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

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

Live Cell Microscopy and Flow Cytometry to Study Streptolysin S-Mediated Erythrocyte Hemolysis.

The ability to induce hemolysis, the rupturing of erythrocytes with the consequent release of their intracellular contents, is a phenotypic hallmark of a number of microbial toxins. Streptococcus pyogenes or Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of diseases from mild pharyngitis to severe conditions such as toxic shock syndrome. GAS produces a powerful hemolytic toxin called streptolysin S (SLS). Herein, we describe a procedure for the preparation of SLS toxin and the use of two complementary approaches, live microscopy and flow cytometry, to study the effects of the SLS toxin on erythrocytes. In addition to providing insights into SLS-mediated hemolysis, these assays have the potential to be modified for the study of other hemolytic toxins and compounds.

Pneumolysin and the bacterial capsule of Streptococcus pneumoniae cooperatively inhibit taxis and motility of microglia.

BACKGROUND: Streptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis. METHODS: We utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis. RESULTS: In brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures. CONCLUSIONS: Our data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.

Streptococcus pyogenes translocates across an epithelial barrier.

Streptococcus pyogenes is a ß-hemolytic organism responsible for a wide variety of human diseases that commonly occur as self-limiting purulent diseases of the pharynx and skin. Although the occurrence of invasive infections by S. pyogenes is rare, mortality rates remain high even with progressive medical therapy. As a prerequisite for causing the severe invasive disease, S. pyogenes must invade underlying sterile tissues by translocating across the epithelial barrier. In this study, streptolysin S and SpeB were identified as the novel factors that facilitate bacterial translocation via degradation of intercellular junctions. Furthermore, we found that S. pyogenes exploits host plasminogen for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. Here, I would like to show our study on bacterial translocation across the epithelial barrier through paracellular route.

Pneumolysin mediates heterotypic aggregation of neutrophils and platelets in vitro.

OBJECTIVES: Platelets orchestrate the inflammatory activities of neutrophils, possibly contributing to pulmonary and myocardial damage during severe pneumococcal infection. This study tested the hypothesis that the pneumococcal toxin, pneumolysin (Ply), activates production of platelet-activating factor (PAF) and thromboxane A2 (TxA2) by neutrophils, these bioactive lipids being potential mediators of neutrophil:platelet (NP) networking. METHODS: The effects of recombinant Ply (10-80 ng mL-1) on the production of PAF and TxA2 by isolated neutrophils were measured using ELISA procedures, and NP aggregation by flow cytometry. RESULTS: Exposure of neutrophils to Ply induced production of PAF and, to a lesser extent, TxA2, achieving statistical significance at ≥20 ng mL-1 of the toxin. In the case of NP interactions, Ply promoted heterotypic aggregation which was dependent on upregulation of P-selectin (CD62P) and activation of protease-activated receptor 1 (PAR1), attaining statistical significance at ≥10 ng mL-1 of the toxin, but did not involve either PAF or TxA2. CONCLUSION: Ply induces synthesis of PAF and TxA2, by human neutrophils, neither of which appears to contribute to the formation of NP heterotypic aggregates in vitro, a process which is seemingly dependent on CD62P and PAR1. These pro-inflammatory activities of Ply may contribute to the pathogenesis of pulmonary and myocardial injury during severe pneumococcal infection.

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin.

RATIONALE: Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly. OBJECTIVES: To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice. METHODS: Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis. MEASUREMENTS AND MAIN RESULTS: We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-ß1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFß1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFß1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFß1-exposed mice. CONCLUSIONS: Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice.

The biology of pneumolysin.

Cholesterol dependent cytolysins are important in the ability of some bacteria to cause disease in man and animals. Pneumolysin (PLY) plays a key role in the diseases caused by Streptococcus pneumoniae (the pneumococcus). This chapter describes the role of PLY in some of the key process in disease. These include induction of cell death by pore formation and toxin-induced apoptosis as well as more subtle effects on gene expression of host cells including epigenetic effects of the toxin. The use of bacterial mutants that either do not express the toxin or express altered versions in biological systems is described. Use of isolated tissue and whole animal systems to dissect the structure/function relationships of the toxin as well as the role played by different activities in the pathogenesis of infection are described. The role of PLY in meningitis and the associated deafness is discussed as well as the role of the toxin in promoting increased lung permeability and inflammation during pneumococcal pneumonia. Different clinical strains of the pneumococcus produce different forms of PLY and the impact of this on disease caused by these strains is discussed. Finally, the impact of this knowledge on the development of treatment and prevention strategies for pneumococcal disease is discussed.

