Anticytotoxin Effects of Amentoflavone to Pneumolysin
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Pneumolysin (PLY) is a devastating bacterial protein toxin of Streptococcus pneumoniae that punctures the cytomembrane, leading to pathological reactions, such as cell disruption and inflammation. Drugs capable of closely impacting the toxin are considered advantageous in the treatment of bacterial infections. Amentoflavone (AMF) is a chemical substance extracted from traditional Chinese herbs. Previous studies have demonstrated that AMF has multiple pharmacological effects and mentioned without attenuating pneumolysin-mediated cytotoxicity. This work focuses on the influence of AMF on inhibitory hemolytic mechanisms. AMF interacts with the toxin at Ser254, Glu277, Arg359, and effectively weakens the oligomerization of wild-type PLY and provides considerable protection against pneumolysin-mediated human alveolar epithelial (A549) cell damage. The results of our study demonstrate that AMF could be a candidate against pneumolysin-related injury.Keywords:
Pneumolysin
Amentoflavone
Pneumolysin (PLY) is a devastating bacterial protein toxin of Streptococcus pneumoniae that punctures the cytomembrane, leading to pathological reactions, such as cell disruption and inflammation. Drugs capable of closely impacting the toxin are considered advantageous in the treatment of bacterial infections. Amentoflavone (AMF) is a chemical substance extracted from traditional Chinese herbs. Previous studies have demonstrated that AMF has multiple pharmacological effects and mentioned without attenuating pneumolysin-mediated cytotoxicity. This work focuses on the influence of AMF on inhibitory hemolytic mechanisms. AMF interacts with the toxin at Ser254, Glu277, Arg359, and effectively weakens the oligomerization of wild-type PLY and provides considerable protection against pneumolysin-mediated human alveolar epithelial (A549) cell damage. The results of our study demonstrate that AMF could be a candidate against pneumolysin-related injury.
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Abstract Streptococcus pneumoniae (S. pneumoniae) is a significant Gram-positive opportunistic pathogen responsible for a variety of lethal infections. This bacterium accounts for more deaths from diseases than any other single pathogen worldwide. Distinctively, these symptoms arise despite effective antibiotic therapy. This study unveiled a novel mechanism of resistance to S. pneumoniae infection by targeting pneumolysin (PLY) and sortase A (Srt A), the key virulence factors of S. pneumoniae. Through protein phenotype assays, we found alnustone to be a potent drug that inhibits both PLY and Srt A. Using a PLY-mediated hemolysis assay, we found that albumin can effectively reduce Srt A peptidase activity by blocking PLY oligomerization, thereby directly inhibiting PLY-expressing cytolysis. Co-incubation of S. pneumoniae D39 Srt A with small-molecule inhibitors reduces cell wall-bound Nan A (pneumococcal-anchored surface protein Srt A), inhibits biofilm formation, and significantly reduces biomass. But more interestingly, the protective effect of invasive pneumococcal disease (IPD) on murine streptococcus pneumoniae was further demonstrated. Our study proposes a detailed bacteriostatic mechanism of pneumococcal and highlights the major translational potential of targeting circulating PLY and Srt A to protect against pneumococcal infections. Our results suggest that the antiviral strategy of directly targeting PLY and Srt A with alnustone is a promising treatment option for Streptococcus pneumoniae and that alnustone can be used as an effective inhibitor of PLY and Srt A.
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Pneumococcal infections
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ABSTRACT Streptococcus pneumoniae is an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms by S. pneumoniae is important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence of S. pneumoniae and is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that a luxS knockout mutant was unable to form early biofilms and expressed less ply mRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation. S. pneumoniae ply knockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply’s role in biofilm formation appears to be independent of its hemolytic activity, as S. pneumoniae serotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly of S. pneumoniae biofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development. IMPORTANCE The bacterium Streptococcus pneumoniae (commonly known as the pneumococcus) is commonly carried in the human nasopharynx and can spread to other body sites to cause disease. In the nasopharynx, middle ear, and lungs, the pneumococcus forms multicellular surface-associated structures called biofilms. Pneumolysin is an important toxin produced by almost all S. pneumoniae strains, extensively studied for its ability to cause damage to human tissue. In this paper, we demonstrate that pneumolysin has a previously unrecognized role in biofilm formation by showing that strains without pneumolysin are unable to form the same amount of biofilm on plastic and human cell substrates. Furthermore, we show that the role of pneumolysin in biofilm formation is separate from the hemolytic activity responsible for tissue damage during pneumococcal diseases. This novel role for pneumolysin suggests that pneumococcal vaccines directed against this protein should be investigated for their potential impact on biofilms formed during carriage and disease.
