediated astrocytes retraction and reshaping of focal adhesions, which are underlined by cytoskeleton reorganization (101, 103). In primary mouse astrocytes and neuronal cells, sub-lytic concentrations of PLY binding to cholesterol promotes the formation of actin stress fibers, filopodia, and lamellipodia through the activation of RhoA and Rac1 GTPases (Figure 3, right) (114, 115).

3.3 Activation of Cell Survival Pathways

PLY binding to the PM triggers activation of cell survival pathways such as the mitogen-activated protein kinase p38 (p38/MAPK) (116, 117), which is a conserved response to sub-lytic doses of PFTs dependent on K⁺ efflux (118), that can stimulate cell survival pathways (117). In epithelial cells, activation of p38/MAPK by sub-lytic concentrations of PLY (119, 120) triggers the production of pro-inflammatory cytokines (e.g., IL-8) to attract neutrophils, thus promoting an effective immune response early in infection when bacterial numbers are low (Figure 3, right) (119). In macrophages, PLY-induced p38/MAPK activation and cytokine production take place at the PM upon pore formation (119, 120) and has been suggested to promote Sp clearance (121). Later in infection, following Sp uptake by macrophages, PLY induces the trafficking of pneumococcal cell wall components to the host cell cytosol, presumably through phagosomal damage (122). If p38/MAPK activation is blocked, leakage of Sp cell wall components to the host cell cytosol is exacerbated and results in macrophage cell death. Thus, PLY-induced p38/MAPK activation protects phagosomal integrity and limits the release of bacterial components, likely modulating the recognition of pathogen-associated molecular patterns by the host surveillance systems (Figure 3, right) (122, 123).

The timing of the MAPK response to PFTs can influence tissue injury. Although MAPK activation can promote cell survival responses upon intoxication by PFTs, its subsequent modulation by protein phosphatases PP1 and PP2A, observed in epithelial cells (120), can prevent excessive inflammatory responses that could lead to irreversible tissue damage. Conversely, the transient nature of p38/MAPK activation might not be sufficient to block cell death triggered by the recognition of Sp components in the macrophage cytosol (122).

Finally, in some cell types, activation of p38/MAPK is detrimental. In SH-SY5Y neuronal cells, p38/MAPK is associated with increased neuronal cell death and neurotoxic effects (124), and in microglial, PLY-mediated MAPK activation increases ROS production and promotes senescence (Figure 3, left) (125).

3.4 Activation of Plasma Membrane Repair Mechanisms

Supernatants from stationary phase cultures of Sp produce sufficient amounts of PLY to permeabilize cells (126); however, during infection, the majority of these perforated cells are able to recover from damage and survive. PLY pore assembly renders host cell PM permeable to ions and small molecules (116, 127, 128). An increase of Ca²⁺ concentrations to above 20 μM impairs host-cell signaling and engages cell death pathways (116, 129). However, an initial increase in intracellular Ca²⁺ levels act as a danger signal and activates PM repair mechanisms to prevent cell lysis (119, 124, 130–132). Thus, at otherwise sub-lytic PLY concentrations, reduced extracellular Ca²⁺ enhances PLY toxicity and result in lysis (133, 134). In the presence of extracellular Ca²⁺, PLY-induced Ca²⁺ influx triggers the recruitment of annexins, cytoplasmic Ca²⁺ responsive proteins that bind to negatively charged phospholipids at the sites of PM injury (135). Annexin A2, which displays the highest Ca²⁺ sensitivity, is the first to translocate to the site of damage, followed by annexin A6 and A1 (Figure 3, right). Annexin translocation fosters the formation of plasmalemmal nanotubes at the pore site that culminates in the release of microvesicles enriched in PLY, annexins, actin-binding and Ca²⁺ regulated proteins, and ESCRT components (Figure 3, right) (126, 134), thus shedding the PLY pore and modulating the rise in intracellular Ca²⁺ (129). This repair process has potential immunological implications for Sp infection, as PLY-containing microvesicles induce macrophage polarization that enhances the immune response towards molecular patterns of Gram-positive bacteria (136). By contrast, in alveolar epithelial cells, human lung explants and infected mice, PLY mediates the release of microvesicles containing mitochondrial cargo that, when uptake by neutrophils suppress their ability to release ROS and thus impair efficient immune response against Sp (137). The differential PLY sensitivity of immune cells is thought to be due to differential efficiency in PM repair (138). Myeloid cells, the first-line defenders, show enhanced shedding of PLY-containing microvesicles and increased resistance to PLY. In contrast, lymphoid cells are enriched in cholesterol-containing lipid rafts, which facilitate PLY binding and pore formation, and are impaired in microvesicles formation and PM resealing activity, leading to high PLY susceptibility (138).

4 PLY Interactions With the Immune System: Pro- or Anti- Inflammatory Responses

PLY can either exacerbate or mitigate damage during infection depending on the state of the immune system as well as the infection site, dose, timing, and interacting host cell type (26, 139). It can trigger inflammation-mediated tissue damage and promote bacterial dissemination (18, 140, 141). However, PLY can elicit host-protective responses in the innate and adaptive branches of the immune system, including activation and stimulation of cytokine production by macrophages, neutrophils, endothelial, epithelial, and dendritic cells (22, 142–144). The multifaceted nature of PLY-induced immune responses may account for some of the disparate outcomes of infection by PLY-deficient strains in different infection models (Figure 4 and Table 1). This also reflects the dual relationship of Sp with host cells, sometimes invasive and inducing severe disease, and other times remaining localized as an asymptomatic colonizer (as discussed in sections 1 and 2.4.).

Figure 4

4.1 Pro-Inflammatory Properties

4.1.1 Epithelial Cell Activation

Epithelial cells are a first line of defense against Sp, in part by functioning as a physical barrier against invasion and, in the lower respiratory tract, maintaining the mucociliary elevator that removes microbes from the lung. PLY both disrupts epithelial cell junctions, compromising this barrier, and interfering with the number and organization of cilia in human and mouse airway epithelium-derived air-liquid interface organoid cultures (145, 146), crippling the mucociliary elevator. In addition, as the sentinels for infection, the mucosal epithelium produces a range of antimicrobial peptides and pro-inflammatory signaling molecules to eliminate invading pathogens and alert immune cells to response to invasion. PLY induces β-defensin 2 secretion in A549 human lung epithelial cells and in human middle-ear cells (147). PLY, is also a potent activator of cytokine and chemoattractant secretion through pore forming insult to airway epithelial cells (94, 119, 148) (Figure 4, left). PLY induces epithelial cell release of IL-1β, TNF-α (148), and the neutrophil chemoattractants IL-8 (119) and hepoxilin A₃ (HXA₃) (94, 149, 150). Accumulation of inflammatory cytokines and chemokines facilitate the recruitment of leukocytes responding to and clearing Sp. Nevertheless, in both a lung epithelial tissue culture and a mouse pulmonary infection model, HXA₃-mediated basolateral-to-apical neutrophil transmigration exacerbates damage to epithelial barrier and promotes lethal Sp systemic spread (151, 152).

4.1.2 Neutrophil Activation

As described above, neutrophil infiltration and the associated epithelial damage promote bacterial dissemination into the bloodstream, resulting in lethal systemic disease (151). While neutrophil recruitment to the lungs during early stages of infection aids Sp clearance, overtime, reduction in adenosine production by lowered CD73 expression correlated with loss of neutrophil antimicrobial efficacy (153). Extracellular ATP is shown to neutralize PLY-mediated neutrophil activation and may explain this shift in neutrophil response profile (154). Excessive neutrophil activities can be harmful to the host (140). Indeed, the prolonged accumulation of PMNs in a murine model of pneumococcal pneumonia results in increased burden and high tissue damage (153). Upon extravasation and encountering invading bacteria, exposure of PMNs to PLY can trigger the release of additional neutrophil-recruiting signals, including Ca²⁺-dependent increases in IL-8, f-MLP, phospholipase A, A2, prostaglandin E₂, and leukotriene B₄ (155, 156), reinforcing neutrophil-direction inflammation. Neutrophils at the site of infection can be further activated by Sp and PLY to produce reactive oxygen species (ROS) (93, 142, 157), degranulate, and form neutrophil extracellular traps (NETs) (Figure 4, left).

ROS have multiple immune-modulatory functions during bacterial infection: in addition to direct antimicrobial activity and signaling to modulate immune cell function, ROS can also cause damage to host cells and tissues (158). Interestingly, pretreating neutrophils with PLY promotes sensitivity to f-MLP (157), which conditions neutrophils to release higher and more sustained levels of ROS, and become more prone to degranulation (Figure 4, left) (142).

During degranulation, neutrophils secrete proteolytic enzymes capable of propagating tissue damage. These include serine proteases neutrophil elastase (NE), cathepsin G and proteinase-3, and neutrophil metalloproteinases (MMPs). PLY can trigger NE release either by degranulation of primary granules or by direct cell lysis (93, 142). Although NE degrades pneumococcal cell wall-localized aminopeptidase N and can facilitate opsonophagocytic killing in the presence of an antiphagocytic capsule (93, 159–161), excessive NE activity is detrimental to the host (162). NE has potent catalytic activity against a range of extracellular matrix proteins, including elastin, proteoglycan, fibronectin, and several collagen types (163), surfactant proteins (162), and alveolar epithelial cell junction protein such as E-cadherin (164). In addition, NE stimulates the lung epithelium to release proinflammatory cytokines and induces epithelial apoptosis (165–167). Severe pneumonia in human patients and experimental animals is associated with increased NE levels in lung bronchoalveolar lavage fluid (168, 169) and plasma (170). NE may thus be a critical inducer of epithelial permeability during pneumococcal pneumonia, compromising lung epithelial barrier and promoting bacterial dissemination (171). Cathepsin G and proteinase-3, which like NE are stored in azurophilc granules, also contribute to infection-related lung injury, albeit to a much lesser degree than NE (172). Metalloproteinases (MMPs) are neutrophil granule components that are also released following the increase in cytosolic Ca²⁺ levels resulting from PLY-mediated pore formation and can be augmented by concurrent stimulation by f-MLP (173). MMPs, especially MMP-8 and -9, have been correlated with tissue injury in pneumococcal pneumonia and meningitis (174–176). Thus, the PLY-mediated release of neutrophil granule components contributes to inflammation and pathogenesis, especially during severe forms of Sp disease.

Finally, pneumococcal capsule and PLY work in synergy to promote neutrophil extracellular trap (NET) production (162), a vital neutrophil effector function for trapping and killing extracellular microbes (Figure 4, left). While NETs exhibit significant antibacterial activity against Sp (177), their efficacy is counteracted by Sp-secreted endonucleases EndA and TatD that digest NETs and facilitate escape (162). NETs are intercalated with NE and other proteases that can damage host cells just as they can a pathogen, and excessive NETosis against Sp is implicated in promoting lung injury (178), sepsis (179), and increasing mortality (180). PLY is thus a potent activator of multiple neutrophil effector functions, most of which can propagate inflammatory tissue damage in the lung and are associated with severe pathology.

4.1.3 Inflammasome Activation

PLY induces inflammasome-dependent IL-1β secretion and initiates the pro-inflammatory cascade associated with Sp infection (181–183). PLY-mediated K⁺ efflux, which is sufficient to trigger NLRP3 activation (184), results in caspase-1 cleavage and the consequent production and secretion of IL-1β (181, 185). An alternative inflammasome activation pathway aided by PLY is the AIM2 inflammasome pathway. AIM2 senses double-stranded DNA from lysed bacteria, which can localize to host cell cytosol after PLY-mediated phagosomal membrane disruption (186, 187). PLY-deficient Sp induces less inflammasome-dependent cytokines production (including IL-1α, IL-1β, and IL-18), which influence downstream cytokine and chemokine responses to Sp. Indeed, inflammasome-dependent cytokines promote secretion of IL-17A, IFN-γ, and neutrophil chemoattractants, each of which aids bacterial clearance (123, 182, 188). Inflammasome activation is host protective in mouse and human macrophages ex vivo and in mouse pneumococcal infection models (182, 188, 189). Sp strains impaired in inflammasome activation, including some serotype 1, serotype 8, serotype 7F, and strains expressing the nonhemolytic allele 5 of PLY, tend to cause more invasive disease (189–191) and chronic infection (17). In contrast, in pneumococcal meningitis models, PLY-dependent inflammasome activation increasing neurotoxicity and pathology (192), possibly because PLY-triggered inflammasome activation results in rapid pyroptotic death in microglial cells (193). Thus, while inflammasome activation is in general a protective response against Sp infection, tissue types such as the brain can be susctiple to pathologies due to pyroptotic death (Figure 4, left).

4.1.4 Macrophage Activation

PLY activates macrophages to release of IL-1β, IL-6, TNF-α, IFN-β, IL-23, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor (MIF), and nitric oxide (NO) (194–197). Many of these pro-inflammaroty and chemotactic responses rely on p38 or inflammasome activation. Some PLY-induced macrophages responses require additional Sp factors as co-stimulants (198). For instance, phagosomal membrane disruption by PLY allows for pneumococcal DNA to enter the cell cytoplasm, triggerring cytosolic DNA sensors for IFN-β production (113). PLY can also modulate macrophage activation when sequestered into vesicles shed during membrane repair; interaction with PLY-containing vesicles increases macrophage IL-1β, TNFα, CCL5, CCL8, and CCL1 (136).

