Melbourne Dental School - Research Publications
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ItemStreptococcus mutans biofilm disruption by κ-casein glycopeptide(ELSEVIER SCI LTD, 2013-06)UNLABELLED: Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities. OBJECTIVES: To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions. METHODS: Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn). RESULTS: CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37μm and a biovolume of 3.88μm(3)/μm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity. CONCLUSIONS: KCG and KCG-Zn may have potential as natural biofilm disruptive agents.
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ItemDifferential Roles of the Protein Corona in the Cellular Uptake of Nanoporous Polymer Particles by Monocyte and Macrophage Cell Lines(AMER CHEMICAL SOC, 2013-12)Many biomolecules, mainly proteins, adsorb onto polymer particles to form a dynamic protein corona in biological environments. The protein corona can significantly influence particle-cell interactions, including internalization and pathway activation. In this work, we demonstrate the differential roles of a given protein corona formed in cell culture media in particle uptake by monocytes and macrophages. By exposing disulfide-stabilized poly(methacrylic acid) nanoporous polymer particles (PMASH NPPs) to complete cell growth media containing 10% fetal bovine serum, a protein corona, with the most abundant component being bovine serum albumin, was characterized. Upon adsorption onto the PMASH NPPs, native bovine serum albumin (BSA) was found to undergo conformational changes. The denatured BSA led to a significant decrease in internalization efficiency in human monocytic cells, THP-1, compared with the bare particles, due to reduced cell membrane adhesion. In contrast, the unfolded BSA on the NPPs triggered class A scavenger receptor-mediated phagocytosis in differentiated macrophage-like cells (dTHP-1) without a significant impact on the overall internalization efficiency. Taken together, this work demonstrates the disparate effects of a given protein corona on particle-cell interactions, highlighting the correlation between protein corona conformation in situ and relevant biological characteristics for biological functionalities.
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ItemMaculatin 1.1 Disrupts Staphylococcus aureus Lipid Membranes via a Pore Mechanism(AMER SOC MICROBIOLOGY, 2013-08)Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.
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ItemClinical isolates and laboratory reference Candida species and strains have varying abilities to form biofilms(OXFORD UNIV PRESS, 2013-11)Candida biofilms are a major virulence trait for this yeast. In this study, the biofilm-forming ability of the major medically important clinical and laboratory reference strains was compared. Biofilms were quantified using traditional methods, that is, crystal violet (CV), tetrazolium (XTT) reduction and colony-forming unit assays (CFU), and two new methods: an automated cell counter (ACC) and biofilm suspension turbidity (BST) method. Biofilms could be categorized based on biofilm biomass (high, medium and low) and growth state (high and low). Candida albicans genotypes, A, B and C, showed medium biofilm mass and low growth rate, and only one C. albicans laboratory strain, ATCC MYA-2719, matched this biofilm category. Of all non-albicans Candida species tested, only Candida dubliniensis and Candida glabrata laboratory and clinical isolates had similar biofilm development. The ACC and BST methods for measuring biofilm significantly correlated with CV and CFU biofilm mass measurements. Thus, biofilm mass can be rapidly assessed using biofilm disruptive/cellular nondestructive methods allowing yeast biofilm cells to be used for further analysis. In conclusion, Candida laboratory reference strains and clinical isolates have been shown to form biofilms at different rates; hence for validity, the selection of laboratory reference strains in biofilm studies may be critical for virulence assessment.
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ItemNo Preview AvailablePolymerisation of a T Cell Epitope with an Immunostimulatory C3d Peptide Sequence Enhances Antigen Specific T Cell Responses(SPRINGER, 2013-03)The complement protein C3d and C3d derived peptides that bind CD21 are known to enhance immunity to co-immunised antigens. In this study we have synthesised the minimal CD21 binding sequence of C3d (1227LYNVEA 1232) as mono, di and tri tandem repeats and derivatised the N-terminus with an acryloyl moiety. These acryloyl-(C3d)n peptides were co-polymerised with a acryloyl-T cell epitope (PAS1K) from the Porphyromonas gingivalis antigen the RgpA–Kgp proteinase–adhesin complex. The ability of C3d containing polymers to enhance T cell immunity in vitro and in vivo was evaluated. When used to stimulate in vitro PAS1K-primed or RgpA–Kgp complex-primed T cells the C3d containing PAS1K polymers induced a mixed and significantly (p\0.05) higher IL-4 and IFNc T cell response compared to that induced by the PAS1K peptide or polymer. PAS1K polymers containing tandem repeats of C3d induced a significantly (p\0.05) stronger maximal proliferative response, at the same antigenic dose, compared to that induced by the PAS1K peptide or polymer. When used as immunogens to prime T cells all of the C3d containing PAS1K polymers induced a dominant IFNc T cell response and reduced the antigen dose required for maximal proliferation 150-fold compared to that required for the PAS1K-peptide or polymer primed T cells. In conclusion, the 6 residue sequence LYNVEA from C3d is sufficient to enhance immunity to an antigen and that the effect is more pronounced when C3d is part of the immunising antigen rather than an in vitro stimulating antigen.
