The Yin and Yang of Pneumolysin During Pneumococcal Infection.

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.

The TCM prescription Ma-xing-shi-gan-tang inhibits Streptococcus pneumoniae pathogenesis by targeting pneumolysin.

ETHNOPHARMACOLOGICAL RELEVANCE: Ma-xing-shi-gan-tang (MXSGT), which is documented in the Treatise on Febrile Diseases and is a therapeutic drug, is a well-known classic prescription in China and has been widely studied. Previous studies have shown that MXSGT has various pharmacological activities, including anti-influenza virus activity, and ameliorates microvascular hyperpermeability and inflammatory reactions. However, no study has reported the effect of MXSGT in the treatment of bacterial pneumonia. AIM OF THE STUDY: In this study, the potential inhibition of MXSGT against the virulence of S. pneumoniae by targeting PLY was investigated. MATERIALS AND METHODS: First, HPLC analysis was used to determine the main components of MXSGT. Then PLY protein was constructed and used for hemolysis assay and western blot to test the ability of MXSGT to inhibit PLY activity, production and widowed characteristics. The growth curve of S. pneumoniae was drawled with or without MXSGT treatment. In addition, the inhibition of MXSGT against PLY-mediated A549 cell death was examined by cytotoxicity assay. Finally, the mouse experiment was used to verify the effect of MXSGT on mouse lungs. RESULTS: This work has discovered that MXSGT, a TCM prescription, is an effective inhibitor of PLY, an important virulence factor that is essential for S. pneumoniae pathogenicity. MXSGT inhibits the oligomerization of PLY without affecting S. pneumoniae growth and PLY production. In addition, experimental MXSGT treatment was effective against S. pneumoniae infection both in vitro and in vivo. CONCLUSION: These findings directly demonstrate the potential mechanism of the Chinese herbal formula MXSGT in the treatment of pneumococcal disease and provide additional evidence for promotion of the wide use of MXSGT in the clinic.

Inhibitory Effect of the Traditional Chinese Medicine Ephedra sinica granules on Streptococcus pneumoniae Pneumolysin.

Streptococcus pneumoniae (S. pneumoniae) is an opportunistic pathogen that causes pneumonia, meningitis and bacteremia in humans and animals. Pneumolysin (PLY), a major pore-forming toxin that is important for S. pneumoniae pathogenicity, is a promising target for the development of anti-infective agents. Ephedra sinica granules (ESG) is one of the oldest medical preparation with multiple biological activities (such as a divergent wind and cold effect); however, the detailed mechanism remains unknown. In this study, we found that ESG treatment significantly inhibited the oligomerization of PLY and then reduced the activity of PLY without affecting S. pneumoniae growth and PLY production. In a PLY and A549 cell co-incubation system, the addition of ESG resulted in significant protection against PLY-mediated cell injury. Furthermore, S. pneumoniae-infected mice showed decreased mortality, and alleviated tissue damage and inflammatory reactions following treatment with ESG. Our results indicate that ESG is a potential candidate treatment for S. pneumoniae infection that targets PLY. This finding partially elucidates the mechanism of the Chinese herbal formula ESG in the treatment of pneumococcal disease.

Engineering detoxified pneumococcal pneumolysin derivative ΔA146PLY for self-biomineralization of calcium phosphate: Assessment of their protective efficacy in murine infection models.

Vaccine design ushered in the era of nanotechnology, as the vaccine is being developed toward particulate formulation. We have previously shown that the attenuated pneumolysin mutant (ΔA146PLY) was a safe and effective pneumococcal vaccine candidate. Here, to further optimize the formulation, we fused calcium phosphate (CaP) binding domains with ΔA146PLY so that the biocompatible CaP can mineralize with the protein automatically, allowing simple production of nanoparticle antigen during preparation. We fabricated four different nanoparticles, and then we compared the characteristics of different CaP-ΔA146PLY nanoparticles and demonstrated the influence of CaP binding domains on the size, shape and surface calcium content of the nanoparticles. It was found that these self-biomineralized CaP-ΔA146PLY nanoparticles varied in their capacity to induce BMDCs and splenocytes production of cytokines. We further demonstrated that, compared to free proteins, nanoparticle antigens induced more efficient humoral and cellular immune responses which was strong enough to protect mice from both pneumonia and sepsis infection. Also, the integration of CaP to protein has no significant impairment on body weight of animals, and subcutaneous injection of ΔA146PLY-peptides@CaP nanoparticles did not lead to permanent formation of nodules in the skin relative to Alum adjuvant formulated antigens. Together, our data sufficiently suggest that soluble ΔA146PLY vaccine candidate could be processed into nanoparticles by self-biomineralization of CaP, the immunogenicity of which could be efficiently improved by the CaP binding domains and biomineralization.

Magnesium therapy improves outcome in Streptococcus pneumoniae meningitis by altering pneumolysin pore formation.

BACKGROUND AND PURPOSE: Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults and is characterized by high lethality and substantial cognitive disabilities in survivors. Here, we have studied the capacity of an established therapeutic agent, magnesium, to improve survival in pneumococcal meningitis by modulating the neurological effects of the major pneumococcal pathogenic factor, pneumolysin. EXPERIMENTAL APPROACH: We used mixed primary glial and acute brain slice cultures, pneumolysin injection in infant rats, a mouse meningitis model and complementary approaches such as Western blot, a black lipid bilayer conductance assay and live imaging of primary glial cells. KEY RESULTS: Treatment with therapeutic concentrations of magnesium chloride (500 mg·kg-1 in animals and 2 mM in cultures) prevented pneumolysin-induced brain swelling and tissue remodelling both in brain slices and in animal models. In contrast to other divalent ions, which diminish the membrane binding of pneumolysin in non-therapeutic concentrations, magnesium delayed toxin-driven pore formation without affecting its membrane binding or the conductance profile of its pores. Finally, magnesium prolonged the survival and improved clinical condition of mice with pneumococcal meningitis, in the absence of antibiotic treatment. CONCLUSIONS AND IMPLICATIONS: Magnesium is a well-established and safe therapeutic agent that has demonstrated capacity for attenuating pneumolysin-triggered pathogenic effects on the brain. The improved animal survival and clinical condition in the meningitis model identifies magnesium as a promising candidate for adjunctive treatment of pneumococcal meningitis, together with antibiotic therapy.

