Background and Purpose
L. monocytogenes remain a leading cause of foodborne infection. Listeriolysin O (LLO), an indispensable virulence determinant involved in diverse pathogenic mechanisms of L. ...monocytogenes infection, represents a promising therapeutic target. In this study, we sought to identify an effective inhibitor of LLO pore formation and its mechanism of action in the treatment of L. monocytogenes infection.
Experimental Approach
Haemolysis assays were carried out to screen an effective LLO inhibitor. The interaction between candidate and LLO was investigated using surface plasmon resonance and molecular docking. The effect of candidate on LLO‐mediated cytotoxicity, barrier disruption and immune response were investigated. Finally, the in vivo effect of candidate on mice challenged with L. monocytogenes was examined.
Key results
Amentoflavone, a natural flavone present in traditional Chinese herbs, effectively inhibited LLO pore formation by engaging the residues Lys93, Asp416, Tyr469 and Lys505 in LLO. Amentoflavone dose‐dependently reduced L. monocytogenes‐induced cell injury in an LLO‐dependent manner. In the Caco‐2 monolayer model, amentoflavone maintained the integrity of the epithelial barrier exposed to LLO. Amentoflavone inhibited the inflammatory response evoked by L. monocytogenes in an LLO‐dependent manner, and inhibition was attributed to ability to block perforation‐associated K+ efflux and Ca2+ influx. In the mouse infection model, amentoflavone treatment significantly reduced bacterial burden and pathological lesions in target organs, with a significant increase in survival rate.
Conclusions and Implications
Amentoflavone reduced the pathogenicity of L. monocytogenes by specifically inhibiting LLO pore formation, and this may represent a potential treatment for L. monocytogenes infection.
Listeria monocytogenes is the causative agent of listeriosis, which is dangerous for pregnant women, the elderly or individuals with a weakened immune system. Individuals with leukaemia, cancer, ...HIV/AIDS, kidney transplant and steroid therapy suffer from immunological damage are menaced. World Health Organization (WHO) reports that human listeriosis has a high mortality rate of 20–30% every year. To date, no vaccine is available to treat listeriosis. Thereby, it is high time to design novel vaccines against L. monocytogenes. Here, we present computational approaches to design an antigenic, stable and safe vaccine against the L. monocytogenes that could help to control the infections associated with the pathogen. Three vital pathogenic proteins of L. monocytogenes, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were selected using a subtractive proteomics approach to design the multi-epitope vaccine (MEV). A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. To design the multi-epitope vaccine (MEV) from the selected proteins, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Additionally, a human β-defensin-3 (hBD-3) adjuvant was added to the N-terminal side of the final MEV construct to increase the immune response to the vaccine. The final MEV was predicted to be antigenic, non-allergen and non-toxic in nature. Physicochemical property analysis suggested that the MEV construct is stable and could be easily purified through the E. coli expression system. This in-silico study showed that MEV has a robust binding interaction with Toll-like receptor 2 (TLR2), a key player in the innate immune system. Current subtractive proteomics and immunoinformatics study provides a background for designing a suitable, safe and effective vaccine against pathogenic L. monocytogenes.
•Three pathogenic proteins, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were chosen through a subtractive proteomics pipeline.•A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from the 3 selected proteins.•The designed multiepitope vaccine (MEV) was found to be stable, highly antigenic, non-allergenic and non-toxic.•Codon optimization and in-silico cloning verified the translation efficiency and successful expression of the designed MEV in E. coli.•Additionally, the in-silico immune simulation study exhibited a significant immunogenic response due to the MEV administration.
Genkwa flos (yuanhua in Chinese), the dried flower buds of the plant Daphne genkwa Siebold & Zucc., as a traditional herb widely used for the treatment of inflammation-related symptoms and diseases, ...with the efficacies of diuretic, phlegm-resolving and cough suppressant.
Traditional Chinese Medicine (TCM) is presumed to be of immense potential against pathogens infection. Whereas, the potential efficacy and mechanisms of Genkwa flos against L. monocytogenes infection has not been extensively explored. The present study aimed to identify the bioactive ingredients of Genkwa flos against L. monocytogenes infection and to delineate the underlying mechanisms of action.