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ItemAcute phase protein and cytokine levels in serum and saliva: A comparison of detectable levels and correlations in a depressed and healthy adolescent sample(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013-11)Recent research has examined associations between inflammation and mental health, and has increasingly focused on utilising younger samples to characterise the temporal relationship between inflammatory responses and the emergence of other symptoms. These studies have typically used blood to measure inflammation, although rates of detection for many inflammatory markers appear to be low. Saliva is a safe and low-cost alternative, and adult research has shown that levels of some salivary markers correlate well with those in serum. However, no research has examined this association in young people. This study examined 16 inflammatory markers in serum and saliva in 17 depressed adolescents and 18 healthy controls, aged 13-18 years. In general, detection rates were higher in saliva compared to in serum. When non-detectable levels were excluded, serum levels of C-reactive protein (CRP) correlated with salivary CRP (r=0.424, p=0.015), and this correlation appeared to only exist for those individuals with high levels of serum CRP (r=0.599, p=0.014). However, when non-detectable levels were included as zero, salivary levels of CRP, interleukin (IL)-2, IL-12p70, and interferon (IFN)-γ correlated with their serum counterparts. No significant clinical group differences in any acute phase proteins or cytokines were present. This study suggests that saliva can be used to measure inflammation in studies with adolescent participants, especially CRP, as it appears to correlate with systemic inflammation for those individuals who are expected to have high levels of inflammation. Implications for future directions in research on salivary inflammatory markers are discussed.
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ItemKappacin, a novel antibacterial peptide from bovine milk(AMER SOC MICROBIOLOGY, 2001-08)Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.
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ItemDivalent metal cations increase the activity of the antimicrobial peptide kappacin(AMER SOC MICROBIOLOGY, 2005-06)Kappacin, nonglycosylated kappa-casein(106-169), is a novel antimicrobial peptide produced from kappa-casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, kappa-casein(138-158), of these forms, we have shown that the Asp148 to Ala148 substitution is responsible for the lesser antibacterial activity of kappa-casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 microM at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a kappa-casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl2 reduced bacterial viability by 3 log10 and suppressed recovery of viability. In contrast 20 mM ZnCl2 alone reduced bacterial viability by approximately 1 log10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.
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ItemAn immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss(AMER ASSOC IMMUNOLOGISTS, 2005-09-15)Porphyromonas gingivalis, a pathogen associated with periodontitis, bound to fibrinogen, fibronectin, hemoglobin, and collagen type V with a similar profile to that of its major virulence factor, the cell surface RgpA-Kgp proteinase-adhesin complex. Using peptide-specific, purified Abs in competitive inhibition ELISAs and epitope mapping assays, we have identified potential adhesin binding motifs (ABMs) of the RgpA-Kgp complex responsible for binding to host proteins. The RgpA-Kgp complex and synthetic ABM and proteinase active site peptides conjugated to diphtheria toxoid, when used as vaccines, protected against P. gingivalis-induced periodontal bone loss in the murine periodontitis model. The most efficacious peptide and protein vaccines were found to induce a high-titer IgG1 Ab response. Furthermore, mice protected in the lesion and periodontitis models had a predominant P. gingivalis-specific IL-4 response, whereas mice with disease had a predominant IFN-gamma response. The peptide-specific Abs directed to the ABM2 sequence (EGLATATTFEEDGVA) protected against periodontal bone loss and inhibited binding of the RgpA-Kgp complex to fibrinogen, fibronectin, and collagen type V. Furthermore, the peptide-specific Abs directed to the ABM3 sequence (GTPNPNPNPNPNPNPGT) protected against periodontal bone loss and inhibited binding to hemoglobin. However, the most protective Abs were those directed to the active sites of the RgpA and Kgp proteinases. The results suggest that when the RgpA-Kgp complex, or functional binding motif or active site peptides are used as a vaccine, they induce a Th2 response that blocks function of the RgpA-Kgp complex and protects against periodontal bone loss.
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ItemA novel Porphyromonas gingivalis FeoB plays a role in manganese accumulation(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005-07-29)FeoB is an atypical transporter that has been shown to exclusively mediate ferrous ion transport in some bacteria. Unusually the genome of the periodontal pathogen Porphyromonas gingivalis has two genes (feoB1 and feoB2) encoding FeoB homologs, both of which are expressed in bicistronic operons. Kinetic analysis of ferrous ion transport by P. gingivalis W50 revealed the presence of a single, high affinity system with a K(t) of 0.31 microM. FeoB1 was found to be solely responsible for this transport as energized cells of the isogenic FeoB1 mutant (W50FB1) did not transport radiolabeled iron, while the isogenic FeoB2 mutant (W50FB2) transported radiolabeled iron at a rate similar to wild type. This was reflected in the iron content of W50FB1 grown in iron excess conditions which was approximately half that of the wild type and W50FB2. The W50FB1 mutant had increased sensitivity to both oxygen and hydrogen peroxide and was avirulent in an animal model of infection whereas W50FB2 exhibited the same virulence as the wild type. Analysis of manganous ion uptake using inductively coupled plasma-mass spectrometry revealed a greater than 3-fold decrease in intracellular manganese accumulation in W50FB2 which was also unable to grow in manganese-limited media. The protein co-expressed with FeoB2 appears to be a novel FeoA-MntR fusion protein that exhibits homology to a manganese-responsive, DNA-binding metalloregulatory protein. These results indicate that FeoB2 is not involved in iron transport but plays a novel role in manganese transport.