Cellular response to PorA-Loop4-derived peptides of Neisseria meningitidis

Neisseria meningitidis (the meningococcus) is a major meningitis-causing bacterium and is known for its ability to breach the blood-brain barrier (BBB). Meningococci bind to the Laminin receptor (LAMR) on the surface of the vascular endothelium, which is part of the BBB. In a previous study, the meningococcal surface proteins PorA and PilQ were identified as the bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). PorA preferentially targets the 37 kDa laminin receptor precursor (37LRP) rather than the 67 kDa laminin receptor form (67LR) on the cell surface. It was confirmed that the PorA:37LRP interaction is mediated by PorA-Loop4 as deletion of this loop abrogates recruitment of 37LRP under meningococcal colonies. Using a cyclised peptide corresponding to PorA-Loop4 from N. meningitidis MC58, the PorA-Loop4:37LRP interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1-phase cell cycle arrest. The interaction of PorA-Loop4:37LRP was cytostatic, not cytotoxic. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells at S-phase and a corresponding increase in the G1 population. Based on these results, it is hypothesised that PI3K/Akt pathway was affected by the interaction resulting in G1 arrest. The current study aimed to investigate the perturbations of key proteins’ expression and activation involved in the PI3K/Akt pathway (including Akt, GSK-3β, Cyclin D1, and CDK4) after the treatment of PorA-Loop4 peptide in HBMECs. The work indicated that PorA-Loop4 caused perturbations of Cyclin D1/CDK4 as cell cycle regulators at G1 phase. Transcriptome analysis using qRT-PCR showed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunoblotting confirmed these results as the 24 h-treatment of PorA-Loop4 peptide caused the downregulation of Cyclin D1 and the upregulation of pCDK4 (Thr172). PorA-Loop4 caused a blockade of PI3K/Akt signalling. Using ZSTK474 as a PI3K inhibitor, we observed that the effect of PorA-Loop4 on Cyclin D1 and pCDK4 (Thr172) was PI3K-dependent at both mRNA and protein levels. Exposure of HBMECs to PorA-Loop4 peptide caused a downregulation of pAkt (Ser473) and an upregulation of pGSK-3β (Ser9) at the protein level 24 h post treatment. The upregulation of pGSK-3β (Ser9) was dependent to Akt, and the downregulation of Cyclin D1 was independent of GSK-3β. Using the whole bacterial cells of N. meningitidis wild-type MC58 and its mutants lacking of PorA-Loop4, PorA, and/or PilQ, the role of PorA-Loop4, PorA and PilQ in influencing the PI3K/Akt pathway were investigated. Similar perturbations of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) occurred in PorA-Loop4 peptide treated cells and wild-type infected cells while the abrogation of the PorA-Loop4 erased the perturbations in HBMECs. Both PorA and PilQ have significant roles in the perturbation of pAkt (Ser473), pGSK-3β (Ser9), and Cyclin D1. However, the upregulation of pCDK4 (Thr172) was mediated by Loop4, not by other parts of the PorA, nor by PilQ. Confocal microscopy showed the localisation of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the uninfected, wild-type-infected, and PorA-Loop4 mutant-infected HBMECs. The pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1 were localised mainly in the cytoplasm, while the pCDK4 (Thr172) was localised in cytoplasmic and nuclear regions. Confocal microscopy confirmed the immunoblotting results, as the diffuse pattern of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the wild-type infected cells were in contrast with the uninfected and the PorA-Loop4 mutant infected cells. In summary, PorA-Loop4 causes the perturbation of PI3K/Akt pathway via Akt/GSK-3β/Cyclin D1/CDK4. The data presented in this thesis has extended knowledge of meningococcal-host pathogen interaction. A thorough understanding of PorA-Loop4 interaction with LAMR and its effect in the PI3K/Akt pathway may provide another strategy for the development of interventions against the meningococcus and perhaps other Gram-negative pathogens

IN VITRO AND IN VIVO STUDIES OF ANTIHYPERURICEMIC AND ANTIOXIDANT ACTIVITY FROM BULBS OF BAWANG TIWAI (ELEUTHERINE PALMIFOLIA (L.) MERR.) FROM INDONESIA

