Melbourne Dental School - Research Publications

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    Architectural Effects of Star-Shaped "Structurally Nanoengineered Antimicrobial Peptide Polymers" (SNAPPs) on Their Biological Activity
    Shirbin, SJ ; Insua, I ; Holden, JA ; Lenzo, JC ; Reynolds, EC ; O'Brien-Simpson, NM ; Qiao, GG (WILEY, 2018-11-01)
    In this work, the effect of two key structural parameters, number of arms and arm length, of star-shaped "structurally nanoengineered antimicrobial peptide polymers" (SNAPPs) on their antimicrobial activity and biocompatibility, is investigated. A library of star-shaped SNAPPs is prepared, containing varying arm numbers and arm lengths. Antimicrobial assays are then performed to assess the capacity of the SNAPPs to disrupt the membrane, inhibit the growth, and kill pathogenic bacteria. A major finding of the study is that increasing arm number and length of SNAPPs enhanced antimicrobial activity, which can be respectively attributed to the higher local concentrations of polypeptide arms and increased α-helical content. SNAPP architecture is shown to affect the bacteria membrane state and therefore mechanism of killing. Two more potent structures with up to twice the antimicrobial activity of the previously reported SNAPP are discovered in this process. Toxicities of the SNAPPs also increase with arm number and arm length, however therapeutic index calculations identified a 16-arm SNAPP and an easier to prepare 4-arm SNAPP as the best therapeutic agents. The biocompatibility of the SNAPP with the best biological activity is also evaluated in vivo, showing no markers of systemic damage in mice.
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    Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo
    Cecil, JD ; O'Brien-Simpson, NM ; Lenzo, JC ; Holden, JA ; Singleton, W ; Perez-Gonzalez, A ; Mansell, A ; Reynolds, EC (FRONTIERS MEDIA SA, 2017-08-25)
    Outer membrane vesicles (OMVs) are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. In this study, we investigated the immune-modulatory effects of P. gingivalis, T. denticola, and T. forsythia OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naïve) and M(IFNγ) macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1β cytokine secretion. P. gingivalis OMVs were also found to induce anti-inflammatory IL-10 secretion. Although unprimed monocytes and macrophages were resistant to OMV-induced cell death, lipopolysaccharide or OMV priming resulted in a significantly reduced cell viability. P. gingivalis, T. denticola, and T. forsythia OMVs all activated inflammasome complexes, as monitored by IL-1β secretion and ASC speck formation. ASC was critical for OMV-induced inflammasome formation, while AIM2-/- and Caspase-1-/- cells had significantly reduced inflammasome formation and NLRP3-/- cells exhibited a slight reduction. OMVs were also found to provide both priming and activation of the inflammasome complex. High-resolution microscopy and flow cytometry showed that P. gingivalis OMVs primed and activated macrophage inflammasomes in vivo with 80% of macrophages exhibiting inflammasome complex formation. In conclusion, periodontal pathogen OMVs were found to have significant immunomodulatory effects upon monocytes and macrophages and should therefore influence pro-inflammatory host responses associated with disease.
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    Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2
    Holden, JA ; O'Brien-Simpson, NM ; Lenzo, JC ; Orth, RKH ; Mansell, A ; Reynolds, EC ; McCormick, B (AMER SOC MICROBIOLOGY, 2017-09-01)
    Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2-/-, and TLR4-/- macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals.
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    Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis
    Lam, RS ; O'Brien-Simpson, NM ; Holden, JA ; Lenzo, JC ; Fong, SB ; Reynolds, EC ; Yilmaz, Ö (PUBLIC LIBRARY SCIENCE, 2016-07-06)
    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis.
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    A therapeutic Porphyromonas gingivalis gingipain vaccine induces neutralising IgG1 antibodies that protect against experimental periodontitis
    O'Brien-Simpson, NM ; Holden, JA ; Lenzo, JC ; Tan, Y ; Brammar, GC ; Walsh, KA ; Singleton, W ; Orth, RKH ; Slakeski, N ; Cross, KJ ; Darby, IB ; Becher, D ; Rowe, T ; Morelli, AB ; Hammet, A ; Nash, A ; Brown, A ; Ma, B ; Vingadassalom, D ; McCluskey, J ; Kleanthous, H ; Reynolds, EC (SPRINGERNATURE, 2016-12-01)
    Porphyromonas gingivalis infected mice with an established P. gingivalis-specific inflammatory immune response were protected from developing alveolar bone resorption by therapeutic vaccination with a chimera (KAS2-A1) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. Protection was characterised by an antigen-specific IgG1 isotype antibody and Th2 cell response. Adoptive transfer of KAS2-A1-specific IgG1 or IgG2 expressing B cells confirmed that IgG1-mediated protection. Furthermore, parenteral or intraoral administration of KAS2-A1-specific polyclonal antibodies protected against the development of P. gingivalis-induced bone resorption. The KAS2-A1-specific antibodies neutralised the gingipains by inhibiting: proteolytic activity, binding to host cells/proteins and co-aggregation with other periodontal bacteria. Combining key gingipain sequences into a chimera vaccine produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis.