In the monocyte-derived THP-1 cell line, PLY is responsible for the majority of gene modulations upon exposure to Sp (194). Upregulated genes include those encoding proinflammatory molecules such as IL-8 and monocyte chemotactic protein 3 (MCP-3), and cell surface receptors that impact inflammation, such as macrophage inflammatory protein 1β (MIP-1β), IL-2 receptor β (IL-2Rβ), IL-15 receptor α (IL-15Rα), and interferon receptor 2, promoting pro-inflammatory cytokine propagation (Figure 4, left).

4.1.5 Necroptosis of Multiple Cell Types

PLY induces necroptosis, an inflammatory pathway of programmed cell death, in the lungs (90) and heart (199). In the lungs, PLY-mediated necroptosis of alveolar macrophages (90) and epithelial cells (90) is triggered in response to loss of ion homeostasis, ATP depletion, and ROS generation (90), and depends on the phosphorylation of MLKL (200), a master effector of necroptosis. The rapid depletion of alveolar macrophages by necroptosis may greatly contribute to extensive lung damage (Figure 4, left). In the heart, PLY-mediated necroptosis occurs in macrophages that infiltrate the infected myocardium and in cardiomyocytes, thereby suppressing the anti-Sp immune response at that site (199) and contributing to cardiac injury (201). The use of pharmacological inhibitors of necroptosis reduces acute injury in the lungs and heart during Sp infection, providing a novel therapeutic target to overcome infection (201). Interestingly, PLY-induced necroptosis was also reported in nasopharyngeal epithelial cells during Sp asymptomatic colonization and was associated with the increased production of anti-pneumococcal antibodies (202), suggesting a role in the development of protective immunity against Sp and highlighting the infection contex dependent nature of PLY-host interactions.

4.2 Anti-Inflammatory Properties

4.2.1 Epithelial Cell Tolerization

As a prerequisite to becoming a deadly pathogen, Sp asymptomatically colonizes the human nasopharynx. This immune-silent colonization and long-term Sp carriage are thought to be sustained by anti-inflammatory responses, which prevent the disruption of the epithelial barrier and the infiltration of neutrophils (203). After nasopharyngeal inoculation of 10⁵Sp in mice, a dose that results in asymptomatic carriage, the production of PLY is associated with higher levels of the immunosuppressive cytokine TGF-β1 and immunomodulatory T regulatory cells (Tregs) and lower levels of neutrophil recruitment (Figure 4, right). Sp infection of human nasopharyngeal epithelial cells and fibroblasts resulted in PLY-dependent secretion of TGF-β1. Treatment of these cells with purified PLY likewise results in TGF-β1 secretion (203). In contrast, compared to the 10⁵ dose, nasopharyngeal inoculation of 10⁷Sp, which leads to bacterial clearance, results in the production of lower levels of TGF-β1 and Tregs, and higher levels of IFN-γ and neutrophil infiltration (203). These findings indicate that PLY is capable of fostering nasopharyngeal carriage by limiting proinflammatory responses.

Interestingly, compared to other bacterial respiratory pathogens, such as Haemophilus influenzae, Sp induced cytokine production by epithelial cells in vitro appears delayed (148), consistent with the low numbers of infiltrated neutrophils detected in early stages of lobular pneumonia in human patients, despite bacterial load (204). At these early infection stages, PLY promotes the expression of MAPK phosphatase 1, which dephosphorylates p38 to suppress TNF-α production (205) (Figure 4, right). Later during infection, secretion of pro-inflammatory cytokines by epithelial cells increase (148). This delay in the initiation of epithelial cell-mediated inflammation may delay immune cell infiltration and provide Sp with a window of unchecked growth to establish infection. In addition, PLY downregulated many binding receptors that aid Sp detection by macrophages, including complement component receptor 2/CD21, platelet-activating factor acetylhydrolase, and oxidized low-density lipoprotein receptor 1 (OLR1). Thus, on top of directly targeting epithelial cells for tolerization, PLY evades surveilling immune cells by downregulating critical receptors for Sp recognition.

4.2.2 MRC-1 as a Mediator of Anti-Inflammatory Response

As mentioned, PLY was recently described to directly interact with MRC-1, which is expressed by DCs and alveolar macrophages, two cell types that represent a first line of defense mounted against Sp in the lungs (61). The PLY-MRC1 interaction impairs inflammatory response, limiting inflammatory cytokine secretion through the cytokine suppressor SOCS1, as well as neutrophil infiltration (Figure 4, right) (61). Consistent with its role as a phagocytic receptor, MRC-1 expressed on DCs binds to sub-lytic concentrations of PLY and promotes internalization of PLY-producing Sp. In mouse alveolar macrophages, internalized PLY-producing Sp colocalizes with MRC-1, whereas PLY-deficient Sp colocalizes with lysosomes (Figure 4, right). Upon pulmonary challenge of mice, PLY production is associated with lower levels of TNF-α and greater numbers of bacteria in lavage fluid. Similarly, genetic ablation or antibody inhibition of MRC-1 results in higher levels of TNF-α and lower bacterial load (61).

These findings opened new therapeutic strategies to fight pneumococcus infection. Indeed, molecular docking approaches identified MRC-1-derived peptides that neutralize PLY-MRC-1 interaction that impair Sp internalization and promote pathogen killing by autophagy (206). Furthermore, MRC-1-derived peptides were shown to inhibit PLY-driven cell lysis, inflammation, and lung epithelium damage by limiting PLY interaction with cells and decreasing IL-8 and TNF-α secretion (206). In zebrafish and mouse models of pneumococcal infection, MRC-1-derived peptides reduce disease development, promote host survival, and decrease bacterial burden (206).

4.2.3 Triggering of Autophagic Processes

Autophagy is a natural self-degradative mechanism that is activated in epithelial and immune cells to degrade cytoplasmic content. Autophagy also plays major roles in pathogen elimination controlling both inflammation and adaptive immune response (207). Sp activates autophagy, which functions as a host protective mechanism by promoting Sp clearance (208). As anticipated by a membrane-damaging agent, PLY was shown to initiate autophagy in a variety of cells such as human alveolar epithelial cells (208), murine microglia (193), and osteoblast cells (209). ROS generation triggered by PLY leads to the inhibition of the PI3K/AKT/mTOR pathway and the consequent activation of canonical autophagy (193, 208), which degrades intracellular Sp and limits infection in host cells (208). In murine microglia, autophagy activated by Sp transiently blocks caspase-1 activation and IL-1β secretion, delaying pyroptosis, the caspase-1-dependent inflammatory cell death pathway (193). In these cells, Sp infection increases the expression of autophagy-related genes in the early phase of infection as a host protective mechanism before pyroptosis and concomitant extensive tissue damage can occur (Figure 4, right). However, at later stages of infection, sustained PLY-mediated high-level ROS generation activates caspase-1 and microglia pyroptosis (193). In osteoblasts, PLY-mediated activation of autophagy impairs differentiation by regulating the expression of differentiation-related genes, which require mTOR signaling (209), a finding with potential relevance to Sp osteomyelitis. Finally, PLY induces selective autophagy promoting the delivery of Sp entrapped in autophagosomes to lysosomes for further degradation (210). In fibroblasts, PLY triggers (LC3)-associated phagocytosis (LAP), followed by canonical autophagy activation, suggesting a hierarchical autophagy activation process leading to Sp clearance (211). In fibroblasts, the activation of canonical autophagy is required for Sp degradation, but in bone-marrow-derived macrophages (BMDMs), PLY-induced LAP is sufficient for bacterial clearance, allowing degradation to occur much more rapidly (212). In aged BMDMs, LAP is compromised, and cells display reduced Sp killing capacity and increased expression of proinflammatory cytokines (Figure 4, right) (212).

4.2.4 Complement Subversion

Complement activation has a crucial role in host protection against pathogens, but Sp utilizes PLY-mediated complement activation to sequester complement components away from Sp surface, thus protecting Sp from host defenses and facilitating bacterial spread and survival. Purified PLY activates the human complement cascade via the classical pathway independently of its lytic activity (62, 213). Homology domains shared with CRP, mediate the PLY binding to the Fc portion of immunoglobulins, which in turn recruit and activate C1q, and are required for PLY-triggered complement activation (62, 63). While in serum from C1q KO mice PLY fails to trigger complement activation, in human C1q-depleted serum, PLY still activates the complement and C3b deposition is observed (214), suggesting that in humans, PLY may stimulate complement through C1q-independent pathways that directly target C3. PLY was further shown to trigger lectin pathway by binding to L-ficolin with high affinity (214). However, PLY-triggered complement activation decreased the serum opsonic activity for Sp both in vitro and in vivo in mouse models, reducing bacterial uptake by neutrophils and impairing the recruitment of T cells to the sites of infection in the lung (62, 64, 213).

4.2.5 Induction of Apoptosis

Pathogen-induced apoptosis, a non-inflammatory programmed cell death pathway, plays an important role in tissue damage caused by infectious diseases and constitutes an important mechanism of protection from invasive disease. PLY cytolytic activity can prompt cells to engage in apoptosis by different mechanisms depending on PLY concentration and the cell type involved (Figure 4, right). One of the first studies connecting PLY to apoptosis showed that in neurons, PLY pore-forming activity triggers Ca²⁺ influx and mitochondrial damage which results in the release of pro-apoptotic factor (AIF) and induces apoptosis in a caspase-independent manner (110, 111). The same mechanism was described in brain microvascular endothelial cells, inner cochlear hair cells, and DCs (77, 91, 215). In addition, intracellular PLY induces caspase-dependent apoptosis in Sp-infected human dendritic cells, whereby blocking their maturation and the production of inflammatory cytokines, thus inhibiting dendritic cell-mediated inflammatory responses (92). Caspase-dependent apoptosis, triggered by PLY-induced phagolysosomal membrane permeabilization, also occurs in human monocyte‐derived macrophages (216, 217). In endothelial cells, PLY-triggered caspase activation and apoptosis depend on the activation of p38/MAPK and suppression of extracellular signaling regulation kinase (ERK)1/2 (218). Further supporting PLY’s ability to trigger apoptosis, in human epithelial lung alveolar cells PLY causes double-stranded DNA breaks, which led to cell cycle arrest followed by non-homologous end joining DNA repair or, if DNA damage persists, by engagement in apoptosis (219). Clinical isolates deficient for PLY are unable to induce DCs apoptosis and trigger a strong proinflammatory response leading to excessive lung inflammation (92). Thus, the deleterious effect of PLY on immune cells, blocking their maturation, inhibiting the secretion of inflammatory cytokines, and inducing apoptosis, promotes bacterial evasion from immune detection and allows for immune-silent colonization.

5 Future Perspectives

A better understanding of the complex “Yin” and “Yang” properties of PLY will facilitate the translation of critical Sp pathogenesis mechanisms into clinical intervention strategies. PLY stands as an attractive therapeutic target to complement classical antibiotic therapy, which, besides fostering the development of resistance, can trigger bacterial lysis and consequent release of PLY that alone can have deleterious effects on cells and the immune system (25). Several repurposed drugs and PLY-neutralizing compounds have been investigated. Statins, which inhibit cholesterol production, administered prior to infection both in vitro or in vivo confer significant resistance to PLY cytotoxicity by impairing binding (220, 221). Also, some natural compounds were shown to target the oligomerization process by binding to specific D3 and 4 residues, resulting in reduced cytolytic activity (222–226). The use of artificial liposomes was also suggested as a way to sequester PLY. The administration of liposomes prior to pneumococcal infection reduced septicemia and invasive disease in a mouse model (171). Recent efforts have been made to find novel therapeutic tools to inhibit PLY release, and recently, a study demonstrated that clarithromycin downregulates ply transcription in vitro and in vivo and consequently reduces PLY production by bacteria (227), proposing a new strategy to treat pneumococcal disease. The clinical potential of current PLY-neutralizing therapeutic strategies and their limitations have been extensively reviewed elsewhere (22, 25).

Since PLY is also a potent trigger for anti-Sp immune responses, harnessing the immunogenic potentials of PLY has long been of therapeutic interest. PLY-immunized mice displayed significantly increased survival upon infection, and thus suggested that PLY should be considered for inclusion in a human vaccine (228). Further studies using different animal models aimed to obtain an active immunization using a genetic toxoid derivative of PLY, alone or in combination with other proteins (229–234). In fact, PLY toxoid demonstrated potential effectiveness as an immunogenic component and in controlling bacteremia and bacterial colonization (229–235). Phase I clinical trials concerning the use of PLY toxoid vaccine formulations were performed in adults and children, and results demonstrated that it is well-tolerated and immunogenic when administered as individual protein vaccines or combined with capsule polysaccharide conjugates (230, 236, 237). Experimental evolution studies revealed the emergence of variants that produce low levels of PLY showing decreased virulence but increased persistence (238). Such lineages have been proposed to serve as starting point to the development of live-attenuated pneumococcal vaccines.

To use PLY-targeting therapies and treatment tailored to the stage and severity of pneumococcal infection, requires a better elucidation of the role of PLY in pathogenesis. As highlighted in this review, PLY induces multiple contradicting responses. The parameters that dictate the exact mechanisms triggered in the host cells during pore-formation are still elusive. It is known that PLY is present in circulation during early pneumococcus infection and sub-lytic doses induced toxicity and modulated host immune response; furthermore, host cells can trigger PM repair mechanisms to recover from damage and get rid of the toxin (116, 127, 130, 239). However, it is still not clear which molecular mechanisms and host proteins are involved during the process of cell survival and the importance of these events in the context of infection. Cells can expel the toxin through vesicle shedding, but whether they can function as a danger signal to neighboring cells or as a modulator of the immune response remains unclear (126, 129, 136, 138). PLY can also have several effects in intracellular signaling and modulate host cytoskeleton but remains uncertain how these events can help cells to survive and repair the damage (101, 103, 114, 115, 240). Furthermore, the use of complex models like 3D cell culture or in vivo models should now be considered to clarify the crosstalk between different host cell types and their microenvironment. Insights into the mechanistic control for cellular response to PLY pores may also help resolve the apparent contradictory inflammatory and anti-inflammatory implications under different infection contexts. The discovery of new mechanisms of host PM repair and survival may bring new insights to the development of therapies against the injurious actions of PLY during pneumococcus infection.