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.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. In this study, we examine an innate immune recognition pathway that senses pneumococcal infection, triggers type I IFN production, and regulates RANTES production. We found that human and murine alveolar macrophages as well as murine bone marrow macrophages, but not alveolar epithelial cells, produced type I IFNs upon infection with S. pneumoniae. This response was dependent on the pore-forming toxin pneumolysin and appeared to be mediated by a cytosolic DNA-sensing pathway involving the adapter molecule STING and the transcription factor IFN regulatory factor 3. Indeed, DNA was present in the cytosol during pneumococcal infection as indicated by the activation of the AIM2 inflammasome, which is known to sense microbial DNA. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated gene expression and RANTES production in macrophages and cocultured alveolar epithelial cells in vitro. Moreover, type I IFNs controlled RANTES production during pneumococcal pneumonia in vivo. In conclusion, we identified an immune sensing pathway detecting S. pneumoniae that triggers a type I IFN response and positively regulates RANTES production.

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins.

Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca(2+) signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca(2+) signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca(2+) oscillations via IP(3) -mediated depletion of intracellular stores and activation of store-operated Ca(2+) (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca(2+) add-back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca(2+) oscillations were comparatively short-lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short-lived oscillatory response into a long-lived one. Thus, we conclude that SLO-mediated Ca(2+) signalling involves Ca(2+) release from intracellular stores and STIM1/Orai1-dependent SOC entry. The phenotype of Ca(2+) signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry-associated proteins in S. pyogenes-induced lung infection and pneumonia.

Deletion of a cation transporter promotes lysis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant human pathogen which causes respiratory and serious invasive diseases. Mg(2+) is essential for life, and its concentration varies throughout the human body. Magnesium uptake plays an important role in the virulence of many bacterial pathogens. To study the Mg(2+) uptake of S. pneumoniae strain D39, a mutant was generated in SPD1383, a P-type ATPase with homology to the Salmonella Mg(2+) transporter MgtA, which has also been shown to be a Ca(2+) exporter in strain TIGR4. Under low-Ca(2+) conditions, mutation led to a growth defect in complex medium and the gene was nearly essential for growth under low-Mg(2+) conditions. Addition of Mg(2+) restored the normal growth of the mutant in all cases, but the addition of other divalent cations had no effect. Addition of Ca(2+), Mn(2+), and Zn(2+) in the presence of high Mg(2+) concentrations inhibited restoration of growth. The mutant was unable to proliferate in blood, which was also alleviated by the addition of Mg(2+). The protein was located in the membrane and produced in various S. pneumoniae strains and pathogenic streptococcal species. Surprisingly, mutation of the gene led to an elevated toxicity for endothelial cells. This was caused by an increased amount of pneumolysin in the medium, mediated by elevated lysis of the mutant. Thus, in this study, we uncovered a role for SPD1383 in Mg(2+) uptake and hypothesize that the protein is a Mg(2+/)Ca(2+) antiporter. Furthermore, a disturbance in Mg(2+) homeostasis seems to promote lysis of S. pneumoniae.

Phosphatase-dependent regulation of epithelial mitogen-activated protein kinase responses to toxin-induced membrane pores.

Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This activation was tightly regulated, and dephosphorylation of both MAPK occurred within 60 min following exposure. Pretreatment of epithelial cells with inhibitors of cellular phosphatases, including sodium orthovanadate, calyculin A, and okadaic acid, prolonged and intensified MAPK activation. Specific inhibition of MAPK phosphatase-1 did not affect the kinetics of MAPK activation in PFT-exposed epithelial cells, but siRNA-mediated knockdown of serine/threonine phosphatases PP1 and PP2A were potent inhibitors of MAPK dephosphorylation. These results indicate an important role for PP1 and PP2A in termination of epithelial responses to PFT and only a minor contribution of dual-specificity phosphatases, such as MAPK phosphatase-1, which are the major regulators of MAPK signals in other cell types. Epithelial regulation of MAPK signaling in response to membrane disruption involves distinct pathways and may require different strategies for therapeutic interventions.