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    Porphyromonas gulae Has Virulence and Immunological Characteristics Similar to Those of the Human Periodontal Pathogen Porphyromonas gingivalis
    Lenzo, JC ; O'Brien-Simpson, NM ; Orth, RK ; Mitchell, HL ; Dashper, SG ; Reynolds, EC ; McCormick, BA (AMER SOC MICROBIOLOGY, 2016-09-01)
    Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human periodontal pathogen Porphyromonas gingivalis is the lysyl- and arginyl-specific proteolytic activity of the gingipains. We screened several Porphyromonas species isolated from companion animals-P. asaccharolytica, P. circumdentaria, P. endodontalis, P. levii, P. gulae, P. macacae, P. catoniae, and P. salivosa-for Lys- and Arg-specific proteolytic activity and compared the epithelial and macrophage responses and induction of alveolar bone resorption of the protease active species to that of Porphyromonas gingivalis Only P. gulae exhibited Lys-and Arg-specific proteolytic activity. The genes encoding the gingipains (RgpA/B and Kgp) were identified in the P. gulae strain ATCC 51700 and all publicly available 12 draft genomes of P. gulae strains. P. gulae ATCC 51700 induced levels of alveolar bone resorption in an animal model of periodontitis similar to those in P. gingivalis W50 and exhibited a higher capacity for autoaggregation and binding to oral epithelial cells with induction of apoptosis. Macrophages (RAW 264.7) were found to phagocytose P. gulae ATCC 51700 and the fimbriated P. gingivalis ATCC 33277 at similar levels. In response to P. gulae ATCC 51700, macrophages secreted higher levels of cytokines than those induced by P. gingivalis ATCC 33277 but lower than those induced by P. gingivalis W50, except for the interleukin-6 response. Our results indicate that P. gulae exhibits virulence characteristics similar to those of the human periodontal pathogen P. gingivalis and therefore may play a key role in the development of periodontitis in companion animals.
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    Rapid Chair-Side Test for Detection of Porphyromonas gingivalis
    O'Brien-Simpson, NM ; Burgess, K ; Lenzo, JC ; Brammar, GC ; Darby, IB ; Reynolds, EC (SAGE PUBLICATIONS INC, 2017-06-01)
    Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis, and its intraoral levels have been shown to predict disease progression (activity). An accurate and sensitive chair-side (point of care) test to determine disease activity is critical for early intervention and clinical management of disease. This study aimed to develop a rapid, chair-side, saliva-based detection of P. gingivalis. Monoclonal antibodies (mAbs) to the A1-adhesin domain of the P. gingivalis RgpA-Kgp proteinase-adhesin complex were screened by enzyme-linked immunosorbent assay and microbial flow cytometry, with 2 mAbs shown to recognize all laboratory and clinical strains tested, without significantly cross-reacting with other oral bacteria tested. With these mAbs, an immunochromatographic device was produced and shown in preclinical studies to detect, in inoculated saliva, all P. gingivalis laboratory strains and clinical isolates tested. The device was able to detect ≥1 × 105 P. gingivalis cells/mL. In a patient age- and sex-matched control clinical cohort, P. gingivalis levels in saliva-as measured by real-time polymerase chain reaction-positively correlated with P. gingivalis levels in subgingival plaque ( r = 0.819, P < 0.01) and clinical parameters of disease ( r = 0.633, P < 0.01). A positive device result strongly correlated with P. gingivalis levels >1 × 105 cells/mL in saliva ( r = 0.778, P < 0.001) and subgingival plaque ( r = 0.715, P < 0.001) with sensitivity, specificity, positive/negative predictive values, and accuracy levels of 95.0%, 93.3%, 90.5%, 96.6%, and 94.0%, respectively. The device result also positively correlated ( r = 0.695, P < 0.01) with disease severity as measured by probing depth. Detection of P. gingivalis in saliva was found to be rapid, taking 3 min from sample collection.
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    Differential Roles of the Protein Corona in the Cellular Uptake of Nanoporous Polymer Particles by Monocyte and Macrophage Cell Lines
    Yan, Y ; Gause, KT ; Kamphuis, MMJ ; Ang, C-S ; O'Brien-Simpson, NM ; Lenzo, JC ; Reynolds, EC ; Nice, EC ; Caruso, F (AMER CHEMICAL SOC, 2013-12-01)
    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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    Codelivery of NOD2 and TLR9 Ligands via Nanoengineered Protein Antigen Particles for Improving and Tuning Immune Responses
    Gause, KT ; Yan, Y ; O'Brien-Simpson, NM ; Cui, J ; Lenzo, JC ; Reynolds, EC ; Caruso, F (WILEY-V C H VERLAG GMBH, 2016-11-02)
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    Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors
    Veith, PD ; Chen, Y-Y ; Chen, D ; O'Brien-Simpson, NM ; Cecil, JD ; Holden, JA ; Lenzo, JC ; Reynolds, EC (AMER CHEMICAL SOC, 2015-12-01)
    Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.