Funding

SS research receives funds from FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-030863 (PTDC/BIA-CEL/30863/2017). Research in JL’s laboratory is supported by NIH R21 AG071268-01. JMP is the recipient of an FCT fellowship (SFRH/BD/143940/2019). SS received support from FCT in the framework of CEEC-Institutional 2017 program.

Publisher’s Note

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References

• 1

  WeiserJNFerreiraDMPatonJC. Streptococcus Pneumoniae: Transmission, Colonization and Invasion. Nat Rev Microbiol (2018) 16:355–67. doi: 10.1038/s41579-018-0001-8

  • CrossRef
  • Google Scholar

• 2

  TroegerCBlackerBFKhalilIARaoPCCaoSZimsenSRMet al. Estimates of the Global, Regional, and National Morbidity, Mortality, and Aetiologies of Lower Respiratory Infections in 195 Countries, 1990-2016: A Systematic Analysis for the Global Burden of Disease Study 2016. Lancet Infect Dis (2018) 18:1191–210. doi: 10.1016/S1473-3099(18)30310-4

  • CrossRef
  • Google Scholar

• 3

  TongSAmandCKiefferAKyawMH. Trends in Healthcare Utilization and Costs Associated With Pneumonia in the United States During 2008–2014. BMC Health Serv Res (2018) 18:715. doi: 10.1186/s12913-018-3529-4

  • CrossRef
  • Google Scholar

• 4

  ZhangDPetigaraTYangX. Clinical and Economic Burden of Pneumococcal Disease in US Adults Aged 19–64 Years With Chronic or Immunocompromising Diseases: An Observational Database Study. BMC Infect Dis (2018) 18:436. doi: 10.1186/s12879-018-3326-z

  • CrossRef
  • Google Scholar

• 5

  KellerLERobinsonDAMcDanielLS. Nonencapsulated Streptococcus Pneumoniae: Emergence and Pathogenesis. mBio (2016) 7:e01792. doi: 10.1128/mBio.01792-15

  • CrossRef
  • Google Scholar

• 6

  WroePCFinkelsteinJARayGTLinderJAJohnsonKMRifas-ShimanSet al. Aging Population and Future Burden of Pneumococcal Pneumonia in the United States. J Infect Dis (2012) 205:1589–92. doi: 10.1093/infdis/jis240

  • CrossRef
  • Google Scholar

• 7

  NegashAAAsratDAbebeWAseffaAVaneechoutteM. Phenotypic and Molecular Characterization of Penicillin and Macrolide-Resistant Streptococcus Pneumoniae Serotypes Among Pediatric Patients in Addis Ababa, Ethiopia. Infect Drug Resist (2021) 14:1765–72. doi: 10.2147/IDR.S309876

  • CrossRef
  • Google Scholar

• 8

  JonesRNSaderHSMendesREFlammRK. Update on Antimicrobial Susceptibility Trends Among Streptococcus Pneumoniae in the United States: Report of Ceftaroline Activity From the SENTRY Antimicrobial Surveillance Program (1998–2011). Diagn Microbiol Infect Dis (2013) 75:107–9. doi: 10.1016/j.diagmicrobio.2012.08.024

  • CrossRef
  • Google Scholar

• 9

  NavaJMBellaFGarauJLiteJMoreraM-AMartíCet al. Predictive Factors for Invasive Disease Due to Penicillin-Resistant Streptococcus Pneumoniae: A Population-Based Study. Clin Infect Dis (1994) 19:884–90. doi: 10.1093/clinids/19.5.884

  • CrossRef
  • Google Scholar

• 10

  MasomianMAhmadZTi GewLPohCL. Development of Next Generation Streptococcus Pneumoniae Vaccines Conferring Broad Protection. Vaccines (2020) 8:132. doi: 10.3390/vaccines8010132

  • CrossRef
  • Google Scholar

• 11

  CremersAJHMobegiFMde JongeMIvan HijumSAFTMeisJFHermansPWMet al. The Post-Vaccine Microevolution of Invasive Streptococcus Pneumoniae. Sci Rep (2015) 5:14952. doi: 10.1038/srep14952

  • CrossRef
  • Google Scholar

• 12

  SubramanianKHenriques-NormarkBNormarkS. Emerging Concepts in the Pathogenesis of the Streptococcus Pneumoniae: From Nasopharyngeal Colonizer to Intracellular Pathogen. Cell Microbiol (2019) 21:e13077. doi: 10.1111/cmi.13077

  • CrossRef
  • Google Scholar

• 13

  ZafarMAWangYHamaguchiSWeiserJN. Host-To-Host Transmission of Streptococcus Pneumoniae Is Driven by Its Inflammatory Toxin, Pneumolysin. Cell Host Microbe (2017) 21:73–83. doi: 10.1016/j.chom.2016.12.005

  • CrossRef
  • Google Scholar

• 14

  BrooksLRKMiasGI. Streptococcus Pneumoniae’s Virulence and Host Immunity: Aging, Diagnostics, and Prevention. Front Immunol (2018) 9. doi: 10.3389/fimmu.2018.01366

  • CrossRef
  • Google Scholar

• 15

  ErcoliGFernandesVEChungWYWanfordJJThomsonSBaylissCDet al. Intracellular Replication of Streptococcus Pneumoniae Inside Splenic Macrophages Serves as a Reservoir for Septicaemia. Nat Microbiol (2018) 3:600–10. doi: 10.1038/s41564-018-0147-1

  • CrossRef
  • Google Scholar

• 16

  CanvinJRMarvinAPSivakumaranMPatonJCBoulnoisGJAndrewPWet al. The Role of Pneumolysin and Autolysin in the Pathology of Pneumonia and Septicemia in Mice Infected With a Type 2 Pneumococcus. J Infect Dis (1995) 172:119–23. doi: 10.1093/infdis/172.1.119

  • CrossRef
  • Google Scholar

• 17

  BentonKAEversonMPBrilesDE. A Pneumolysin-Negative Mutant of Streptococcus Pneumoniae Causes Chronic Bacteremia Rather Than Acute Sepsis in Mice. Infect Immun (1995) 63:448–55. doi: 10.1128/iai.63.2.448-455.1995

  • CrossRef
  • Google Scholar

• 18

  AndersonRNelJGFeldmanC. Multifaceted Role of Pneumolysin in the Pathogenesis of Myocardial Injury in Community-Acquired Pneumonia. Int J Mol Sci (2018) 19:1147–68. doi: 10.3390/ijms19041147

  • CrossRef
  • Google Scholar

• 19

  YauBHuntNHMitchellAJTooLK. Blood−Brain Barrier Pathology and CNS Outcomes in Streptococcus Pneumoniae Meningitis. Int J Mol Sci (2018) 19:3555–76. doi: 10.3390/ijms19113555.

  • CrossRef
  • Google Scholar

• 20

  TettelinHChanceySMitchellTDenapaiteDSchähleYRiegerMet al. Chapter 5 - Genomics, Genetic Variation, and Regions of Differences. In: BrownJHammerschmidtSOrihuelaC, editors. Streptococcus Pneumoniae. Amsterdam: Academic Press (2015). p. 81–107.

  • Google Scholar

• 21

  van PeeKMulvihillEMüllerDJYildizÖ. Unraveling the Pore-Forming Steps of Pneumolysin From Streptococcus Pneumoniae. Nano Lett (2016) 16:7915–24. doi: 10.1021/acs.nanolett.6b04219

  • CrossRef
  • Google Scholar

• 22

  NishimotoATRoschJWTuomanenEI. Pneumolysin: Pathogenesis and Therapeutic Target. Front Microbiol (2020) 11:1543. doi: 10.3389/fmicb.2020.01543

  • CrossRef
  • Google Scholar

• 23

  BabiychukEBDraegerA. Defying Death: Cellular Survival Strategies Following Plasmalemmal Injury by Bacterial Toxins. Semin Cell Dev Biol (2015) 45:39–47. doi: 10.1016/j.semcdb.2015.10.016

  • CrossRef
  • Google Scholar

• 24

  JohnsonMKHobdenJAHagenahMO'CallaghanRJHillJMChenS. The Role of Pneumolysin in Ocular Infections With Streptococcus Pneumoniae. Curr Eye Res (1990) 9:1107–14. doi: 10.3109/02713689008997584

  • CrossRef
  • Google Scholar

• 25

  AndersonRFeldmanC. Pneumolysin as a Potential Therapeutic Target in Severe Pneumococcal Disease. J Infect (2017) 74:527–44. doi: 10.1016/j.jinf.2017.03.005

  • CrossRef
  • Google Scholar

• 26

  MarriottHMMitchellTJDockrellDH. Pneumolysin: A Double-Edged Sword During the Host-Pathogen Interaction. Curr Mol Med (2008) 8:497–509. doi: 10.2174/156652408785747924

  • CrossRef
  • Google Scholar

• 27

  RabesASuttorpNOpitzB. Inflammasomes in Pneumococcal Infection: Innate Immune Sensing and Bacterial Evasion Strategies. Curr Top Microbiol Immunol (2016) 397:215–27. doi: 10.1007/978-3-319-41171-2_11

  • CrossRef
  • Google Scholar

• 28

  SurveMVBanerjeeA. Cell-To-Cell Phenotypic Heterogeneity in Pneumococcal Pathogenesis. Future Microbiol (2019) 14:647–51. doi: 10.2217/fmb-2019-0096

  • CrossRef
  • Google Scholar

• 29

  SurveMVBhutdaSDateyAAnilARawatSPushpakaranAet al. Heterogeneity in Pneumolysin Expression Governs the Fate of Streptococcus Pneumoniae During Blood-Brain Barrier Trafficking. PloS Pathog (2018) 14:e1007168. doi: 10.1371/journal.ppat.1007168

  • CrossRef
  • Google Scholar

• 30

  PanagiotouSChaguzaCYahyaRAudshasaiTBaltazarMResselLet al. Hypervirulent Pneumococcal Serotype 1 Harbours Two Pneumolysin Variants With Differential Haemolytic Activity. Sci Rep (2020) 10:17313. doi: 10.1038/s41598-020-73454-w

  • CrossRef
  • Google Scholar

• 31

  JacquesLCPanagiotouSBaltazarMSenghoreMKhandakerSXuRet al. Increased Pathogenicity of Pneumococcal Serotype 1 is Driven by Rapid Autolysis and Release of Pneumolysin. Nat Commun (2020) 11:1892. doi: 10.1038/s41467-020-15751-6

  • CrossRef
  • Google Scholar

• 32

  PriceKECamilliA. Pneumolysin Localizes to the Cell Wall of Streptococcus Pneumoniae. J Bacteriol (2009) 191:2163–8. doi: 10.1128/JB.01489-08

  • CrossRef
  • Google Scholar

• 33

  JohnsonMK. Cellular Location of Pneumolysin. FEMS Microbiol Lett (1977) 2:243–5. doi: 10.1111/j.1574-6968.1977.tb00951.x

  • CrossRef
  • Google Scholar

• 34

  AlhamdiYNeillDRAbramsSTMalakHAYahyaRBarrett-JolleyRet al. Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury During Pneumococcal Infection. PloS Pathog (2015) 11:e1004836. doi: 10.1371/journal.ppat.1004836

  • CrossRef
  • Google Scholar

• 35

  SpreerAKerstanHBöttcherTGerberJSiemerAZyskGet al. Reduced Release of Pneumolysin by Streptococcus Pneumoniae In Vitro and In Vivo After Treatment With Nonbacteriolytic Antibiotics in Comparison to Ceftriaxone. Antimicrob Agents Chemother (2003) 47:2649–54. doi: 10.1128/AAC.47.8.2649-2654.2003

  • CrossRef
  • Google Scholar

• 36

  PriceKEGreeneNGCamilliA. Export Requirements of Pneumolysin in Streptococcus Pneumoniae. J Bacteriol (2012) 194:3651–60. doi: 10.1128/JB.00114-12

  • CrossRef
  • Google Scholar

• 37

  BerryAMLockRAHansmanDPatonJC. Contribution of Autolysin to Virulence of Streptococcus Pneumoniae. Infect Immun (1989) 57:2324–30. doi: 10.1128/iai.57.8.2324-2330.1989

  • CrossRef
  • Google Scholar

• 38

  BalachandranPHollingsheadSKPatonJCBrilesDE. The Autolytic Enzyme LytA of Streptococcus Pneumoniae is Not Responsible for Releasing Pneumolysin. J Bacteriol (2001) 183:3108–16. doi: 10.1128/JB.183.10.3108-3116.2001

  • CrossRef
  • Google Scholar

• 39

  BandaraMSkehelJMKadiogluACollinsonINobbsAHBlockerAJet al. The Accessory Sec System (SecY2A2) in Streptococcus Pneumoniae is Involved in Export of Pneumolysin Toxin, Adhesion and Biofilm Formation. Microbes Infect (2017) 19:402–12. doi: 10.1016/j.micinf.2017.04.003