Objectives: This study would like to investigate the in vitro antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl assay and in vitro xanthine oxidase activity of the bulbs. This study performs in vivo assays to study the antihyperuricemic activity and antioxidant in the hyperuricemic rat through plasma malondialdehyde measurement. Method: The study was conducted by testing the fresh bulbs of bawang tiwai (Eleutherine palmifolia (L.) Merr. with chemical solvent of ethanol 70% to extract the bulbs. Allopurinol and Vitamin C were used as positive control for the antihyperuricemic assay and antioxidant assay, respectively. Other chemical substances were also used in this study. This study used chicken extract (Brands) 20 ml/kg/body weight to induce the level of uric acid in the blood serum, and potassium oxonate (Sigma 156124) to inhibit the uricase in rats. Results: The results show that the levels of uric acid were measured using spectrophotometer with dichloro-hydroxybenzen sulfonate (Biolabo) a as reagent. The ethanol extract of bawang tiwai (EBT) (E. palmifolia (L.) Merr) was potential to reduce uric acid level at 140, 280, and 560 mg/kg body weight, but possibly without inhibition against xanthine oxydase activity. Conclusion: All doses of EBT could inhibit lipid peroxidation in hyperuricemic condition caused by high purine diet in 14 days

Immunomodulatory effects exerted by extracellular vesicles from <i>Staphylococcus epidermidis</i> and <i>Staphylococcus aureus</i> isolated from bone-anchored prostheses

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties

Role of sodium salicylate in Staphylococcus aureus quorum sensing, virulence, biofilm formation and antimicrobial susceptibility

The widespread threat of antibiotic resistance requires new treatment options. Disrupting bacterial communication, quorum sensing (QS), has the potential to reduce pathogenesis by decreasing bacterial virulence. The aim of this study was to investigate the influence of sodium salicylate (NaSa) on Staphylococcus aureus QS, virulence production and biofilm formation. In S. aureus ATCC 25923 (agr III), with or without serum, NaSa (10 mM) downregulated the agr QS system and decreased the secretion levels of alpha-hemolysin, staphopain A and delta-hemolysin. Inhibition of agr expression caused a downregulation of delta-hemolysin, decreasing biofilm dispersal and increasing biofilm formation on polystyrene and titanium under static conditions. In contrast, NaSa did not increase biofilm biomass under flow but caused one log10 reduction in biofilm viability on polystyrene pegs, resulting in biofilms being twice as susceptible to rifampicin. A concentrationdependent effect of NaSa was further observed, where high concentrations (10 mM) decreased agr expression, while low concentrations (≤0.1 mM) increased agr expression. In S. aureus 8325-4 (agr I), a high concentration of NaSa (10 mM) decreased hla expression, and a low concentration of NaSa (≤1 mM) increased rnaIII and hla expression. The activity of NaSa on biofilm formation was dependent on agr type and material surface. Eight clinical strains isolated from prosthetic joint infection (PJI) or wound infection belonging to each of the four agr types were evaluated. The four PJI S. aureus strains did not change their biofilm phenotype with NaSa on the clinically relevant titanium surface. Half of the wound strains (agr III and IV) did not change the biofilm phenotype in the 3D collagen wound model. In addition, compared to the control, ATCC 25923 biofilms formed with 10 mM NaSa in the collagen model were more susceptible to silver. It is concluded that NaSa can inhibit QS in S. aureus, decreasing the levels of toxin production with certain modulation of biofilm formation. The effect on biofilm formation was dependent on the strain and material surface. It is suggested that the observed NaSa inhibition of bacterial communication is a potential alternative or adjuvant to traditional antibiotics.This research was funded by the Swedish Foundation for Strategic Research (SSF; RMA15-0110 2016), Mölnlycke Health Care AB (Sweden), the European Commission within the H2020-MSCA grant agreement no. 861046 (BIOREMIA-ETN), the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement No: 754412 (MoRE2020— Region Västra Götaland), CARe—Centre for Antibiotic Resistance Research at University of Gothenburg; Swedish Research Council (2018–02891), the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (ALFGBG-725641), the IngaBritt and Arne Lundberg Foundation (LU2021- 0048), the Hjalmar Svensson Foundation; the Doctor Felix Neuberghs Foundation, the Adlerbertska Foundation, and the Area of Advance Materials of Chalmers/GU Biomaterials within the Strategic Research Area initiative launched by the Swedish government

Genomics of Staphylococcus aureus and Staphylococcus epidermidis from periprosthetic joint infections and correlation to clinical outcome