Pyruvate oxidase of Streptococcus pneumoniae contributes to pneumolysin release.

BACKGROUND: Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth. RESULTS: Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis. CONCLUSIONS: Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.

Bacterial cytolysin during meningitis disrupts the regulation of glutamate in the brain, leading to synaptic damage.

Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.

Allicin from garlic neutralizes the hemolytic activity of intra- and extra-cellular pneumolysin O in vitro.

Pneumolysin (PLY) is a key virulence factor contributes to the pathogenesis of Streptococcus pneumoniae. In this study we investigated the effect of allicin and aqueous garlic extracts on hemolytic activity of PLY both in prelysed and intact cells. Additionally the antimicrobial activity of allicin was tested against the bacteria. All tested materials potently inhibited the PLY hemolytic activity. Allicin neutralizes PLY in a concentration- and time-dependent manner. Twenty five minute incubation of PLY (2 HU/mL) with 0.61 µM/mL concentration of allicin, totally inhibited hemolytic activity of PLY (IC50 = 0.28 µM/mL). The inhibitory activity of old extract of garlic was similar to pure allicin (IC50 = 50.46 µL/mL; 0.31 µM/mL; P < 0.05). In contrast fresh extract of garlic inhibits the PLY hemolytic activity at lower concentrations (IC50 = 13.96 µL/mL; 0.08 µM/mL allicin). Exposure of intact cells to allicin (1.8 µM) completely inhibited hemolytic activity of PLY inside bacterial cells. The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 5 mM, proposing that allicin likely inhibits the PLY by binding to cysteinyl residue in the binding site. The MIC value of allicin was determined to be 512 µg/mL (3.15 µM/mL). These results indicate that PLY is a novel target for allicin and may provide a new line of investigation on pneumococcal diseases in the future.

Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance.

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3mug/mL and 43.1mug/mL against sheep and 13.5mug/mL and 8.8mug/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore((R)) technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4mug/mL to 35.4mug/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.

Inhibition of streptolysin O by allicin - an active component of garlic.

Streptolysin O (SLO) is a potent cytolytic toxin produced by almost all strains of group A streptococci and is considered an important virulence factor for this organism. In this study we investigated the effect of allicin and aqueous garlic extracts on the haemolytic activity of SLO. All tested materials potentially inhibited the SLO haemolytic activity. Allicin neutralized SLO in a dose- and time-dependent manner. A 15 min incubation of SLO with 35 microg allicin totally inhibited the haemolytic activity of SLO [IC(50) (concentration necessary to reach half maximum inhibition)=5.97 microg]. The inhibitory activity of an old extract of garlic was equipotent to pure allicin (IC(50)=6.27 microg; P<0.05). In contrast, fresh extract of garlic inhibited the SLO haemolytic activity at lower concentrations (IC(50)=1.59 microl; 1.9 microg allicin). The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 2 mM, suggesting that allicin likely inhibits the SLO by binding to the cysteine residue in the binding site. These results indicate a new activity for allicin and allicin may be a potential alternative drug against streptococcal diseases.

Reduced release of pneumolysin by Streptococcus pneumoniae in vitro and in vivo after treatment with nonbacteriolytic antibiotics in comparison to ceftriaxone.

Pneumolysin, a virulence factor of Streptococcus pneumoniae with cytotoxic and proinflammatory activities, occurs at concentrations from 0.85 to 180 ng/ml in cerebrospinal fluid (CSF) of meningitis patients. In pneumococcal cultures and in a rabbit meningitis model, the concentrations of pneumolysin in supernatant and CSF were lower after addition of nonbacteriolytic bactericidal antibiotics (rifampin and clindamycin) than after incubation with ceftriaxone.

Insertional inactivation of streptolysin S expression is associated with altered riboflavin metabolism in Streptococcus pyogenes.

Transposon Tn916 mutagenesis was used to create a mutant of Streptococcus pyogenes M type 3, designated ISS417, in which the ability to produce streptolysin S (SLS) and several other exoproteins was impaired. Concomitantly, the mutant became dependent upon riboflavin for growth and was able to grow in Todd Hewitt broth (THB) when supplemented with riboflavin or riboflavinrich yeast extract. The parent strain was apparently able to utilize THB-derived components as a substitute for riboflavin, while the mutant was not. Although the parent strain grew well in synthetic medium, it was unable to produce SLS, except when it was supplemented with a small amount of THB. Thus, a component of THB was able to "trigger" SLS formation in the parent strain. The mutant grew well in this medium, but was unable to produce SLS even when it was supplemented with THB. Southern hybridization analysis revealed that the ISS417 mutant harbours a single transposon insertion in its chromosome. Phage transduction experiments showed that the riboflavin dependency and the inability to make SLS phenotypes are co-transducible. The pleotrophic properties of the ISS417 mutant differ from those reported for insertional inactivation of the mga locus which regulates production of a number of surface proteins in S. pyogenes and the sar locus which regulates production of a number of exoproteins in Staphylococcus aureus. In view of the possibility that there exist a genetic linkage between the riboflavin biosynthetic pathway and expression of the oxygen-stable SLS, we hypothesize that SLS has a role in the growth economy of S. pyogenes.