  • CrossRef
  • Google Scholar

• 40

  GreeneNGNarcisoARFilipeSRCamilliA. Peptidoglycan Branched Stem Peptides Contribute to Streptococcus Pneumoniae Virulence by Inhibiting Pneumolysin Release. PloS Pathog (2015) 11:e1004996. doi: 10.1371/journal.ppat.1004996

  • CrossRef
  • Google Scholar

• 41

  BillingtonSJJostBHSongerJG. Thiol-Activated Cytolysins: Structure, Function and Role in Pathogenesis. FEMS Microbiol Lett (2000) 182:197–205. doi: 10.1016/S0378-1097(99)00536-4

  • CrossRef
  • Google Scholar

• 42

  HillJAndrewPWMitchellTJ. Amino Acids in Pneumolysin Important for Hemolytic Activity Identified by Random Mutagenesis. Infect Immun (1994) 62:757–8. doi: 10.1128/iai.62.2.757-758.1994

  • CrossRef
  • Google Scholar

• 43

  MorganPJHarrisonGFreestonePPECraneDRoweAJMitchellTJet al. Structural and Functional Characterisation of Two Proteolytic Fragments of the Bacterial Protein Toxin, Pneumolysin. FEBS Lett (1997) 412:563–7. doi: 10.1016/S0014-5793(97)00838-7

  • CrossRef
  • Google Scholar

• 44

  MarshallJEFarajBHGingrasARLonnenRSheikhMAEl-MezgueldiMet al. The Crystal Structure of Pneumolysin at 2.0 Å Resolution Reveals the Molecular Packing of the Pre-Pore Complex. Sci Rep (2015) 5:13293. doi: 10.1038/srep13293

  • CrossRef
  • Google Scholar

• 45

  BadgujarDCAnilAGreenAESurveMVMadhavanSBeckettAet al. Structural Insights Into Loss of Function of a Pore Forming Toxin and its Role in Pneumococcal Adaptation to an Intracellular Lifestyle. PloS Pathog (2020) 16:e1009016. doi: 10.1371/journal.ppat.1009016

  • CrossRef
  • Google Scholar

• 46

  LawrenceSLFeilSCMortonCJFarrandAJMulhernTDGormanMAet al. Crystal Structure of Streptococcus Pneumoniae Pneumolysin Provides Key Insights Into Early Steps of Pore Formation. Sci Rep (2015) 5:14352. doi: 10.1038/srep14352

  • CrossRef
  • Google Scholar

• 47

  OwenRHGBoulnoisGJAndrewPWMitchellTJ. A Role in Cell-Binding for the C-Terminus of Pneumolysin, the Thiol-Activated Toxin of Streptococcus Pneumoniae. FEMS Microbiol Lett (1994) 121:217–21. doi: 10.1111/j.1574-6968.1994.tb07101.x

  • CrossRef
  • Google Scholar

• 48

  BabaHKawamuraIKohdaCNomuraTItoYKimotoTet al. Essential Role of Domain 4 of Pneumolysin From Streptococcus Pneumoniae in Cytolytic Activity as Determined by Truncated Proteins. Biochem Biophys Res Commun (2001) 281:37–44. doi: 10.1006/bbrc.2001.4297

  • CrossRef
  • Google Scholar

• 49

  de los ToyosJRMéndezFJAparicioJFVázquezFDel Mar García SuárezMFleitesAet al. Functional Analysis of Pneumolysin by Use of Monoclonal Antibodies. Infect Immun (1996) 64:480–4. doi: 10.1128/iai.64.2.480-484.1996

  • CrossRef
  • Google Scholar

• 50

  FarrandAJLaChapelleSHotzeEMJohnsonAETwetenRK. Only Two Amino Acids are Essential for Cytolytic Toxin Recognition of Cholesterol at the Membrane Surface. Proc Natl Acad Sci USA (2010) 107:4341–6. doi: 10.1073/pnas.0911581107

  • CrossRef
  • Google Scholar

• 51

  NöllmannMGilbertRMitchellTSferrazzaMByronO. The Role of Cholesterol in the Activity of Pneumolysin, a Bacterial Protein Toxin. Biophys J (2004) 86:3141–51. doi: 10.1016/S0006-3495(04)74362-3

  • CrossRef
  • Google Scholar

• 52

  TilleySJOrlovaEVGilbertRJCAndrewPWSaibilHR. Structural Basis of Pore Formation by the Bacterial Toxin Pneumolysin. Cell (2005) 121:247–56. doi: 10.1016/j.cell.2005.02.033

  • CrossRef
  • Google Scholar

• 53

  van PeeKNeuhausAD'ImprimaEMillsDJKühlbrandtWYildizÖ. CryoEM Structures of Membrane Pore and Prepore Complex Reveal Cytolytic Mechanism of Pneumolysin. eLife (2017) 6:e23644. doi: 10.7554/eLife.23644

  • CrossRef
  • Google Scholar

• 54

  VögeleMBhaskaraRMMulvihillEvan PeeKYildizÖ.KühlbrandtWet al. Membrane Perforation by the Pore-Forming Toxin Pneumolysin. Proc Natl Acad Sci USA (2019) 116:13352–7. doi: 10.1073/pnas.1904304116

  • CrossRef
  • Google Scholar

• 55

  SonnenAFPPlitzkoJMGilbertRJC. Incomplete Pneumolysin Oligomers Form Membrane Pores. Open Biol (2014) 4:140044–4. doi: 10.1098/rsob.140044

  • CrossRef
  • Google Scholar

• 56

  GilbertRJJiménezJLChenSTickleIJRossjohnJParkerMet al. Two Structural Transitions in Membrane Pore Formation by Pneumolysin, the Pore-Forming Toxin of Streptococcus Pneumoniae. Cell (1999) 97:647–55. doi: 10.1016/S0092-8674(00)80775-8

  • CrossRef
  • Google Scholar

• 57

  JefferiesJMCJohnstonCHGKirkhamL-ASCowanGJMRossKSSmithAet al. Presence of Nonhemolytic Pneumolysin in Serotypes of Streptococcus Pneumoniae Associated With Disease Outbreaks. J Infect Dis (2007) 196:936–44. doi: 10.1086/520091

  • CrossRef
  • Google Scholar

• 58

  LeungCDudkinaNVLukoyanovaNHodelAWFarabellaIPanduranganAPet al. Stepwise Visualization of Membrane Pore Formation by Suilysin, a Bacterial Cholesterol-Dependent Cytolysin. Elife (2014) 3:e04247. doi: 10.7554/eLife.04247

  • CrossRef
  • Google Scholar

• 59

  GilbertRJMikeljMDalla SerraMFroelichCJAnderluhG. Effects of MACPF/CDC Proteins on Lipid Membranes. Cell Mol Life Sci (2013) 70:2083–98. doi: 10.1007/s00018-012-1153-8

  • CrossRef
  • Google Scholar

• 60

  GilbertRJCSonnenAFP. Measuring Kinetic Drivers of Pneumolysin Pore Structure. Eur Biophys J (2016) 45:365–76. doi: 10.1007/s00249-015-1106-x

  • CrossRef
  • Google Scholar

• 61

  SubramanianKNeillDRMalakHASpelminkLKhandakerSDalla Libera MarchioriGet al. Pneumolysin Binds to the Mannose Receptor C Type 1 (MRC-1) Leading to Anti-Inflammatory Responses and Enhanced Pneumococcal Survival. Nat Microbiol (2019) 4:62–70. doi: 10.1038/s41564-018-0280-x

  • CrossRef
  • Google Scholar

• 62

  PatonJCRowan-KellyBFerranteA. Activation of Human Complement by the Pneumococcal Toxin Pneumolysin. Infect Immun (1984) 43:1085–7. doi: 10.1128/iai.43.3.1085-1087.1984

  • CrossRef
  • Google Scholar

• 63

  MitchellTJAndrewPWSaundersFKSmithANBoulnoisGJ. Complement Activation and Antibody Binding by Pneumolysin via a Region of the Toxin Homologous to a Human Acute-Phase Protein. Mol Microbiol (1991) 5:1883–8. doi: 10.1111/j.1365-2958.1991.tb00812.x

  • CrossRef
  • Google Scholar

• 64

  YusteJBottoMPatonJCHoldenDWBrownJS. Additive Inhibition of Complement Deposition by Pneumolysin and PspA Facilitates Streptococcus Pneumoniae Septicemia. J Immunol (2005) 175:1813–9. doi: 10.4049/jimmunol.175.3.1813

  • CrossRef
  • Google Scholar

• 65

  ShewellLKHarveyRMHigginsMADayCJHartley-TassellLEChenAYet al. The Cholesterol-Dependent Cytolysins Pneumolysin and Streptolysin O Require Binding to Red Blood Cell Glycans for Hemolytic Activity. Proc Natl Acad Sci (2014) 111:E5312–20. doi: 10.1073/pnas.1412703111

  • CrossRef
  • Google Scholar

• 66

  HirstRAKadiogluAO'callaghanCAndrewPW. The Role of Pneumolysin in Pneumococcal Pneumonia and Meningitis. Clin Exp Immunol (2004) 138:195–201. doi: 10.1111/j.1365-2249.2004.02611.x

  • CrossRef
  • Google Scholar

• 67

  KadiogluATaylorSIannelliFPozziGMitchellTJAndrewPW. Upper and Lower Respiratory Tract Infection by Streptococcus Pneumoniae is Affected by Pneumolysin Deficiency and Differences in Capsule Type. Infect Immun (2002) 70:2886–90. doi: 10.1128/IAI.70.6.2886-2890.2002

  • CrossRef
  • Google Scholar

• 68

  HotomiMYuasaJBrilesDEYamanakaN. Pneumolysin Plays a Key Role at the Initial Step of Establishing Pneumococcal Nasal Colonization. Folia Microbiol (Praha) (2016) 61:375–83. doi: 10.1007/s12223-016-0445-z

  • CrossRef
  • Google Scholar

• 69

  RubinsJBJanoffEN. Pneumolysin: A Multifunctional Pneumococcal Virulence Factor. J Lab Clin Med (1998) 131:21–7. doi: 10.1016/S0022-2143(98)90073-7

  • CrossRef
  • Google Scholar

• 70

  AllegrucciMHuFZShenKHayesJEhrlichGDPostJCet al. Phenotypic Characterization of Streptococcus Pneumoniae Biofilm Development. J Bacteriol (2006) 188:2325–35. doi: 10.1128/JB.188.7.2325-2335.2006

  • CrossRef
  • Google Scholar

• 71

  VidalJELudewickHPKunkelRMZähnerDKlugmanKP. The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoniae Strain D39. Infect Immun (2011) 79:4050–60. doi: 10.1128/IAI.05186-11

  • CrossRef
  • Google Scholar

• 72

  SchachernPATsuprunVGoetzSCureogluSJuhnSKBrilesDEet al. Viability and Virulence of Pneumolysin, Pneumococcal Surface Protein A, and Pneumolysin/Pneumococcal Surface Protein A Mutants in the Ear. JAMA Otolaryngol–Head Neck Surg (2013) 139:937–43. doi: 10.1001/jamaoto.2013.4104

  • CrossRef
  • Google Scholar

• 73

  KellerLEBradshawJLPipkinsHMcDanielLS. Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated Streptococcus Pneumoniae Mediate Virulence in a Chinchilla Model of Otitis Media. Front Cell Infect Microbiol (2016) 6:55. doi: 10.3389/fcimb.2016.00055

  • CrossRef
  • Google Scholar

• 74

  ComisSDOsborneMPStephenJTarlowMJHaywardTLMitchellTJet al. Cytotoxic Effects on Hair Cells of Guinea Pig Cochlea Produced by Pneumolysin, the Thiol Activated Toxin of Streptococcus Pneumoniae. Acta Otolaryngol (1993) 113:152–9. doi: 10.3109/00016489309135784

  • CrossRef
  • Google Scholar

• 75

  WinterAJComisSDOsborneMPTarlowMJStephenJAndrewPWet al. A Role for Pneumolysin But Not Neuraminidase in the Hearing Loss and Cochlear Damage Induced by Experimental Pneumococcal Meningitis in Guinea Pigs. Infect Immun (1997) 65:4411–8. doi: 10.1128/iai.65.11.4411-4418.1997

  • CrossRef
  • Google Scholar

• 76

  SkinnerLJBeurgMMitchellTJDarrouzetVAranJMDulonD. Intracochlear Perfusion of Pneumolysin, a Pneumococcal Protein, Rapidly Abolishes Auditory Potentials in the Guinea Pig Cochlea. Acta Otolaryngol (2004) 124:1000–7. doi: 10.1080/00016480410017125

  • CrossRef
  • Google Scholar

• 77

  BeurgMHafidiASkinnerLCowanGHondarragueYMitchellTJet al. The Mechanism of Pneumolysin-Induced Cochlear Hair Cell Death in the Rat. J Physiol (2005) 568:211–27. doi: 10.1113/jphysiol.2005.092478

  • CrossRef
  • Google Scholar

• 78

  RecchiaFMBusbeeBGPearlmanRBCarvalho-RecchiaCAHoAC. Changing Trends in the Microbiologic Aspects of Postcataract Endophthalmitis. Arch Ophthalmol (2005) 123:341–6. doi: 10.1001/archopht.123.3.341

  • CrossRef
  • Google Scholar

• 79

  NorcrossEWSandersMEMooreQC3MarquartME. Pathogenesis of A Clinical Ocular Strain of Streptococcus Pneumoniae and the Interaction of Pneumolysin With Corneal Cells. J Bacteriol Parasitol (2011) 2:108–8. doi: 10.4172/2155-9597.1000108

