Biology subjects

Craik, D. J.

Publications and source records attributed to Craik, D. J..

9 recordsLinked to original sources

bioRxiv2026

Selective and Efficient Functionalization of P22 Virus-Like Particles Using an Asparaginyl Ligase

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

biochemistry↗

bioRxiv2026

Bacteriophage P22 virus-like particles as nanoscale protein scaffolds for plant synthetic biology

Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.

synthetic biology↗

bioRxiv2024

A protein cage system for producing bioactive peptides in Escherichia coli

New therapeutics are urgently needed to curb the spread of drug-resistant diseases. Bioactive peptides (BAPs), including antimicrobial peptides, are emerging as an exciting new class of compounds with advantages over current drug modalities, especially small molecule drugs that are prone to resistance development. Here we evaluated a bacteriophage P22 virus-like particle (VLP) system where BAPs are encapsulated as fusion proteins with the P22 scaffold protein (SP) within self-assembling protein cages in Escherichia coli. Representative peptides from three structurally distinct classes of BAPs were successfully encapsulated into P22 VLPs at high cargo to VLP coat protein (CP) ratios that corresponded to interactions between the compact electropositive structures of the SP-BAPs and electronegative regions on the inward facing surface of CP subunits. However, high loading densities did not correspond to improved SP-BAP yields. An unexpected finding of this study was that while encapsulation alleviated negative effects of SP-BAPs on E. coli growth, the P22 scaffold protein acted as a sufficient fusion partner for accumulating BAPs, and co-expression of the CP did not further improve SP-BAP yields. Nevertheless, encapsulation in VLPs provided a useful first step in the purification pipeline for producing both linear and cyclic recombinant (r)BAPs that were functionally equivalent to their synthetic counterparts. Further efforts to optimise expression ratios of CP to SP-BAP fusions will be required to realise the full potential of encapsulation for protecting expression hosts and maximizing rBAP yields.

synthetic biology↗

bioRxiv2023

Seasonal tissue-specific gene expression reveals reproductive and stress-related transcriptional systems in wild crown-of-thorns starfish

Animals are influenced by the season, yet we know little about the changes that occur in most species throughout the year. This is particularly true in tropical marine animals that experience relatively small annual temperature and daylight changes. Like many coral reef inhabitants, the crown-of-thorns starfish (COTS), well known as a notorious consumer of corals and destroyer of coral reefs, reproduces exclusively in the summer. By comparing gene expression in seven somatic tissues procured from wild COTS sampled on the Great Barrier Reef, we identified more than 2,000 protein-coding genes that change significantly between summer and winter. COTS genes that appear to mediate conspecific communication - including both signalling factors released into the surrounding sea water and cell surface receptors - are upregulated in external secretory and sensory tissues in the summer, often in a sex-specific manner. The sexually dimorphic gene expression appears to be underpinned by sex- and season-specific transcription factors (TFs) and gene regulatory programs. There are over 100 TFs that are seasonally expressed, 86% of which are significantly upregulated in the summer. Six nuclear receptors are upregulated in all tissues in the summer, suggesting that systemic seasonal changes are hormonally controlled, as occurs in vertebrates. Unexpectedly, there is a suite of stress-related chaperone proteins and TFs, including HIF, ATF3, C/EBP, CREB and NF-{kappa}B, that are uniquely co-expressed in gravid females. The upregulation of these stress proteins in the summer suggests the demands of oogenesis in this highly fecund starfish affects protein stability and turnover in somatic cells. Together, these circannual changes in COTS gene expression provide novel insights into seasonal changes in this coral reef pest and have the potential to identify vulnerabilities for targeted biocontrol.

zoology↗

bioRxiv2022

Membrane-active peptides escape drug-resistance in cancer

Acquired drug-resistance is a recurring problem in cancer treatment, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. In the current study, we explored the use of membrane-active peptides as an alternative therapeutic modality to target drug-resistant melanoma cells. We produced slow-cycling and drug-resistant melanoma cells using dabrafenib, a small molecule drug that targets tumor cells with BRAF V600E mutation, and characterised their lipidome and proteome to investigate the role of membrane lipids in acquired drug-resistance. Despite some changes in the lipid composition, tested anti-melanoma membrane-active cyclic peptides (cTI and cGm) killed melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Importantly, melanoma cells did not develop resistance to cTI or cGm, nor changed their lipid composition with long-term peptide treatment. Therefore, these peptides are well suited as templates to design therapeutic leads to target drug-resistant metastatic melanoma cells and/or as co-treatment with small molecule drugs.

cancer biology↗

bioRxiv2022

Crown-of-thorns starfish in captivity experience sustained large-scale changes in gene expression

Marine animals in the wild are often difficult to access, so that biologists have to extrapolate from the study of animals in captivity. However, the implicit assumption that physiological and cellular processes of animals in artificial environments are not significantly different from those in the wild has rarely been tested. Here we investigate the extent to which the biological state of an animal is impacted by captivity by comparing global gene expression in wild and captive crown-of-thorns starfish (COTS). We compare transcriptomes of three external tissues obtained from wild COTS with captive COTS maintained in aquaria for at least one week. On average, an astonishingly large 24% of the coding sequences in the genome are differentially expressed. Comparing transcriptomes from coelomocytes - cells in internal coelomic fluid - in wild and captive COTS, we find that 20% of the coding sequences in the genome rapidly change expression. These captive transcriptomes remained markedly different from the wild ones for more than 30 days in captivity, and showed no indication of reverting back to a wild state. Genes consistently upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in intercellular signalling. These extensive changes in gene expression in captive COTS suggest that captivity has a profound and sustained impact on the physiology, behaviour and health of these echinoderms. The potential for such dramatic changes should be accounted for when designing studies seeking to understand wild animals.

molecular biology↗

bioRxiv2021

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

synthetic biology↗

bioRxiv2021

Modified defence peptides from horseshoe crab target and kill bacteria inside host cells

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two {beta}-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

microbiology↗

bioRxiv2020

Drug susceptibility profiling of Australian Burkholderia species as models for developing melioidosis therapeutics

SynopsisO_ST_ABSBackgroundC_ST_ABSMelioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current treatment regimens are prolonged and costly, and acquired antimicrobial resistance has been reported for all currently used antibiotics. ObjectivesEfforts to develop new treatments for melioidosis are hampered by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with Biosafety Level (BSL) 3 containment. Closely related Burkholderia species that are less pathogenic can be investigated under less stringent BSL 2 containment. We hypothesized that near-neighbour Burkholderia species could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. MethodsWe used microbroth dilution assays to compare the susceptibility of three Australian B. pseudomallei isolates and five near-neighbour Burkholderia species - B. humptydooensis, B. thailandensis, B. oklahomensis, B territorii and B. stagnalis - to antibiotics currently used to treat melioidosis, and general-use antibacterial agents. We also established the susceptibility profiles of B. humptydooensis and B. territorii to 400 compounds from the Medicines for Malaria Venture Pathogen Box. ResultsFrom these comparisons, we observed a high degree of similarity in the susceptibility profiles of B. pseudomallei and near-neighbour species B. humptydooensis, B. thailandensis, B. oklahomensis and B. territorii. ConclusionsLess pathogenic Australian Burkholderia species B. humptydooensis, B. thailandensis, B. oklahomensis and B. territorii are excellent model organisms for developing potential new therapies for melioidosis.

microbiology↗