Peptide-mediated delivery of gold nanoparticles and proteins into lysosomes of mammalian cells

Dr Chaitali Dekiwadia has developed a therapeutic approach of targeting gold nanoparticles and proteins to the lysosomes mediated through peptides. She designed and synthesised peptides to deliver a protein-nanoparticle conjugates to the lysosomes penetrating the challenging cell membrane barrier. This novel work has shown for the first time that protein-nanoparticle conjugates could be targeted to lysosomes using short peptides. This peptide-mediated approach could underpin the development of a novel strategy thereby widening the pharmaceutical index for current lysosomal storage diseases

Subcompartmentalisation of Proteins in the Rhoptries Correlates with Ordered Events of Erythrocyte Invasion by the Blood Stage Malaria Parasite

Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion. © 2012 Zuccala et al

Super-Resolution Dissection of Coordinated Events during Malaria Parasite Invasion of the Human Erythrocyte

Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion. © 2011 Elsevier Inc

Thermal Degradation and Fire Properties of Fungal Mycelium and Mycelium: Biomass Composite Materials

Mycelium and mycelium-biomass composites are emerging as new sustainable materials with useful flame-retardant potentials. Here we report a detailed characterisation of the thermal degradation and fire properties of fungal mycelium and mycelium-biomass composites. Measurements and analyses are carried out on key parameters such as decomposition temperatures, residual char, and gases evolved during pyrolysis. Pyrolysis flow combustion calorimetry (PCFC) evaluations reveal that the corresponding combustion propensity of mycelium is significantly lower compared to poly(methyl methacrylate) (PMMA) and polylactic acid (PLA), indicating that they are noticeably less prone to ignition and flaming combustion, and therefore safer to use. The hyphal diameters of mycelium decrease following pyrolysis. Cone calorimetry testing results show that the presence of mycelium has a positive influence on the fire reaction properties of wheat grains. This improvement is attributable to the relatively higher charring tendency of mycelium compared to wheat grain, which reduces the heat release rate (HRR) by acting as a thermal insulator and by limiting the supply of combustible gases to the flame front. The mycelium growth time has been found to yield no significant improvements in the fire properties of mycelium-wheat grain composites

Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu

Plasmodium falciparum Merozoite Invasion Is Inhibited by Antibodies that Target the PfRh2a and b Binding Domains

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes

An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process

Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes

There is considerable interest in the sub cellular targeting and delivery of biomolecules therapeutic and imaging agents and nanoparticles and nanoparticle conjugates into organelles for therapeutic and imaging purposes. To date a number of studies have used sorting peptides for targeted delivery of cargo into different cell organelles but not into lysosomes. In this study the delivery of 13 nm gold nanoparticles across the cell membrane followed by targeted localisation into the lysosomes of a mammalian cell line was examined using novel combinations of cell penetrating peptides and lysosomal sorting peptides conjugated to the nanoparticles. Using a combination of fluorescence spectroscopy fluorescence microscopy and transmission electron microscopy techniques we show that these nanoconjugates were efficiently and selectively delivered into the lysosomes with minimal cytotoxic effects. This novel targeted delivery system may underpin the development of a new strategy for the treatment of lysosomal storage diseases by exploiting the large surface area of nanoparticles to deliver drugs or replacement enzymes directly to the lysosome

Acoustically-mediated intracellular delivery

Recent breakthroughs in gene editing have necessitated practical ex vivo methods to rapidly and efficiently re-engineer patient-harvested cells. Many physical membrane-disruption or pore-forming techniques for intracellular delivery, however, result in poor cell viability, while most carrier-mediated techniques suffer from suboptimal endosomal escape and hence cytoplasmic or nuclear targeting. In this work, we show that short exposure of cells to high frequency (>10 MHz) acoustic excitation facilitates temporal reorganisation of the lipid structure in the cell membrane that enhances translocation of gold nano- particles and therapeutic molecules into the cell within just ten minutes. Due to its transient nature, rapid cell self-healing is observed, leading to high cellular viabilities (>97%). Moreover, the internalised cargo appears to be uniformly distributed throughout the cytosol, circumventing the need for strategies to facilitate endosomal escape. In the case of siRNA delivery, the method is seen to enhance gene silencing by over twofold, demonstrating its potential for enhancing therapeutic delivery into cells

Heparin assisted assembly of somatostatin amyloid nanofibrils results in disordered precipitates by hindrance of protofilaments interactions

Amyloid nanofibrils are β-sheet rich protein or peptide assemblies that have pathological roles in over 20 neurodegenerative diseases, but also can have essential physiological roles. This wide variety of functions is likely to be due to subtle differences in amyloid structure and assembly mechanisms. Glycosaminoglycans (GAGs), like heparin, are frequently used in vitro to increase the kinetics of assembly of amyloid fibrils. However, little is known about the effects of adding large polymeric sugars on assembly mechanisms and amyloid nanostructures. Here, we provide insights into the kinetics, assembly mechanisms and structural effects of heparin on the self-assembly of a functional-amyloid forming neuropeptide hormone, somatostatin-14. We show that pure somatostatin-14 self-assembles into amyloid fibrils via the formation of antiparallel β-sheet networks, in a typical amyloid aggregation process. These fibrils then laterally assemble into ordered liquid crystalline structures through the generation of further parallel β-sheet networks. If heparin molecules are present, they intercalate between the peptide assemblies during the initial stages of aggregation. This intercalation screens electrostatic repulsions hindering the lateral association of protofilaments, preventing liquid crystal formation and resulting in the rapid formation of disordered micron scale precipitates. Our results show that aggregation promotors like heparin can have large effects not just on the kinetics of aggregation but also on assembly mechanisms, and the architecture of amyloid assemblies. Thus highlighting the dangers of using such polymeric sugars in fundamental studies of amyloid aggregation, especially when drawing conclusions on structure-function relationships or when investigating amyloid-based nanostructures as bionanomaterials