  • CrossRef
  • Google Scholar

• 80

  NgEWSamiyNRubinsJBCousinsFVRuoffKLBakerASet al. Implication of Pneumolysin as a Virulence Factor in Streptococcus Pneumoniae Endophthalmitis. Retina (1997) 17:521–9. doi: 10.1097/00006982-199711000-00006

  • CrossRef
  • Google Scholar

• 81

  SandersMENorcrossEWMooreQC3FratkinJThompsonHMarquartME. Immunization With Pneumolysin Protects Against Both Retinal and Global Damage Caused by Streptococcus Pneumoniae Endophthalmitis. J Ocul Pharmacol Ther (2010) 26:571–7. doi: 10.1089/jop.2010.0077

  • CrossRef
  • Google Scholar

• 82

  TaylorSDSandersMETullosNAStraySJNorcrossEWMcDanielLSet al. The Cholesterol-Dependent Cytolysin Pneumolysin From Streptococcus Pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells. PloS One (2013) 8:e61300. doi: 10.1371/journal.pone.0061300

  • CrossRef
  • Google Scholar

• 83

  SteinfortCWilsonRMitchellTFeldmanCRutmanAToddHet al. Effect of Streptococcus Pneumoniae on Human Respiratory Epithelium In Vitro. Infect Immun (1989) 57:2006–13. doi: 10.1128/iai.57.7.2006-2013.1989

  • CrossRef
  • Google Scholar

• 84

  RubinsJBDuanePGClawsonDCharboneauDYoungJNiewoehnerDE. Toxicity of Pneumolysin to Pulmonary Alveolar Epithelial Cells. Infect Immun (1993) 61:1352–8. doi: 10.1128/iai.61.4.1352-1358.1993

  • CrossRef
  • Google Scholar

• 85

  RubinsJBDuanePGCharboneauDJanoffEN. Toxicity of Pneumolysin to Pulmonary Endothelial Cells In Vitro. Infect Immun (1992) 60:1740–6. doi: 10.1128/iai.60.5.1740-1746.1992

  • CrossRef
  • Google Scholar

• 86

  NelJGDurandtCMitchellTJFeldmanCAndersonRTintingerGR. Pneumolysin Mediates Platelet Activation In Vitro. Lung (2016) 194:589–93. doi: 10.1007/s00408-016-9900-5

  • CrossRef
  • Google Scholar

• 87

  JahnKHandtkeSPalankarRWeißmüllerSNouaillesGKohlerTPet al. Pneumolysin Induces Platelet Destruction, Not Platelet Activation, Which can be Prevented by Immunoglobulin Preparations In Vitro. Blood Adv (2020) 4:6315–26. doi: 10.1182/bloodadvances.2020002372

  • CrossRef
  • Google Scholar

• 88

  MausUASrivastavaMPatonJCMackMEverhartMBBlackwellTSet al. Pneumolysin-Induced Lung Injury is Independent of Leukocyte Trafficking Into the Alveolar Space. J Immunol (2004) 173:1307–12. doi: 10.4049/jimmunol.173.2.1307

  • CrossRef
  • Google Scholar

• 89

  García-Suárez MdelMFlórezNAstudilloAVázquezFVillaverdeRFabrizioKet al. The Role of Pneumolysin in Mediating Lung Damage in a Lethal Pneumococcal Pneumonia Murine Model. Respir Res (2007) 8:3. doi: 10.1186/1465-9921-8-3

  • CrossRef
  • Google Scholar

• 90

  González-JuarbeNGilleyRPHinojosaCABradleyKMKameiAGaoGet al. Pore-Forming Toxins Induce Macrophage Necroptosis During Acute Bacterial Pneumonia. PloS Pathog (2015) 11:e1005337. doi: 10.1371/journal.ppat.1005337

  • CrossRef
  • Google Scholar

• 91

  ColinoJSnapperCM. Two Distinct Mechanisms For Induction of Dendritic Cell Apoptosis in Response to Intact Streptococcus Pneumoniae. J Immunol (2003) 171:2354–65. doi: 10.4049/jimmunol.171.5.2354

  • CrossRef
  • Google Scholar

• 92

  LittmannMAlbigerBFrentzenANormarkSHenriques-NormarkBPlantL. Streptococcus Pneumoniae Evades Human Dendritic Cell Surveillance by Pneumolysin Expression. EMBO Mol Med (2009) 1:211–22. doi: 10.1002/emmm.200900025

  • CrossRef
  • Google Scholar

• 93

  DomonHOdaMMaekawaTNagaiKTakedaWTeraoY. Streptococcus Pneumoniae Disrupts Pulmonary Immune Defence via Elastase Release Following Pneumolysin-Dependent Neutrophil Lysis. Sci Rep (2016) 6:38013. doi: 10.1038/srep38013

  • CrossRef
  • Google Scholar

• 94

  AdamsWBhowmickRBou GhanemENWadeKShchepetovMWeiserJNet al. Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration During Streptococcus Pneumoniae Infection. J Immunol (2020) 204:101–11. doi: 10.4049/jimmunol.1800748

  • CrossRef
  • Google Scholar

• 95

  MorelandJGBaileyG. Neutrophil Transendothelial Migration In Vitro to Streptococcus Pneumoniae is Pneumolysin Dependent. Am J Physiol-Lung Cell Mol Physiol 290 (2006) 5:L833–40. doi: 10.1152/ajplung.00333.2005

  • CrossRef
  • Google Scholar

• 96

  BrownAOMannBGaoGHankinsJSHumannJGiardinaJet al. Streptococcus Pneumoniae Translocates Into the Myocardium and Forms Unique Microlesions That Disrupt Cardiac Function. PloS Pathog (2014) 10:e1004383. doi: 10.1371/journal.ppat.1004383

  • CrossRef
  • Google Scholar

• 97

  BrissacTShenoyATPattersonLAOrihuelaCJ. Cell Invasion and Pyruvate Oxidase-Derived H(2)O(2) Are Critical for Streptococcus Pneumoniae-Mediated Cardiomyocyte Killing. Infect Immun 86 (2018) 86:e00569-17. doi: 10.1128/IAI.00569-17

  • CrossRef
  • Google Scholar

• 98

  ShenoyATBrissacTGilleyRPKumarNWangYGonzalez-JuarbeNet al. Streptococcus Pneumoniae in the Heart Subvert the Host Response Through Biofilm-Mediated Resident Macrophage Killing. PloS Pathog (2017) 13:e1006582. doi: 10.1371/journal.ppat.1006582

  • CrossRef
  • Google Scholar

• 99

  TabusiMThorsdottirSLysandrouMNarcisoARMinoiaMSrambickalCVet al. Neuronal Death in Pneumococcal Meningitis is Triggered by Pneumolysin and RrgA Interactions With β-Actin. PloS Pathog (2021) 17:e1009432. doi: 10.1371/journal.ppat.1009432

  • CrossRef
  • Google Scholar

• 100

  BelloCSmailYSainte-RoseVPodglajenIGilbertAMoreiraVet al. Role of Astroglial Connexin 43 in Pneumolysin Cytotoxicity and During Pneumococcal Meningitis. PloS Pathog (2020) 16:e1009152. doi: 10.1371/journal.ppat.1009152

  • CrossRef
  • Google Scholar

• 101

  HuppSHeimerothVWippelCFörtschCMaJMitchellTJet al. Astrocytic Tissue Remodeling by the Meningitis Neurotoxin Pneumolysin Facilitates Pathogen Tissue Penetration and Produces Interstitial Brain Edema. Glia (2012) 60:137–46. doi: 10.1002/glia.21256

  • CrossRef
  • Google Scholar

• 102

  IlievAIDjannatianJROpazoFGerberJNauRMitchellTJet al. Rapid Microtubule Bundling and Stabilization by the Streptococcus Pneumoniae Neurotoxin Pneumolysin in a Cholesterol-Dependent, non-Lytic and Src-Kinase Dependent Manner Inhibits Intracellular Trafficking. Mol Microbiol (2009) 71:461–77. doi: 10.1111/j.1365-2958.2008.06538.x

  • CrossRef
  • Google Scholar

• 103

  FörtschCHuppSMaJMitchellTJMaierEBenzRet al. Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity. Toxins (Basel) (2011) 3:43–62. doi: 10.3390/toxins3010043

  • CrossRef
  • Google Scholar

• 104

  ZyskGSchneider-WaldBKHwangJHBejoLKimKSMitchellTJet al. Pneumolysin is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by Streptococcus Pneumoniae. Infect Immun (2001) 69:845–52. doi: 10.1128/IAI.69.2.845-852.2001

  • CrossRef
  • Google Scholar

• 105

  HuppSGrandgirardDMitchellTJLeibSLHathawayLJIlievAI. Pneumolysin and the Bacterial Capsule of Streptococcus Pneumoniae Cooperatively Inhibit Taxis and Motility of Microglia. J Neuroinflamm (2019) 16:105. doi: 10.1186/s12974-019-1491-7

  • CrossRef
  • Google Scholar

• 106

  HirstRARutmanASikandKAndrewPWMitchellTJO'CallaghanC. Effect of Pneumolysin on Rat Brain Ciliary Function: Comparison of Brain Slices With Cultured Ependymal Cells. Pediatr Res (2000) 47:381–4. doi: 10.1203/00006450-200003000-00016

  • CrossRef
  • Google Scholar

• 107

  MohammedBJMitchellTJAndrewPWHirstRAO'CallaghanC. The Effect of the Pneumococcal Toxin, Pneumolysin on Brain Ependymal Cilia. Microbial Pathog (1999) 27:303–9. doi: 10.1006/mpat.1999.0306

  • CrossRef
  • Google Scholar

• 108

  HirstRASikandKSRutmanAMitchellTJAndrewPWO'CallaghanC. Relative Roles of Pneumolysin and Hydrogen Peroxide From Streptococcus Pneumoniae in Inhibition of Ependymal Ciliary Beat Frequency. Infect Immun (2000) 68:1557–62. doi: 10.1128/IAI.68.3.1557-1562.2000

  • CrossRef
  • Google Scholar

• 109

  HirstRAGosaiBRutmanAAndrewPWO'CallaghanC. Streptococcus Pneumoniae Damages the Ciliated Ependyma of the Brain During Meningitis. Infect Immun (2003) 71:6095–100. doi: 10.1128/IAI.71.10.6095-6100.2003

  • CrossRef
  • Google Scholar

• 110

  BraunJSSublettJEFreyerDMitchellTJClevelandJLTuomanenEIet al. Pneumococcal Pneumolysin and H(2)O(2) Mediate Brain Cell Apoptosis During Meningitis. J Clin Invest (2002) 109:19–27. doi: 10.1172/JCI12035

  • CrossRef
  • Google Scholar

• 111

  BraunJSHoffmannOSchickhausMFreyerDDagandEBermpohlDet al. Pneumolysin Causes Neuronal Cell Death Through Mitochondrial Damage. Infect Immun (2007) 75:4245–54. doi: 10.1128/IAI.00031-07

  • CrossRef
  • Google Scholar

• 112

  NerlichAMiethMLetsiouEFatykhovaDZscheppangKImai-MatsushimaAet al. Pneumolysin Induced Mitochondrial Dysfunction Leads to Release of Mitochondrial DNA. Sci Rep (2018) 8:182. doi: 10.1038/s41598-017-18468-7

  • CrossRef
  • Google Scholar

• 113

  HuXPengXLuCZhangXGanLGaoYet al. Type I IFN Expression is Stimulated by Cytosolic MtDNA Released From Pneumolysin-Damaged Mitochondria via the STING Signaling Pathway in Macrophages. FEBS J (2019) 286:4754–68. doi: 10.1111/febs.15001

  • CrossRef
  • Google Scholar

• 114

  HuppSFörtschCWippelCMaJMitchellTJIlievAI. Direct Transmembrane Interaction Between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin. J Mol Biol (2013) 425:636–46. doi: 10.1016/j.jmb.2012.11.034

  • CrossRef
  • Google Scholar

• 115

  IlievAIDjannatianJRNauRMitchellTJWoutersFS. Cholesterol-Dependent Actin Remodeling via RhoA and Rac1 Activation by the Streptococcus Pneumoniae Toxin Pneumolysin. Proc Natl Acad Sci USA (2007) 104:2897–902. doi: 10.1073/pnas.0608213104

  • CrossRef
  • Google Scholar

• 116

  BritoCCabanesDSarmento MesquitaFSousaS. Mechanisms Protecting Host Cells Against Bacterial Pore-Forming Toxins. Cell Mol Life Sci (2019) 76:1319–39. doi: 10.1007/s00018-018-2992-8

  • CrossRef
  • Google Scholar

• 117

  PortaHCancino-RodeznoASoberónMBravoA. Role of MAPK P38 in the Cellular Responses to Pore-Forming Toxins. Peptides (2011) 32:601–6. doi: 10.1016/j.peptides.2010.06.012

  • CrossRef
  • Google Scholar

• 118

  KloftNBuschTNeukirchCWeisSBoukhalloukFBobkiewiczWet al. Pore-Forming Toxins Activate MAPK P38 by Causing Loss of Cellular Potassium. Biochem Biophys Res Commun (2009) 385:503–6. doi: 10.1016/j.bbrc.2009.05.121

  • CrossRef
  • Google Scholar

• 119

  RatnerAJHippeKRAguilarJLBenderMHNelsonALWeiserJN. Epithelial Cells are Sensitive Detectors of Bacterial Pore-Forming Toxins. J Biol Chem (2006) 281:12994–8. doi: 10.1074/jbc.M511431200