The approach of sequencing or genotyping to characterize the pathogenic potential of staphylococci from orthopedic device-related infection (ODRI) has been applied in recent studies. These studies described the genomic carriage of virulence in clinical strains and compared it with those in commensal strains. Only a few studies have directly correlated genomic profiles to patient outcome and phenotypic virulence properties in periprosthetic joint infections (PJIs). We investigated the association between genomic variations and virulence-associated phenotypes (biofilm-forming ability and antimicrobial resistance) in 111 staphylococcal strains isolated from patients with PJI and the infection outcome (resolved/unresolved). The presence of a strong biofilm phenotype in Staphylococcus aureus and an antibiotic-resistant phenotype in Staphylococcus epidermidis were both associated with treatment failure of PJI. In S. epidermidis, multidrug resistance (MDR) and resistance to rifampicin were associated with unresolved infection. Sequence type 45 (ST45) and ST2 were particularly enriched in S. aureus and S. epidermidis, respectively. S. epidermidis ST2 caused the majority of relapses and was associated with MDR and strong biofilm production, whereas ST215 correlated with MDR and non/weak biofilm production. S. aureus agr II correlated with resolved infection, while S. epidermidis agr I was associated with strong biofilm production and agr III with non/weak production. Collectively, our results highlight the importance of careful genomic and phenotypic characterization to anticipate the probability of the strain causing treatment failure in PJI. Due to the high rate of resistant S. epidermidis strains identified, this study provides evidence that the current recommended treatment of rifampicin and a fluoroquinolone should not be administered without knowledge of the resistance pattern

Cellular response to PorA-Loop4-derived peptides of Neisseria meningitidis

Neisseria meningitidis (the meningococcus) is a major meningitis-causing bacterium and is known for its ability to breach the blood-brain barrier (BBB). Meningococci bind to the Laminin receptor (LAMR) on the surface of the vascular endothelium, which is part of the BBB. In a previous study, the meningococcal surface proteins PorA and PilQ were identified as the bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). PorA preferentially targets the 37 kDa laminin receptor precursor (37LRP) rather than the 67 kDa laminin receptor form (67LR) on the cell surface. It was confirmed that the PorA:37LRP interaction is mediated by PorA-Loop4 as deletion of this loop abrogates recruitment of 37LRP under meningococcal colonies. Using a cyclised peptide corresponding to PorA-Loop4 from N. meningitidis MC58, the PorA-Loop4:37LRP interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1-phase cell cycle arrest. The interaction of PorA-Loop4:37LRP was cytostatic, not cytotoxic. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells at S-phase and a corresponding increase in the G1 population. Based on these results, it is hypothesised that PI3K/Akt pathway was affected by the interaction resulting in G1 arrest. The current study aimed to investigate the perturbations of key proteins’ expression and activation involved in the PI3K/Akt pathway (including Akt, GSK-3β, Cyclin D1, and CDK4) after the treatment of PorA-Loop4 peptide in HBMECs. The work indicated that PorA-Loop4 caused perturbations of Cyclin D1/CDK4 as cell cycle regulators at G1 phase. Transcriptome analysis using qRT-PCR showed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunoblotting confirmed these results as the 24 h-treatment of PorA-Loop4 peptide caused the downregulation of Cyclin D1 and the upregulation of pCDK4 (Thr172). PorA-Loop4 caused a blockade of PI3K/Akt signalling. Using ZSTK474 as a PI3K inhibitor, we observed that the effect of PorA-Loop4 on Cyclin D1 and pCDK4 (Thr172) was PI3K-dependent at both mRNA and protein levels. Exposure of HBMECs to PorA-Loop4 peptide caused a downregulation of pAkt (Ser473) and an upregulation of pGSK-3β (Ser9) at the protein level 24 h post treatment. The upregulation of pGSK-3β (Ser9) was dependent to Akt, and the downregulation of Cyclin D1 was independent of GSK-3β. Using the whole bacterial cells of N. meningitidis wild-type MC58 and its mutants lacking of PorA-Loop4, PorA, and/or PilQ, the role of PorA-Loop4, PorA and PilQ in influencing the PI3K/Akt pathway were investigated. Similar perturbations of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) occurred in PorA-Loop4 peptide treated cells and wild-type infected cells while the abrogation of the PorA-Loop4 erased the perturbations in HBMECs. Both PorA and PilQ have significant roles in the perturbation of pAkt (Ser473), pGSK-3β (Ser9), and Cyclin D1. However, the upregulation of pCDK4 (Thr172) was mediated by Loop4, not by other parts of the PorA, nor by PilQ. Confocal microscopy showed the localisation of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the uninfected, wild-type-infected, and PorA-Loop4 mutant-infected HBMECs. The pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1 were localised mainly in the cytoplasm, while the pCDK4 (Thr172) was localised in cytoplasmic and nuclear regions. Confocal microscopy confirmed the immunoblotting results, as the diffuse pattern of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the wild-type infected cells were in contrast with the uninfected and the PorA-Loop4 mutant infected cells. In summary, PorA-Loop4 causes the perturbation of PI3K/Akt pathway via Akt/GSK-3β/Cyclin D1/CDK4. The data presented in this thesis has extended knowledge of meningococcal-host pathogen interaction. A thorough understanding of PorA-Loop4 interaction with LAMR and its effect in the PI3K/Akt pathway may provide another strategy for the development of interventions against the meningococcus and perhaps other Gram-negative pathogens