Melbourne Dental School - Research Publications

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    Maculatin 1.1 Disrupts Staphylococcus aureus Lipid Membranes via a Pore Mechanism
    Sani, M-A ; Whitwell, TC ; Gehman, JD ; Robins-Browne, RM ; Pantarat, N ; Attard, TJ ; Reynolds, EC ; O'Brien-Simpson, NM ; Separovic, F (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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    Polymerisation of a T Cell Epitope with an Immunostimulatory C3d Peptide Sequence Enhances Antigen Specific T Cell Responses
    O'Brien-Simpson, NM ; Attard, TJ ; Zheng, B ; Walsh, KA ; Reynolds, EC (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.