  • CrossRef
  • Google Scholar

• 120

  AguilarJLKulkarniRRandisTMSomanSKikuchiAYinYet al. Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores. PloS One (2009) 4:e8076. doi: 10.1371/journal.pone.0008076

  • CrossRef
  • Google Scholar

• 121

  DasRLaRoseMIHergottCBLengLBucalaRWeiserJN. Macrophage Migration Inhibitory Factor Promotes Clearance of Pneumococcal Colonization. J Immunol (2014) 193:764–72. doi: 10.4049/jimmunol.1400133

  • CrossRef
  • Google Scholar

• 122

  LemonJKWeiserJN. Degradation Products of the Extracellular Pathogen Streptococcus Pneumoniae Access the Cytosol via its Pore-Forming Toxin. mBio 6 (2015) 6:e02110–14. doi: 10.1128/mBio.02110-14

  • CrossRef
  • Google Scholar

• 123

  LemonJKMillerMRWeiserJN. Sensing of Interleukin-1 Cytokines During Streptococcus Pneumoniae Colonization Contributes to Macrophage Recruitment and Bacterial Clearance. Infect Immun (2015) 83:3204–12. doi: 10.1128/IAI.00224-15

  • CrossRef
  • Google Scholar

• 124

  StringarisAKGeisenhainerJBergmannFBalshüsemannCLeeUZyskGet al. Neurotoxicity of Pneumolysin, a Major Pneumococcal Virulence Factor, Involves Calcium Influx and Depends on Activation of P38 Mitogen-Activated Protein Kinase. Neurobiol Dis (2002) 11:355–68. doi: 10.1006/nbdi.2002.0561

  • CrossRef
  • Google Scholar

• 125

  KwonISKimJRheeDKKimBOPyoS. Pneumolysin Induces Cellular Senescence by Increasing ROS Production and Activation of MAPK/NF-κb Signal Pathway in Glial Cells. Toxicon (2017) 129:100–12. doi: 10.1016/j.toxicon.2017.02.017

  • CrossRef
  • Google Scholar

• 126

  WolfmeierHRadeckeJSchoenauerRKoeffelRBabiychukVSDrückerPet al. Active Release of Pneumolysin Prepores and Pores by Mammalian Cells Undergoing a Streptococcus Pneumoniae Attack. Biochim Biophys Acta (2016) 1860:2498–509. doi: 10.1016/j.bbagen.2016.07.022

  • CrossRef
  • Google Scholar

• 127

  BischofbergerMIacovacheIvan der GootFG. Pathogenic Pore-Forming Proteins: Function and Host Response. Cell Host Microbe (2012) 12:266–75. doi: 10.1016/j.chom.2012.08.005

  • CrossRef
  • Google Scholar

• 128

  Dal PeraroMvan der GootFG. Pore-Forming Toxins: Ancient, But Never Really Out of Fashion. Nat Rev Microbiol (2016) 14:77–92. doi: 10.1038/nrmicro.2015.3

  • CrossRef
  • Google Scholar

• 129

  WolfmeierHSchoenauerRAtanassoffAPNeillDRKadiogluADraegerAet al. Ca²⁺-Dependent Repair of Pneumolysin Pores: A New Paradigm for Host Cellular Defense Against Bacterial Pore-Forming Toxins. Biochim Biophys Acta (2015) 1853:2045–54. doi: 10.1016/j.bbamcr.2014.09.005

  • CrossRef
  • Google Scholar

• 130

  JimenezAJPerezF. Plasma Membrane Repair: The Adaptable Cell Life-Insurance. Curr Opin Cell Biol (2017) 47:99–107. doi: 10.1016/j.ceb.2017.03.011

  • CrossRef
  • Google Scholar

• 131

  BouillotSReboudEHuberP. Functional Consequences of Calcium Influx Promoted by Bacterial Pore-Forming Toxins. Toxins (2018) 10:387. doi: 10.3390/toxins10100387

  • CrossRef
  • Google Scholar

• 132

  EtxanizAGonzález-BullónDMartínCOstolazaH. Membrane Repair Mechanisms Against Permeabilization by Pore-Forming Toxins. Toxins (2018) 10:234. doi: 10.3390/toxins10060234

  • CrossRef
  • Google Scholar

• 133

  WippelCFörtschCHuppSMaierEBenzRMaJet al. Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue. J Infect Dis (2011) 204:930–6. doi: 10.1093/infdis/jir434

  • CrossRef
  • Google Scholar

• 134

  MaurerJHuppSPillichHMitchellTJChakrabortyTIlievAI. Missing Elimination via Membrane Vesicle Shedding Contributes to the Diminished Calcium Sensitivity of Listeriolysin O. Sci Rep (2018) 8:15846. doi: 10.1038/s41598-018-34031-4

  • CrossRef
  • Google Scholar

• 135

  PotezSLuginbühlMMonastyrskayaKHostettlerADraegerABabiychukEB. Tailored Protection Against Plasmalemmal Injury by Annexins With Different Ca²⁺ Sensitivities. J Biol Chem (2011) 286:17982–91. doi: 10.1074/jbc.M110.187625

  • CrossRef
  • Google Scholar

• 136

  KoffelRWolfmeierHLarpinYBesanconHSchoenauerRBabiychukVSet al. Host-Derived Microvesicles Carrying Bacterial Pore-Forming Toxins Deliver Signals to Macrophages: A Novel Mechanism of Shaping Immune Responses. Front Immunol (2018) 9:1688. doi: 10.3389/fimmu.2018.01688

  • CrossRef
  • Google Scholar

• 137

  LetsiouETeixeira AlvesLGFatykhovaDFeltenMMitchellTJMüller-RedetzkyHCet al. Microvesicles Released From Pneumolysin-Stimulated Lung Epithelial Cells Carry Mitochondrial Cargo and Suppress Neutrophil Oxidative Burst. Sci Rep (2021) 11:9529. doi: 10.1038/s41598-021-88897-y

  • CrossRef
  • Google Scholar

• 138

  LarpinYBesançonHIacovacheMIBabiychukVSBabiychukEBZuberBet al. Bacterial Pore-Forming Toxin Pneumolysin: Cell Membrane Structure and Microvesicle Shedding Capacity Determines Differential Survival of Cell Types. FASEB J (2020) 34:1665–78. doi: 10.1096/fj.201901737RR

  • CrossRef
  • Google Scholar

• 139

  NeillDRMitchellTJKadiogluA. Pneumolysin. Streptococcus Pneumoniae Elsevier (2015) 257–75. doi: 10.1016/B978-0-12-410530-0.00014-4

  • CrossRef
  • Google Scholar

• 140

  PechousRD. With Friends Like These: The Complex Role of Neutrophils in the Progression of Severe Pneumonia. Front Cell Infect Microbiol (2017) 7:160. doi: 10.3389/fcimb.2017.00160

  • CrossRef
  • Google Scholar

• 141

  MitchellTJDalzielCE. The Biology of Pneumolysin. Subcell Biochem (2014) 80:145–60. doi: 10.1007/978-94-017-8881-6_8

  • CrossRef
  • Google Scholar

• 142

  CockeranRTheronAJSteelHCMatlolaNMMitchellTJFeldmanCet al. Proinflammatory Interactions of Pneumolysin With Human Neutrophils. J Infect Dis (2001) 183:604–11. doi: 10.1086/318536

  • CrossRef
  • Google Scholar

• 143

  ThapaRRaySKeyelPA. Interaction of Macrophages and Cholesterol-Dependent Cytolysins: The Impact on Immune Response and Cellular Survival. Toxins (Basel) 12 (2020) 12:531–62 doi: 10.3390/toxins12090531

  • CrossRef
  • Google Scholar

• 144

  RubinsJBMitchellTJAndrewPWNiewoehnerDE. Pneumolysin Activates Phospholipase A in Pulmonary Artery Endothelial Cells. Infect Immun (1994) 62:3829–36. doi: 10.1128/iai.62.9.3829-3836.1994

  • CrossRef
  • Google Scholar

• 145

  RaynerCFJacksonADRutmanADewarAMitchellTJAndrewPWet al. Interaction of Pneumolysin-Sufficient and -Deficient Isogenic Variants of Streptococcus Pneumoniae With Human Respiratory Mucosa. Infect Immun (1995) 63:442–7. doi: 10.1128/iai.63.2.442-447.1995

  • CrossRef
  • Google Scholar

• 146

  FliegaufMSonnenAFKremerBHennekeP. Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection. PloS One (2013) 8:e59925. doi: 10.1371/journal.pone.0059925

  • CrossRef
  • Google Scholar

• 147

  KimY-JShinH-SLeeJ-HJungYWKimH-BHaU-H. Pneumolysin-Mediated Expression of β-Defensin 2 is Coordinated by P38 MAP Kinase-MKP1 in Human Airway Cells. J Microbiol (2013) 51:194–9. doi: 10.1007/s12275-013-2579-x

  • CrossRef
  • Google Scholar

• 148

  YooIHShinHSKimYJKimHBJinSHaUH. Role of Pneumococcal Pneumolysin in the Induction of an Inflammatory Response in Human Epithelial Cells. FEMS Immunol Med Microbiol (2010) 60:28–35. doi: 10.1111/j.1574-695X.2010.00699.x

  • CrossRef
  • Google Scholar

• 149

  BhowmickRClarkSBonventreJVLeongJMMcCormickBA. Cytosolic Phospholipase a(2)α Promotes Pulmonary Inflammation and Systemic Disease During Streptococcus Pneumoniae Infection. Infect Immun 85 (2017) 85:e00280-17 doi: 10.1128/IAI.00280-17

  • CrossRef
  • Google Scholar

• 150

  ZukauskasAMrsnyRJCortés BarrantesPTurnerJRLeongJMMcCormickBA. Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration During Streptococcus Pneumoniae Lung Infection. mSphere 3 (2018) 3:e00303-18 doi: 10.1128/mSphere.00303-18

  • CrossRef
  • Google Scholar

• 151

  BhowmickRMaungNHurleyBPGhanemEBGronertKMcCormickBAet al. Systemic Disease During Streptococcus Pneumoniae Acute Lung Infection Requires 12-Lipoxygenase-Dependent Inflammation. J Immunol (2013) 191:5115–23. doi: 10.4049/jimmunol.1300522

  • CrossRef
  • Google Scholar

• 152

  BhowmickRClarkSBonventreJVLeongJMMcCormickBA. Cytosolic Phospholipase A2alpha Promotes Pulmonary Inflammation and Systemic Disease During Streptococcus Pneumoniae Infection. Infect Immun 85 (2017) 85:e00280-17. doi: 10.1128/IAI.00280-17

  • CrossRef
  • Google Scholar

• 153

  Bou GhanemENClarkSRoggensackSEMcIverSRAlcaidePHaydonPGet al. Extracellular Adenosine Protects Against Streptococcus Pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment. PloS Pathog (2015) 11:e1005126. doi: 10.1371/journal.ppat.1005126

  • CrossRef
  • Google Scholar

• 154

  CuypersFKlabundeBGesell SalazarMSurabhiSSkorkaSBBurchhardtGet al. Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation. J Infect Dis (2020) 222:1702–12. doi: 10.1093/infdis/jiaa277

  • CrossRef
  • Google Scholar

• 155

  FicklHCockeranRSteelHCFeldmanCCowanGMitchellTJet al. Pneumolysin-Mediated Activation of NFkappaB in Human Neutrophils is Antagonized by Docosahexaenoic Acid. Clin Exp Immunol (2005) 140:274–81. doi: 10.1111/j.1365-2249.2005.02757.x

  • CrossRef
  • Google Scholar

• 156

  CockeranRSteelHCMitchellTJFeldmanCAndersonR. Pneumolysin Potentiates Production of Prostaglandin E(2) and Leukotriene B(4) by Human Neutrophils. Infect Immun (2001) 69:3494–6. doi: 10.1128/IAI.69.5.3494-3496.2001

  • CrossRef
  • Google Scholar

• 157

  MartnerADahlgrenCPatonJCWoldAE. Pneumolysin Released During Streptococcus Pneumoniae Autolysis is a Potent Activator of Intracellular Oxygen Radical Production in Neutrophils. Infect Immun (2008) 76:4079–87. doi: 10.1128/IAI.01747-07

  • CrossRef
  • Google Scholar

• 158

  SegalAW. How Neutrophils Kill Microbes. Annu Rev Immunol (2005) 23:197–223. doi: 10.1146/annurev.immunol.23.021704.115653

  • CrossRef
  • Google Scholar

• 159

  NganjeCNHaynesSAQabarCMLentRCBou GhanemENShainheitMG. PepN is a non-Essential, Cell Wall-Localized Protein That Contributes to Neutrophil Elastase-Mediated Killing of Streptococcus Pneumoniae. PloS One (2019) 14:e0211632. doi: 10.1371/journal.pone.0211632

  • CrossRef
  • Google Scholar

• 160

  StandishAJWeiserJN. Human Neutrophils Kill Streptococcus Pneumoniae via Serine Proteases. J Immunol (2009) 183:2602–9. doi: 10.4049/jimmunol.0900688

  • CrossRef
  • Google Scholar

• 161

  BelaaouajAMcCarthyRBaumannMGaoZLeyTJAbrahamSNet al. Mice Lacking Neutrophil Elastase Reveal Impaired Host Defense Against Gram Negative Bacterial Sepsis. Nat Med (1998) 4:615–8. doi: 10.1038/nm0598-615