Bioinformatics approach at protein network level was employed to investigate the therapeutic mechanisms of Genkwa flos against L. monocytogenes infection. And hemolysis inhibition assay, cytoprotection test, western blotting, oligomerization assay and molecular docking analysis were applied to substantiate the multiple efficacies of Genkwa flos and the bioactive ingredient genkwanin. Histopathological analysis and biochemistry detection were conducted to evaluate the in vivo protective effect of genkwanin.
Network pharmacology and experimental validation revealed that Traditional Chinese Medicine (TCM) Genkwa flos exhibited anti-L. monocytogenes potency and was found to inhibit the hemolytic activity of LLO. Bioactive ingredient genkwanin interfered with the pore-forming activity of LLO by engaging the active residues Tyr414, Tyr98, Asn473, Val100, Tyr440 and Val438, and thereby attenuated LLO-mediated cytotoxicity. Consistent with the bioinformatics prediction, exposed to genkwanin could upregulate the Nrf2 level and promote the translocation of Nrf2. In vivo, genkwanin oral administration (80 mg/kg) significantly protected against systemic L. monocytogenes infection, as evidenced by reduced myeloperoxidase (MPO) and malondialdehyde (MDA) levels, increased mice survival rate by 30% and decreased pathogen colonization.
Our study demonstrated that Genkwa flos is a potential anti-L. monocytogenes TCM, highlighted the therapeutic potential of Genkwa flos active ingredient genkwanin by targeting the pore-forming cytolysin LLO and acting as a promising antioxidative candidate against L. monocytogenes infection.
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•Uncovering the therapeutic mechanisms of Genkwa flos against L. monocytogenes infection.•Genkwa flos and genkwanin were identified as LLO-targeted promising candidates.•Genkwanin exhibited notable treatment benefits against L. monocytogenes in vivo.
Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to ...evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes-induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.
Listeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many infections, including meningitis and abortion in humans and animals; thus, L. ...monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The isolation rate of Listeria monocytogenes in fishery products has always been high. And the pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes. LLO can promote cytosolic bacterial proliferation and help the pathogen evade attacks from the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions.
Here, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization. We also found that morin can effectively alleviate the inflammation induced by Listeria in vivo and in vitro and exerted an obvious protective effect on infected cells and mice.
Morin does not possess anti-Listeria activity, neither does it interfere with secretion of LLO. However, morin inhibits oligomerisation of LLO and morin does reduce the inflammation caused during Listeria infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This study aimed to evaluate the effect of dietary chitosan and listeriolysin O (LLO) on the immune response against Listeria monocytogenes infection in mice. L. monocytogenes isolate was obtained ...from the unit of Zoonotic Disease, College of Veterinary Medicine, University of Baghdad. It was reactivated and cultured on PALCAM agar and exposed to a confirmatory diagnostic test. listeriolysin O (LLO) was extracted and purified. Sixty mice were used and divided into four groups each one involving 15 mice; the first group was fed on diet supplemented with a chitosan for 4 weeks. At the end of the 2nd week, normal saline was injected S/C; the second group was fed on a normal diet without any addition. At the end of 2nd week, 0.3 ml of LLO was injected S/C, then the booster dose of LLO was given after 14 days of 1st injection; the third group was fed on a diet supplemented with chitosan as in G1, and treated as in G2 and the fourth group fed on a normal diet without any addition and treated as in G1. A skin test was performed on 5 mice of each group while a Challenge test by injection of 0.2ml of 1x109 CFU/ml of viable L. monocytogenes intraperitoneally was performed on other mice. The concentration of IgG titer and IL6 were measured. The results revealed that the third group recorded significantly higher values in the skin thickness, IgG, and IL6 concentrations when compared with other groups at different times which indicate that LLO and chitosan may support each other to provide the most beneficial effect by eliciting of good cellular and humeral immunity against listeriosis.
Background
Listeria monocytogenes
(
L. monocytogenes
), as a pandemic foodborne pathogen, severely threatens food security and public health care worldwide, which evolves multiple bacterial virulence ...factors (such as listeriolysin O, LLO) for manipulating the immune response of
L. monocytogenes
-host interactions.