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Pneumolysin, a cytolytic protein produced by Streptococcus pneumoniae, has many properties which suggest it may be an important virulence factor in pneumococcal ocular infections. To directly test this possibility, we have constructed pneumolysin-negative strains of U., pneumoniae and compared their virulence with that of the wild type in a rabbit model of intracorneal infection. A pneumolysin-negative strain produced by chemical mutagenesis (probably a point mutant) was found to be no less virulent than the parent strain. However, a strain bearing a deletion in the pneumolysin gene showed greatly reduced virulence. This strain produced less pathology while showing significantly higher bacterial counts. These results suggest that a property of the pneumolysin molecule other than its cytolytic (hemolytic) activity may be involved in its pathogenic mechanism of action. This property may be the ability to activate complement, known to be a function of pneumolysin, which results in influx of PMNs, reducing the bacterial counts but also producing tissue damage.
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Virulence factor
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Lytic cycle
Pneumococcal infections
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Pneumolysin
Acute Otitis Media
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Pneumococcal infections
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We evaluated the applicability of ply PCR for confirmation of the identification of Streptococcus pneumoniae. lytA PCR, 16S rRNA sequencing, and amplified-fragment length polymorphism were used as reference methods. In contrast to the lytA gene, the ply gene proved to be not specific for S. pneumoniae. The presence of the ply gene in other streptococci, in particular Streptococcus mitis, suggests that pneumolysin plays a pathogenic role.
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Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns expressed by pathogens. Pneumolysin, an intracellular toxin found in all Streptococcus pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by TLR4. Although TLR2 is considered the most important receptor for Gram-positive bacteria, our laboratory previously could not demonstrate a decisive role for TLR2 in host defence against pneumonia caused by a serotype 3 S. pneumoniae. Here we tested the hypothesis that in the absence of TLR2, S. pneumoniae can still be sensed by the immune system through an interaction between pneumolysin and TLR4. C57BL/6 wild-type (WT) and TLR2 knockout (KO) mice were intranasally infected with either WT S. pneumoniae D39 (serotype 2) or the isogenic pneumolysin-deficient S. pneumoniae strain D39 PLN. TLR2 did not contribute to antibacterial defence against WT S. pneumoniae D39. In contrast, pneumolysin-deficient S. pneumoniae only grew in lungs of TLR2 KO mice. TLR2 KO mice displayed a strongly reduced early inflammatory response in their lungs during pneumonia caused by both pneumolysin-producing and pneumolysin-deficient pneumococci. These data suggest that pneumolysin-induced TLR4 signalling can compensate for TLR2 deficiency during respiratory tract infection with S. pneumoniae.
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To investigate the effects of clarithromycin (0.01–0.5 mg/L) alone or in combination with ceftriaxone (0.1 and 0.25 mg/L) on pneumolysin production by both macrolide-susceptible and -resistant [2 erm(B) positive and 2 mef(A) positive] strains of Streptococcus pneumoniae. The bacteria were cultured for 6 h at 37°C/5% CO2 in tryptone soy broth, washed, enumerated and resuspended to 0.5–3 × 108 cfu/mL in tissue culture medium, RPMI 1640. After 16 h of incubation at 37°C / 5% CO2, pneumolysin was assayed in the bacteria-free supernatants, as well as in lysates, using a functional assay based on the influx of calcium into human neutrophils. Exposure of not only macrolide-susceptible strains, but also the macrolide-resistant strains, of S. pneumoniae to sub-MICs of clarithromycin resulted in dose-related inhibition of the pneumolysin production, whereas production of the toxin was unaffected by ceftriaxone. These observations demonstrate that even in the setting of macrolide resistance the production of pneumolysin, a key virulence factor of the pneumococcus, is attenuated by exposure of this microbial pathogen to clarithromycin.
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