  • CrossRef
  • Google Scholar

• 162

  DomonHTeraoY. The Role of Neutrophils and Neutrophil Elastase in Pneumococcal Pneumonia. Front Cell Infect Microbiol (2021) 11:615959. doi: 10.3389/fcimb.2021.615959

  • CrossRef
  • Google Scholar

• 163

  WatanabeHHattoriSKatsudaSNakanishiINagaiY. Human Neutrophil Elastase: Degradation of Basement Membrane Components and Immunolocalization in the Tissue. J Biochem (1990) 108:753–9. doi: 10.1093/oxfordjournals.jbchem.a123277

  • CrossRef
  • Google Scholar

• 164

  BoxioRWartelleJNawrocki-RabyBLagrangeBMalleretLHircheTet al. Neutrophil Elastase Cleaves Epithelial Cadherin in Acutely Injured Lung Epithelium. Respir Res (2016) 17:129. doi: 10.1186/s12931-016-0449-x

  • CrossRef
  • Google Scholar

• 165

  WitherdenIRVanden BonEJGoldstrawPRatcliffeCPastorinoUTetleyTD. Primary Human Alveolar Type II Epithelial Cell Chemokine Release: Effects of Cigarette Smoke and Neutrophil Elastase. Am J Respir Cell Mol Biol (2004) 30:500–9. doi: 10.1165/rcmb.4890

  • CrossRef
  • Google Scholar

• 166

  ChenHCLinHCLiuCYWangCHHwangTHuangTTet al. Neutrophil Elastase Induces IL-8 Synthesis by Lung Epithelial Cells via the Mitogen-Activated Protein Kinase Pathway. J BioMed Sci (2004) 11:49–58. doi: 10.1007/BF02256548

  • CrossRef
  • Google Scholar

• 167

  SuzukiTYamashitaCZemansRLBrionesNVan LindenADowneyGP. Leukocyte Elastase Induces Lung Epithelial Apoptosis via a PAR-1-, NF-kappaB-, and P53-Dependent Pathway. Am J Respir Cell Mol Biol (2009) 41:742–55. doi: 10.1165/rcmb.2008-0157OC

  • CrossRef
  • Google Scholar

• 168

  HagioTKishikawaKKawabataKTasakaSHashimotoSHasegawaNet al. Inhibition of Neutrophil Elastase Reduces Lung Injury and Bacterial Count in Hamsters. Pulm Pharmacol Ther (2008) 21:884–91. doi: 10.1016/j.pupt.2008.10.002

  • CrossRef
  • Google Scholar

• 169

  WilkinsonTSConway MorrisAKefalaKO'KaneCMMooreNRBoothNAet al. Ventilator-Associated Pneumonia is Characterized by Excessive Release of Neutrophil Proteases in the Lung. Chest (2012) 142:1425–32. doi: 10.1378/chest.11-3273

  • CrossRef
  • Google Scholar

• 170

  TagamiTKushimotoSTosaROmuraMYonezawaKAkiyamaGet al. Plasma Neutrophil Elastase Correlates With Pulmonary Vascular Permeability: A Prospective Observational Study in Patients With Pneumonia. Respirology (2011) 16:953–8. doi: 10.1111/j.1440-1843.2011.01997.x

  • CrossRef
  • Google Scholar

• 171

  GinzbergHHCherapanovVDongQCantinAMcCullochCAShannonPTet al. Neutrophil-Mediated Epithelial Injury During Transmigration: Role of Elastase. Am J Physiol Gastrointest Liver Physiol 281 (2001) 3:G705–17. doi: 10.1152/ajpgi.2001.281.3.G705

  • CrossRef
  • Google Scholar

• 172

  MoraesTJZurawskaJHDowneyGP. Neutrophil Granule Contents in the Pathogenesis of Lung Injury. Curr Opin Hematol (2006) 13:21–7. doi: 10.1097/01.moh.0000190113.31027.d5

  • CrossRef
  • Google Scholar

• 173

  CockeranRMitchellTJFeldmanCAndersonR. Pneumolysin Induces Release of Matrix Metalloproteinase-8 and -9 From Human Neutrophils. Eur Respir J (2009) 34:1167–70. doi: 10.1183/09031936.00007109

  • CrossRef
  • Google Scholar

• 174

  HartogCMWermeltJASommerfeldCOEichlerWDalhoffKBraunJ. Pulmonary Matrix Metalloproteinase Excess in Hospital-Acquired Pneumonia. Am J Respir Crit Care Med (2003) 167:593–8. doi: 10.1164/rccm.200203-258OC

  • CrossRef
  • Google Scholar

• 175

  NguyenHXO'BarrTJAndersonAJ. Polymorphonuclear Leukocytes Promote Neurotoxicity Through Release of Matrix Metalloproteinases, Reactive Oxygen Species, and TNF-Alpha. J Neurochem (2007) 102:900–12. doi: 10.1111/j.1471-4159.2007.04643.x

  • CrossRef
  • Google Scholar

• 176

  SchaafBLiebauCKurowskiVDroemannDDalhoffK. Hospital Acquired Pneumonia With High-Risk Bacteria is Associated With Increased Pulmonary Matrix Metalloproteinase Activity. BMC Pulm Med (2008) 8:12. doi: 10.1186/1471-2466-8-12

  • CrossRef
  • Google Scholar

• 177

  MoriYYamaguchiMTeraoYHamadaSOoshimaTKawabataS. Alpha-Enolase of Streptococcus Pneumoniae Induces Formation of Neutrophil Extracellular Traps. J Biol Chem (2012) 287:10472–81. doi: 10.1074/jbc.M111.280321

  • CrossRef
  • Google Scholar

• 178

  NarasarajuTYangESamyRPNgHHPohWPLiewAAet al. Excessive Neutrophils and Neutrophil Extracellular Traps Contribute to Acute Lung Injury of Influenza Pneumonitis. Am J Pathol (2011) 179:199–210. doi: 10.1016/j.ajpath.2011.03.013

  • CrossRef
  • Google Scholar

• 179

  CzaikoskiPGMotaJMNascimentoDCSonegoFCastanheiraFVMeloPHet al. Neutrophil Extracellular Traps Induce Organ Damage During Experimental and Clinical Sepsis. PloS One (2016) 11:e0148142. doi: 10.1371/journal.pone.0148142

  • CrossRef
  • Google Scholar

• 180

  EbrahimiFGiaglisSHahnSBlumCABaumgartnerCKutzAet al. Markers of Neutrophil Extracellular Traps Predict Adverse Outcome in Community-Acquired Pneumonia: Secondary Analysis of a Randomised Controlled Trial. Eur Respir J 51 (2018) 51:1701389–99. doi: 10.1183/13993003.01389-2017

  • CrossRef
  • Google Scholar

• 181

  FangRTsuchiyaKKawamuraIShenYHaraHSakaiSet al. Critical Roles of ASC Inflammasomes in Caspase-1 Activation and Host Innate Resistance to Streptococcus Pneumoniae Infection. J Immunol (2011) 187:4890–9. doi: 10.4049/jimmunol.1100381

  • CrossRef
  • Google Scholar

• 182

  McNeelaEABurkeANeillDRBaxterCFernandesVEFerreiraDet al. Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4. PloS Pathog (2010) 6:e1001191. doi: 10.1371/journal.ppat.1001191

  • CrossRef
  • Google Scholar

• 183

  GreaneyAJLepplaSHMoayeriM. Bacterial Exotoxins and the Inflammasome. Front Immunol (2015) 6:570. doi: 10.3389/fimmu.2015.00570

  • CrossRef
  • Google Scholar

• 184

  Munoz-PlanilloRKuffaPMartinez-ColonGSmithBLRajendiranTMNunezG. K(+) Efflux is the Common Trigger of NLRP3 Inflammasome Activation by Bacterial Toxins and Particulate Matter. Immunity (2013) 38:1142–53. doi: 10.1016/j.immuni.2013.05.016

  • CrossRef
  • Google Scholar

• 185

  KarmakarMKatsnelsonMMalakHAGreeneNGHowellSJHiseAGet al. Neutrophil IL-1β Processing Induced by Pneumolysin is Mediated by the NLRP3/ASC Inflammasome and Caspase-1 Activation and is Dependent on K+ Efflux. J Immunol (2015) 194:1763–75. doi: 10.4049/jimmunol.1401624

  • CrossRef
  • Google Scholar

• 186

  LaRockCNNizetV. Inflammasome/IL-1beta Responses to Streptococcal Pathogens. Front Immunol (2015) 6:518. doi: 10.3389/fimmu.2015.00518

  • CrossRef
  • Google Scholar

• 187

  KoppeUHognerKDoehnJMMullerHCWitzenrathMGutbierBet al. Streptococcus Pneumoniae Stimulates a STING- and IFN Regulatory Factor 3-Dependent Type I IFN Production in Macrophages, Which Regulates RANTES Production in Macrophages, Cocultured Alveolar Epithelial Cells, and Mouse Lungs. J Immunol (2012) 188:811–7. doi: 10.4049/jimmunol.1004143

  • CrossRef
  • Google Scholar

• 188

  ShomaSTsuchiyaKKawamuraINomuraTHaraHUchiyamaRet al. Critical Involvement of Pneumolysin in Production of Interleukin-1alpha and Caspase-1-Dependent Cytokines in Infection With Streptococcus Pneumoniae In Vitro: A Novel Function of Pneumolysin in Caspase-1 Activation. Infect Immun (2008) 76:1547–57. doi: 10.1128/IAI.01269-07

  • CrossRef
  • Google Scholar

• 189

  WitzenrathMPacheFLorenzDKoppeUGutbierBTabelingCet al. The NLRP3 Inflammasome is Differentially Activated by Pneumolysin Variants and Contributes to Host Defense in Pneumococcal Pneumonia. J Immunol (2011) 187:434–40. doi: 10.4049/jimmunol.1003143

  • CrossRef
  • Google Scholar

• 190

  GeldhoffMMook-KanamoriBBBrouwerMCTroostDLeemansJCFlavellRAet al. Inflammasome Activation Mediates Inflammation and Outcome in Humans and Mice With Pneumococcal Meningitis. BMC Infect Dis (2013) 13:358. doi: 10.1186/1471-2334-13-358

  • CrossRef
  • Google Scholar

• 191

  WeinbergerDMHarboeZBSandersEANdirituMKlugmanKPRuckingerSet al. Association of Serotype With Risk of Death Due to Pneumococcal Pneumonia: A Meta-Analysis. Clin Infect Dis (2010) 51:692–9. doi: 10.1086/655828

  • CrossRef
  • Google Scholar

• 192

  HoegenTTremelNKleinMAngeleBWagnerHKirschningCet al. The NLRP3 Inflammasome Contributes to Brain Injury in Pneumococcal Meningitis and is Activated Through ATP-Dependent Lysosomal Cathepsin B Release. J Immunol (2011) 187:5440–51. doi: 10.4049/jimmunol.1100790

  • CrossRef
  • Google Scholar

• 193

  KimJ-YPatonJCBrilesDERheeD-KPyoS. Streptococcus Pneumoniae Induces Pyroptosis Through the Regulation of Autophagy in Murine Microglia. Oncotarget 6 (2015) 6:44161–78. doi: 10.18632/oncotarget.6592

  • CrossRef
  • Google Scholar

• 194

  RogersPDThorntonJBarkerKSMcDanielDOSacksGSSwiatloEet al. Pneumolysin-Dependent and -Independent Gene Expression Identified by cDNA Microarray Analysis of THP-1 Human Mononuclear Cells Stimulated by Streptococcus Pneumoniae. Infect Immun (2003) 71:2087–94. doi: 10.1128/IAI.71.4.2087-2094.2003

  • CrossRef
  • Google Scholar

• 195

  BraunJSNovakRGaoGMurrayPJShenepJL. Pneumolysin, a Protein Toxin of Streptococcus Pneumoniae, Induces Nitric Oxide Production From Macrophages. Infect Immun (1999) 67:3750–6. doi: 10.1128/IAI.67.8.3750-3756.1999

  • CrossRef
  • Google Scholar

• 196

  ParkerDMartinFJSoongGHarfenistBSAguilarJLRatnerAJet al. Streptococcus Pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract. mBio (2011) 2:e00016–11. doi: 10.1128/mBio.00016-11

  • CrossRef
  • Google Scholar

• 197

  HouldsworthSAndrewPWMitchellTJ. Pneumolysin Stimulates Production of Tumor Necrosis Factor Alpha and Interleukin-1 Beta by Human Mononuclear Phagocytes. Infect Immun (1994) 62:1501–3. doi: 10.1128/iai.62.4.1501-1503.1994

  • CrossRef
  • Google Scholar

• 198

  HarveyRMHughesCEPatonAWTrappettiCTwetenRKPatonJC. The Impact of Pneumolysin on the Macrophage Response to Streptococcus Pneumoniae is Strain-Dependent. PloS One (2014) 9:e103625. doi: 10.1371/journal.pone.0103625

  • CrossRef
  • Google Scholar

• 199

  GilleyRPGonzález-JuarbeNShenoyATReyesLFDubePHRestrepoMIet al. Infiltrated Macrophages Die of Pneumolysin-Mediated Necroptosis Following Pneumococcal Myocardial Invasion. Infect Immun (2016) 84:1457–69. doi: 10.1128/IAI.00007-16

  • CrossRef
  • Google Scholar

• 200

  González-JuarbeNBradleyKMShenoyATGilleyRPReyesLFHinojosaCAet al. Pore-Forming Toxin-Mediated Ion Dysregulation Leads to Death Receptor-Independent Necroptosis of Lung Epithelial Cells During Bacterial Pneumonia. Cell Death Differ (2017) 24:917–28. doi: 10.1038/cdd.2017.49

  • CrossRef
  • Google Scholar

• 201

  BenoSMRieglerANGilleyRPBrissacTWangYKruckowKLet al. Inhibition of Necroptosis to Prevent Long-Term Cardiac Damage During Pneumococcal Pneumonia and Invasive Disease. J Infect Dis (2020) 222:1882–93. doi: 10.1093/infdis/jiaa295

  • CrossRef
  • Google Scholar

• 202

  RieglerANBrissacTGonzalez-JuarbeNOrihuelaCJ. Necroptotic Cell Death Promotes Adaptive Immunity Against Colonizing Pneumococci. Front Immunol 10 (2019) 615. doi: 10.3389/fimmu.2019.00615

  • CrossRef
  • Google Scholar

• 203

  NeillDRCowardWRGritzfeldJFRichardsLGarcia-GarciaFJDotorJet al. Density and Duration of Pneumococcal Carriage is Maintained by Transforming Growth Factor β1 and T Regulatory Cells. Am J Respir Crit Care Med (2014) 189:1250–9. doi: 10.1164/rccm.201401-0128OC

  • CrossRef
  • Google Scholar

• 204

  PahalPRajasuryaVSharmaS. Typical Bacterial Pneumonia. Treasure Island (FL: StatPearls (2021).

