Kathy Parisi

Hexima (Australia)

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Nicole L. van der Weerden

La Trobe University

Joanne L. Casey

Monash University

Mark R. Bleackley

La Trobe University

Michael Foley

La Trobe University

Marilyn A. Anderson

La Trobe University

Andrew M. Coley

La Trobe University

Pedro Quimbar

La Trobe University

Thomas Shafee

La Trobe University

James A. McKenna

La Trobe University

Shaily Vasa

La Trobe University

Cooperative Institutions

La Trobe University

University of Melbourne

Hexima (Australia)

Walter and Eliza Hall Institute of Medical Research

Monash University

Australian Research Council

Commonwealth Scientific and Industrial Research Organisation

Australian Regenerative Medicine Institute

Health Sciences and Nutrition

University of Queensland

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Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Molecular Microbiology (2014)

Mark R. Bleackley Brigitte M. E. Hayes Kathy Parisi Tamanna Saiyed Ana Traven

Summary Antimicrobial peptides ( AMPs ) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor ( BPTI ). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of S accharomyces cerevisiae and the human pathogen C andida albicans . Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg 2+ levels. Populations treated with BPTI had fewer cells in S ‐phase of the cell cycle and a corresponding increase of cells in G 0 / G 1 and G 2 phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine( III )cobalt chloride. Analysis of the growth inhibition of C . albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs .

Mode of Action

Growth inhibition

Mechanism of Action

10.1111/mmi.12621

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Citations (23)

32994 Pezadeftide is a potent antifungal peptide with rapid fungicidial activity via a unique mechanism of action

Journal of the American Academy of Dermatology (2022)

Nicole L. van der Weerden James T Mckenna Kathy Parisi

Mode of Action

Mechanism of Action

10.1016/j.jaad.2022.06.401

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Citations (2)

Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection

Protein Engineering Design and Selection (2008)

Joanne L. Casey Andrew M. Coley Kathy Parisi Michael Foley

There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein–Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1–4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV.

Polyclonal antibodies

Epitope mapping

Linear epitope

Conformational epitope

10.1093/protein/gzn076

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Citations (21)

The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis

Journal of Fungi (2023)

Nicole L. van der Weerden Kathy Parisi James A. McKenna Brigitte M. E. Hayes Peta J. Harvey

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

Nail disease

Dermatophyte

Terbinafine

10.3390/jof9111111

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Citations (5)

Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential

Protein Engineering Design and Selection (2011)

Joanne L. Casey Abdulmonem M. Sanalla Debra C. Tamvakis Claudia M. Thalmann E. L. Carroll

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5–87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.

Epitope mapping

10.1093/protein/gzr026

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Citations (6)

The evolution, function and mechanisms of action for plant defensins

Seminars in Cell and Developmental Biology (2018)

Kathy Parisi Thomas Shafee Pedro Quimbar Nicole L. van der Weerden Mark R. Bleackley

Convergent evolution

Protein family

Structural motif

Conserved sequence

10.1016/j.semcdb.2018.02.004

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Citations (187)

Display of a Peptide Mimotope on a Crystalline Bacterial Cell Surface Layer (S-layer) Lattice for Diagnosis of Epstein–Barr Virus Infection

Bioconjugate Chemistry (2008)

Helga Tschiggerl Joanne L. Casey Kathy Parisi Michael Foley Uwe B. Sleytr

Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72/p2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein–Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 individual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.

Mimotope

S-layer

10.1021/bc7003523

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Citations (29)

Ptychographic imaging of NaD1 induced yeast cell death

Biomedical Optics Express (2019)

Nicholas Anthony Connie Darmanin Mark R. Bleackley Kathy Parisi Guido Cadenazzi

Characterising and understanding the mechanisms involved in cell death are especially important to combating threats to human health, particularly for the study of antimicrobial peptides and their effectiveness against pathogenic fungi. However, imaging these processes often relies on the use of synthetic molecules which bind to specific cellular targets to produce contrast. Here we study yeast cell death, induced by the anti-fungal peptide, NaD1. By treating yeast as a model organism we aim to understand anti-fungal cell death processes without relying on sample modification. Using a quantitative phase imaging technique, ptychography, we were able to produce label free images of yeast cells during death and use them to investigate the mode of action of NaD1. Using this technique we were able to identify a significant phase shift which provided a clear signature of yeast cell death. Additionally, ptychography identifies cell death much earlier than a comparative fluorescence study, providing new insights into the cellular changes that occur during cell death. The results indicate ptychography has great potential as a means of providing additional information about cellular processes which otherwise may be masked by indirect labelling approaches.

10.1364/boe.10.004964

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Citations (9)

The Most Polymorphic Residue on Plasmodium falciparum Apical Membrane Antigen 1 Determines Binding of an Invasion-Inhibitory Antibody

Infection and Immunity (2006)

Andrew M. Coley Kathy Parisi Rosella Masciantonio Jeannine Van Hoeck Joanne L. Casey

Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

10.1128/iai.74.5.2628-2636.2006

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Citations (118)

Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications

Antibodies (2013)

Katherine Griffiths Olan Dolezal Kathy Parisi Julie Angerosa Con Dogovski

The single variable new antigen receptor domain antibody fragments (VNARs) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

10.3390/antib2010066

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Citations (59)