Methods
Hemolysis assay was employed to screen a potential LLO inhibitor and the underlying mechanisms were investigated using molecular dynamics (MD) simulation and oligomerization assay. The effects of candidates on immune response were examined by qRT-PCR and immunoblotting analysis. Histological analysis, ELISA assay and biochemistry detection were conducted to assess
in vivo
efficacy of candidates.
Results
In the present study, natural terpenoid atractylodin was characterized as an alternative drug candidate for the treatment of
L. monocytogenes
by the regulation of LLO function and host Nrf2/NLRP3 signaling pathway. Notably,
in vivo
infection model by
L. monocytogenes
also highlighted that atractylodin treatment provided effective therapeutic benefits, as evidenced by decreased bacterial burden and diminished inflammation. Congruently, the survival rate of
L. monocytogenes
-infection mice increased significantly from 10.0% to 40.0% by atractylodin treatment.
Conclusion
Collectively, our study showed for the first time that atractylodin has tremendous potential to attenuate
L. monocytogenes
pathogenicity by blocking LLO pore formation and mediating the suppression of inflammation and oxidative stress, providing a promising therapeutic strategy and broadening the applications of atractylodin against
L. monocytogenes
infection.
Listeriolysin O (LLO) is a pore‐forming toxin that enables survival and cell‐to‐cell spread of foodborne bacterial pathogen Listeria monocytogenes, which is responsible for the life‐threatening ...disease, listeriosis. LLO–membrane interactions are crucial for pathogenicity of Listeria, but remained unexplained in detail at the molecular level. Here we addressed them by means of 2H, 31P, 13C and 19F solid‐state NMR spectroscopy. Different fluid and ordered cholesterol‐rich membrane lipid bilayer systems were prepared and checked for the integrity and properties in the presence of LLO. LLO has significantly changed dynamics of phospholipid acyl chains of more fluid cholesterol‐rich bilayers, whereas the lipid bilayer organization was not affected. LLO has also affected cholesterol dynamics by increasing the intensity of low frequency motions, indicating direct interactions of LLO with cholesterol. Additionally, the LLO protein was shown to interact differently with lipid membranes, depending on the properties of cholesterol‐rich membranes. The presented results, therefore, provide new insights into the interactions of the bacterial toxin LLO with cholesterol‐rich membrane systems.
Listeriolysin O is a cholesterol‐dependent cytolysin, responsible for the escape of pathogenic bacteria, Listeria monocytogenes, from the acidic phagolysosome. Listeriolysin O binds cholesterol‐rich membranes, oligomerizes, and forms pores by inserting parts of its polypeptide chain into the membrane. Solid‐state NMR experiments showed that listeriolysin O preferentially binds and perturbs the hydrophobic core of more fluid cholesterol‐rich membranes.
Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued ...until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.
Limited information is available on the phenotypic characteristics of Listeriamonocytogenes that may affect its virulence of L.monocytogenes. The study focused on comparing the differences between ...two strains of L. monocytogenes, one hemolytic (LLO+) and one non-hemolytic (tLLO). Using Phenotype Microarray (PM) technology, carbon, nitrogen, and phosphorus-sulfur source utilization by the strains was performed and the KEGG pathway involvement in substrate utilization was analyzed. Of the 285 substrates screened, the results suggested that d-gluconic acid and l-serine may play a role in L. monocytogenes hemolytic activity towards LLO production by generating pyruvate as an intermediate and amino acid biosynthesis, which may play an important role in virulence protein translation. d-gluconic acid and l-serine showed no effect on the tLLO strain in PM analysis and validation tests. Notably, the scope of this study was limited to these two strains of L. monocytogenes. While these findings provide valuable insights into the metabolic differences between hemolytic and non-hemolytic strains, further research is necessary to expand this understanding to a broader range of strains and explore additional factors that may influence LLO functionality.
•There are metabolism differences between L. monocytogenes producing active LLO and truncated LLO.•l-serine and d-gluconic acid were identified as key metabolites that may affect hemolytic activity.•l-serine and d-gluconic acid metabolism generates PRPP and pyruvate as intermediates, respectively.•The metabolism of the intermediates synthesized amino acids may be involved in LLO formation.
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