  • Google Scholar

• 205

  ShinHSYooIHKimYJKimHBJinSHaUH. MKP1 Regulates the Induction of Inflammatory Response by Pneumococcal Pneumolysin in Human Epithelial Cells. FEMS Immunol Med Microbiol (2010) 60:171–8. doi: 10.1111/j.1574-695X.2010.00733.x

  • CrossRef
  • Google Scholar

• 206

  SubramanianKIovinoFTsikourkitoudiVMerklPAhmedSBerrySBet al. Mannose Receptor-Derived Peptides Neutralize Pore-Forming Toxins and Reduce Inflammation and Development of Pneumococcal Disease. EMBO Mol Med (2020) 12:e12695. doi: 10.15252/emmm.202012695

  • CrossRef
  • Google Scholar

• 207

  DereticVSaitohTAkiraS. Autophagy in Infection, Inflammation and Immunity. Nat Rev Immunol (2013) 13:722–37. doi: 10.1038/nri3532

  • CrossRef
  • Google Scholar

• 208

  LiPShiJHeQHuQWangYYZhangLJet al. Streptococcus Pneumoniae Induces Autophagy Through the Inhibition of the PI3K-I/Akt/mTOR Pathway and ROS Hypergeneration in A549 Cells. PloS One (2015) 10:e0122753. doi: 10.1371/journal.pone.0122753

  • CrossRef
  • Google Scholar

• 209

  KimJLeeH-WRheeDKPatonJCPyoS. Pneumolysin-Induced Autophagy Contributes to Inhibition of Osteoblast Differentiation Through Downregulation of Sp1 in Human Osteosarcoma Cells. Biochim Biophys Acta (BBA) - Gen Subj (2017) 1861:2663–73. doi: 10.1016/j.bbagen.2017.07.008

  • CrossRef
  • Google Scholar

• 210

  OgawaMMatsudaRTakadaNTomokiyoMYamamotoSShizukuishiSet al. Molecular Mechanisms of Streptococcus Pneumoniae-Targeted Autophagy via Pneumolysin, Golgi-Resident Rab41, and Nedd4-1-Mediated K63-Linked Ubiquitination. Cell Microbiol (2018) 20:e12846. doi: 10.1111/cmi.12846

  • CrossRef
  • Google Scholar

• 211

  OgawaMTakadaNShizukuishiSTomokiyoMChangBYoshidaMet al. Streptococcus Pneumoniae Triggers Hierarchical Autophagy Through Reprogramming of LAPosome-Like Vesicles via NDP52-Delocalization. Commun Biol (2020) 3:25. doi: 10.1038/s42003-020-0753-3

  • CrossRef
  • Google Scholar

• 212

  InomataMXuSChandraPMeydaniSNTakemuraGPhilipsJAet al. Macrophage LC3-Associated Phagocytosis is an Immune Defense Against Streptococcus Pneumoniae That Diminishes With Host Aging. Proc Natl Acad Sci (2020) 117:33561–9. doi: 10.1073/pnas.2015368117

  • CrossRef
  • Google Scholar

• 213

  JounblatRKadiogluAMitchellTJAndrewPW. Pneumococcal Behavior and Host Responses During Bronchopneumonia are Affected Differently by the Cytolytic and Complement-Activating Activities of Pneumolysin. Infect Immun (2003) 71:1813–9. doi: 10.1128/IAI.71.4.1813-1819.2003

  • CrossRef
  • Google Scholar

• 214

  AliYMKenawyHIMuhammadASimRBAndrewPWSchwaebleWJ. Human L-Ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus Pneumoniae. PloS One (2013) 8:e82583. doi: 10.1371/journal.pone.0082583

  • CrossRef
  • Google Scholar

• 215

  BermpohlDHalleAFreyerDDagandEBraunJSBechmannIet al. Bacterial Programmed Cell Death of Cerebral Endothelial Cells Involves Dual Death Pathways. J Clin Invest (2005) 115:1607–15. doi: 10.1172/JCI23223

  • CrossRef
  • Google Scholar

• 216

  MarriottHMAliFReadRCMitchellTJWhyteMKBDockrellDH. Nitric Oxide Levels Regulate Macrophage Commitment to Apoptosis or Necrosis During Pneumococcal Infection. FASEB J (2004) 18:1126–8. doi: 10.1096/fj.03-1450fje

  • CrossRef
  • Google Scholar

• 217

  BewleyMANaughtonMPrestonJMitchellAHolmesAMarriottHMet al. Pneumolysin Activates Macrophage Lysosomal Membrane Permeabilization and Executes Apoptosis by Distinct Mechanisms Without Membrane Pore Formation. mBio (2014) 5:e01710–14. doi: 10.1128/mBio.01710-14

  • CrossRef
  • Google Scholar

• 218

  ZhouAWangHLanKZhangXXuWYinYet al. Apoptosis Induced by Pneumolysin in Human Endothelial Cells Involves Mitogen-Activated Protein Kinase Phosphorylation. Int J Mol Med (2012) 29:1025–30. doi: 10.3892/ijmm.2012.946

  • CrossRef
  • Google Scholar

• 219

  RaiPHeFKwangJEngelwardBPChowVTK. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest. Sci Rep (2016) 6:22972. doi: 10.1038/srep22972

  • CrossRef
  • Google Scholar

• 220

  RoschJWBoydARHinojosaEPestinaTHuYPersonsDAet al. Statins Protect Against Fulminant Pneumococcal Infection and Cytolysin Toxicity in a Mouse Model of Sickle Cell Disease. J Clin Invest (2010) 120:627–35. doi: 10.1172/JCI39843

  • CrossRef
  • Google Scholar

• 221

  StattSRuanJWHungLYChangCYHuangCTLimJHet al. Statin-Conferred Enhanced Cellular Resistance Against Bacterial Pore-Forming Toxins in Airway Epithelial Cells. Am J Respir Cell Mol Biol (2015) 53:689–702. doi: 10.1165/rcmb.2014-0391OC

  • CrossRef
  • Google Scholar

• 222

  ZhaoXLiHWangJGuoYLiuBDengXet al. Verbascoside Alleviates Pneumococcal Pneumonia by Reducing Pneumolysin Oligomers. Mol Pharmacol (2016) 89:376–87. doi: 10.1124/mol.115.100610

  • CrossRef
  • Google Scholar

• 223

  ZhaoXLiuBLiuSWangLWangJ. Anticytotoxin Effects of Amentoflavone to Pneumolysin. Biol Pharm Bull (2017) 40:61–7. doi: 10.1248/bpb.b16-00598

  • CrossRef
  • Google Scholar

• 224

  LiHZhaoXWangJDongYMengSLiRet al. β-Sitosterol Interacts With Pneumolysin to Prevent Streptococcus Pneumoniae Infection. Sci Rep (2015) 5:17668–8. doi: 10.1038/srep17668

  • CrossRef
  • Google Scholar

• 225

  LiHZhaoXDengXWangJSongMNiuXet al. Insights Into Structure and Activity of Natural Compound Inhibitors of Pneumolysin. Sci Rep (2017) 7:42015. doi: 10.1038/srep42015

  • CrossRef
  • Google Scholar

• 226

  LvQZhangPQuanPCuiMLiuTYinYet al. Quercetin, a Pneumolysin Inhibitor, Protects Mice Against Streptococcus Pneumoniae Infection. Microb Pathog (2020) 140:103934. doi: 10.1016/j.micpath.2019.103934

  • CrossRef
  • Google Scholar

• 227

  DomonHIsonoTHiyoshiTTamuraHSasagawaKMaekawaTet al. Clarithromycin Inhibits Pneumolysin Production via Downregulation of Ply Gene Transcription Despite Autolysis Activation. Microbiol Spectr (2021) 9:e0031821. doi: 10.1128/Spectrum.00318-21

  • CrossRef
  • Google Scholar

• 228

  PatonJCLockRAHansmanDJ. Effect of Immunization With Pneumolysin on Survival Time of Mice Challenged With Streptococcus Pneumoniae. Infect Immun (1983) 40:548–52. doi: 10.1128/iai.40.2.548-552.1983

  • CrossRef
  • Google Scholar

• 229

  MannBThorntonJHeathRWadeKRTwetenRKGaoGet al. Broadly Protective Protein-Based Pneumococcal Vaccine Composed of Pneumolysin Toxoid-CbpA Peptide Recombinant Fusion Protein. J Infect Dis (2014) 209:1116–25. doi: 10.1093/infdis/jit502

  • CrossRef
  • Google Scholar

• 230

  AlexanderJELockRAPeetersCCPoolmanJTAndrewPWMitchellTJet al. Immunization of Mice With Pneumolysin Toxoid Confers a Significant Degree of Protection Against at Least Nine Serotypes of Streptococcus Pneumoniae. Infect Immun (1994) 62:5683–8. doi: 10.1128/iai.62.12.5683-5688.1994

  • CrossRef
  • Google Scholar

• 231

  KirkhamL-ASKerrARDouceGRPatersonGKDiltsDALiuD-Fet al. Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines. Infect Immun (2006) 74:586. doi: 10.1128/IAI.74.1.586-593.2006

  • CrossRef
  • Google Scholar

• 232

  OgunniyiADWoodrowMCPoolmanJTPatonJC. Protection Against Streptococcus Pneumoniae Elicited by Immunization With Pneumolysin and CbpA. Infect Immun (2001) 69:5997–6003. doi: 10.1128/IAI.69.10.5997-6003.2001

  • CrossRef
  • Google Scholar

• 233

  DenoëlPPhilippMTDoyleLMartinDCarlettiGPoolmanJT. A Protein-Based Pneumococcal Vaccine Protects Rhesus Macaques From Pneumonia After Experimental Infection With Streptococcus Pneumoniae. Vaccine (2011) 29:5495–501. doi: 10.1016/j.vaccine.2011.05.051

  • CrossRef
  • Google Scholar

• 234

  NelJGTheronAJDurandtCTintingerGRPoolRMitchellTJet al. Pneumolysin Activates Neutrophil Extracellular Trap Formation. Clin Exp Immunol (2016) 184:358–67. doi: 10.1111/cei.12766

  • CrossRef
  • Google Scholar

• 235

  VerhoevenDPerrySPichicheroME. Contributions to Protection From Streptococcus Pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA, PhtD, and PlyD1 in an Infant Murine Model During Challenge. Clin Vaccine Immunol (2014) 21:1037–45. doi: 10.1128/CVI.00052-14

  • CrossRef
  • Google Scholar

• 236

  Leroux-RoelsGMaesCDe BoeverFTraskineMRüggebergJUBorysD. Safety, Reactogenicity and Immunogenicity of a Novel Pneumococcal Protein-Based Vaccine in Adults: A Phase I/II Randomized Clinical Study. Vaccine (2014) 32:6838–46. doi: 10.1016/j.vaccine.2014.02.052

  • CrossRef
  • Google Scholar

• 237

  PrymulaRPazdioraPTraskineMRüggebergJUBorysD. Safety and Immunogenicity of an Investigational Vaccine Containing Two Common Pneumococcal Proteins in Toddlers: A Phase II Randomized Clinical Trial. Vaccine (2014) 32:3025–34. doi: 10.1016/j.vaccine.2014.03.066

  • CrossRef
  • Google Scholar

• 238

  GreenAEHowarthDChaguzaCEchlinHLangendonkRFMunroCet al. Pneumococcal Colonization and Virulence Factors Identified Via Experimental Evolution in Infection Models. Mol Biol Evol (2021) 38:2209–26. doi: 10.1093/molbev/msab018

  • CrossRef
  • Google Scholar

• 239

  ChristieMPJohnstoneBATwetenRKParkerMWMortonCJ. Cholesterol-Dependent Cytolysins: From Water-Soluble State to Membrane Pore. Biophys Rev (2018) 10:1337–48. doi: 10.1007/s12551-018-0448-x

  • CrossRef
  • Google Scholar

• 240

  CargnelloMRouxPP. Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases. Microbiol Mol Biol Rev (2011) 75:50. doi: 10.1128/MMBR.00031-10

  • CrossRef
  • Google Scholar