Towards control of Streptococcus iniae.

Streptococcus iniae is an emerging zoonotic pathogen; such infections generally occur through injuries associated with preparing whole fresh fish for cooking. Those infected to date have been of Asian descent, are usually elderly (average age 68 years), and have had >/=1 underlying conditions that may predispose them to infection. Studies of the foundations of growth characteristics of S. iniae and its interactions with piscine host cells have recently been complemented by molecular studies. Advances in molecular biology have allowed research groups to identify numerous virulence factors and to explore their roles in the progression of S. iniae infection. Many of these virulence factors are homologous to those found in the major human pathogen S. pyogenes. An increased understanding of the properties of these factors and their effect on the success of infection is leading to novel approaches to control S. iniae infection; in particular, vaccination programs at fish farms have reduced the reservoir of infection for additional clinical cases.

Activation of the Nlrp3 inflammasome by Streptococcus pyogenes requires streptolysin O and NF-kappa B activation but proceeds independently of TLR signaling and P2X7 receptor.

Macrophages play a crucial role in the innate immune response against the human pathogen Streptococcus pyogenes, yet the innate immune response against the bacterium is poorly characterized. In the present study, we show that caspase-1 activation and IL-1beta secretion were induced by live, but not killed, S. pyogenes, and required expression of the pore-forming toxin streptolysin O. Using macrophages deficient in inflammasome components, we found that both NLR family pyrin domain-containing 3 (Nlrp3) and apoptosis-associated speck-like protein (Asc) were crucial for caspase-1 activation and IL-1beta secretion, but dispensable for pro-IL-1beta induction, in response to S. pyogenes infection. Conversely, macrophages deficient in the essential TLR adaptors Myd88 and Trif showed normal activation of caspase-1, but impaired induction of pro-IL-1beta and secretion of IL-1beta. Notably, activation of caspase-1 by TLR2 and TLR4 ligands in the presence of streptolysin O required Myd88/Trif, whereas that induced by S. pyogenes was blocked by inhibition of NF-kappaB. Unlike activation of the Nlrp3 inflammasome by TLR ligands, the induction of caspase-1 activation by S. pyogenes did not require exogenous ATP or the P2X7R. In vivo experiments revealed that Nlrp3 was critical for the production of IL-1beta but was not important for survival in a mouse model of S. pyogenes peritoneal infection. These results indicate that caspase-1 activation in response to S. pyogenes infection requires NF-kappaB and the virulence factor streptolysin O, but proceeds independently of P2X7R and TLR signaling.

Streptococcus pneumoniae autolysis prevents phagocytosis and production of phagocyte-activating cytokines.

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-gamma), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-gamma, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-gamma, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.

Structural and functional dissection of the heterocyclic peptide cytotoxin streptolysin S.

The human pathogen Streptococcus pyogenes secretes a highly cytolytic toxin known as streptolysin S (SLS). SLS is a key virulence determinant and responsible for the beta-hemolytic phenotype of these bacteria. Despite over a century of research, the chemical structure of SLS remains unknown. Recent experiments have revealed that SLS is generated from an inactive precursor peptide that undergoes extensive post-translational modification to an active form. In this work, we address outstanding questions regarding the SLS biosynthetic process, elucidating the features of substrate recognition and sites of posttranslational modification to the SLS precursor peptide. Further, we exploit these findings to guide the design of artificial cytolytic toxins that are recognized by the SLS biosynthetic enzymes and others that are intrinsically cytolytic. This new structural information has ramifications for future antimicrobial therapies.

Role of pneumococcal proteins in sensorineural hearing loss due to otitis media.

HYPOTHESIS: Two Streptococcus pneumoniae proteins, pneumococcal surface protein A (PspA) and pneumolysin (Ply), have functional and histopathologic effects on the inner ear. BACKGROUND: Temporary or permanent sensorineural hearing loss is known to be a sequela of pneumococcal otitis media. Several pneumococcal proteins such as PspA and Ply have been shown to contribute to the pathogenesis of the middle ear; however, effects of these proteins on the inner ear and hearing loss are unknown. METHODS: Middle ears of chinchillas were inoculated with either wild-type S. pneumoniae or its mutants, deficient in PspA or Ply proteins. After 28 days, auditory brainstem response of animals was tested, and their bullae were processed for histopathologic analysis by light microscopy. RESULTS: Twenty-eight days after instillation of 20 colony-forming units of wild-type pneumococci, auditory brainstem response test showed threshold changes of 10 to 15 dB for 4 to 32 kHz and more than 20 dB for 1 to 2 kHz. No significant hearing loss was observed after instillation of the same or even higher doses of isogenic S. pneumoniae mutants of PspA or Ply proteins, or saline injection, after the same period. Histologic analysis showed no fluid, inflammatory cells, or bacteria in the middle ear, indicating that hearing loss was sensorineural. Inner ear morphology showed pathologic changes in the stria vascularis, suggesting it as the target of otitis media-induced damage, which may lead to sensorineural hearing loss. CONCLUSION: The virulence PspA and Ply proteins of S. pneumoniae affect the inner ear and auditory function.

Pneumolysin causes neuronal cell death through mitochondrial damage.

Bacterial toxins such as pneumolysin are key mediators of cytotoxicity in infections. Pneumolysin is a pore-forming toxin released by Streptococcus pneumoniae, the major cause of bacterial meningitis. We found that pneumolysin is the pneumococcal factor that accounts for the cell death pathways induced by live bacteria in primary neurons. The pore-forming activity of pneumolysin is essential for the induction of mitochondrial damage and apoptosis. Pneumolysin colocalized with mitochondrial membranes, altered the mitochondrial membrane potential, and caused the release of apoptosis-inducing factor and cell death. Pneumolysin induced neuronal apoptosis without activating caspase-1, -3, or -8. Wild-type pneumococci also induced apoptosis without activation of caspase-3, whereas pneumolysin-negative pneumococci activated caspase-3 through the release of bacterial hydrogen peroxide. Pneumolysin caused upregulation of X-chromosome-linked inhibitor of apoptosis protein and inhibited staurosporine-induced caspase activation, suggesting the presence of actively suppressive mechanisms on caspases. In conclusion, our results indicate additional functions of pneumolysin as a mitochondrial toxin and as a determinant of caspase-independent apoptosis. Considering this, blocking of pneumolysin may be a promising cytoprotective strategy in pneumococcal meningitis and other infections.

Pneumolysin, PspA, and PspC contribute to pneumococcal evasion of early innate immune responses during bacteremia in mice.

The pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a Deltaply PspA(-) PspC(-) mutant. Also, Ply, PspA, and PspC expression resulted in C3 degradation in vitro and in vivo. Finally, blood clearance assays demonstrated that there was enhanced clearance of Deltaply PspA(-) PspC(-) pneumococci compared to the clearance of nonencapsulated pneumococci.

Contributions of pneumolysin, pneumococcal surface protein A (PspA), and PspC to pathogenicity of Streptococcus pneumoniae D39 in a mouse model.

Successful colonization of the upper respiratory tract by Streptococcus pneumoniae is an essential first step in the pathogenesis of pneumococcal disease. However, the bacterial and host factors that provoke the progression from asymptomatic colonization to invasive disease are yet to be fully defined. In this study, we investigated the effects of single and combined mutations in genes encoding pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface protein C (PspC, also known as choline-binding protein A) on the pathogenicity of Streptococcus pneumoniae serotype 2 (D39) in mice. Following intranasal challenge with D39, stable colonization of the nasopharynx was maintained over a 7-day period at a level of approximately 10(5) bacteria per mouse. The abilities of the mutant deficient in PspA to colonize the nasopharynx and to cause lung infection and bacteremia were significantly reduced. Likewise, the PspC mutant and, to a lesser extent, the Ply mutant also had reduced abilities to colonize the nasopharynx. As expected, the double mutants colonized less well than the parent to various degrees and had difficulty translocating to the lungs and blood. A significant additive attenuation was observed for the double and triple mutants in pneumonia and systemic disease models. Surprisingly, the colonization profile of the derivative lacking all three proteins was similar to that of the wild type, indicating virulence gene compensation. These findings further demonstrate that the mechanism of pneumococcal pathogenesis is highly complex and multifactorial but ascribes a role for each of these virulence proteins, alone or in combination, in the process.