Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to ß-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced ß-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of ß-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

The adhesion capability of Staphylococcus aureus cells is heterogeneously distributed over the cell envelope.

Understanding and controlling microbial adhesion is a critical challenge in biomedical research, given the profound impact of bacterial infections on global health. Many facets of bacterial adhesion, including the distribution of adhesion forces across the cell wall, remain poorly understood. While a recent 'patchy colloid' model has shed light on adhesion in Gram-negative Escherichia coli cells, a corresponding model for Gram-positive cells has been elusive. In this study, we employ single cell force spectroscopy to investigate the adhesion force of Staphylococcus aureus. Normally, only one contact point of the entire bacterial surface is measured. However, by using a sine-shaped surface and recording force-distance curves along a path perpendicular to the rippled structures, we can characterize almost a hemisphere of one and the same bacterium. This unique approach allows us to study a greater number of contact points between the bacterium and the surface compared to conventional flat substrata. Distributed over the bacterial surface, we identify sites of higher and lower adhesion, which we call 'patchy adhesion', reminiscent of the patchy colloid model. The experimental results show that only some cells exhibit particularly strong adhesion at certain locations. To gain a better understanding of these locations, a geometric model of the bacterial cell surface was created. The experimental results were best reproduced by a model that features a few (5-6) particularly strong adhesion sites (diameter about 250 nm) that are widely distributed over the cell surface. Within the simulated patches, the number of molecules or their individual adhesive strength is increased. A more detailed comparison shows that simple geometric considerations for interacting molecules are not sufficient, but rather strong angle-dependent molecule-substratum interactions are required. We discuss the implications of our results for the development of new materials and the design and analysis of future studies.

Exploring the impact of fungicide exposure and nutritional stress on the microbiota and immune response of the Cape honey bee (Apis mellifera capensis).

Honey bees (Apis mellifera) harbour a stable core microbial community within their gut, that is suggested to play a role in metabolic functioning, immune regulation, and host homeostasis. This microbiota presents a unique opportunity to observe the effects of stressors on honey bee health. We examined the effects of two common honey bee stressors: indirect fungicide contamination and nutrient limitation. These effects were observed through changes in their hind- and midgut microbiota using Automated Ribosomal Intergenic Spacer Analysis (ARISA), alongside high-throughput amplicon sequencing. Expression of the honey bees' immune response was examined through the expression of three immune-related genes, namely, immune deficiency (imd), proPhenolOxidase (proPO), and spaetzle (spz). Additionally, longevity of the honey bees was monitored through observation of the expression levels of Vitellogenin (Vg). Both treatment groups were compared to a negative control, and a diseased positive control. There was no effect on the hindgut microbiota due to the stressors, while significant changes in the midgut was observed. This was also observed in the expression of the immune-related genes within the treatment groups. The Imd pathway was substantially downregulated, with upregulation in the prophenoloxidase pathway. However, no significant effect was observed in the expression of spz, and only the pollen treatment group showed reduced longevity through a downregulation of Vg. Overall, the effect of these two common stressors indicate a compromise in honey bee immunity, and potential vulnerabilities within the immune defence mechanisms.

Hydrophobin Bilayer as Water Impermeable Protein Membrane.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

Surface Modification of Brass via Ultrashort Pulsed Direct Laser Interference Patterning and Its Effect on Bacteria-Substrate Interaction.

In recent decades, antibiotic resistance has become a crucial challenge for human health. One potential solution to this problem is the use of antibacterial surfaces, i.e., copper and copper alloys. This study investigates the antibacterial properties of brass that underwent topographic surface functionalization via ultrashort pulsed direct laser interference patterning. Periodic line-like patterns in the scale range of single bacterial cells were created on brass with a 37% zinc content to enhance the contact area for rod-shaped Escherichia coli (E. coli). Although the topography facilitates attachment of bacteria to the surface, reduced killing rates for E. coli are observed. In parallel, a high-resolution methodical approach was employed to explore the impact of laser-induced topographical and chemical modifications on the antibacterial properties. The findings reveal the underlying role of the chemical modification concerning the antimicrobial efficiency of the Cu-based alloy within the superficial layers of a few hundred nanometers. Overall, this study provides valuable insight into the effect of alloy composition on targeted laser processing for antimicrobial Cu-surfaces, which facilitates the thorough development and optimization of the process concerning antimicrobial applications.

Hericium ophelieae sp. nov., a novel species of Hericium (Basidiomycota: Russulales, Hericiaceae) from the Southern Afrotemperate forests of South Africa.

A novel species of Hericium was recently collected in the Afrotemperate forests (Knysna - Amatole region) of Southern Africa. The novel species shares many similar, dentate features common to other species in Hericium, and its basidiome first appears stark white and yellows with age. However, the substrate choice and gloeocystidia and basidiospore sizes of the specimens collected were distinct from other Hericium species. This was confirmed by sequencing the ITS and 28S genetic markers, respectively. The novel species is described as Hericium ophelieae sp. nov. and appears unique as it grows on hardwoods indigenous to Southern Africa. The species has larger basidiospores and wider gloeocystidia compared to its closest relative. H. ophelieae sp. nov. is the first endemic species of the medicinal mushroom genus Hericium to be described from Southern Africa, and the second to be described from Africa, after its closest relative, H. bembedjaense, which was isolated in Cameroon. Although this is the first Hericium to be described from the Southern African region, there are likely others to be discovered, and this study highlights the need for further research into the fungal diversity of Afrotemperate environments.

Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2.

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Strong and Elastic Membranes via Hydrogen Bonding Directed Self-Assembly of Atomically Precise Nanoclusters.

2D nanomaterials have provided an extraordinary palette of mechanical, electrical, optical, and catalytic properties. Ultrathin 2D nanomaterials are classically produced via exfoliation, delamination, deposition, or advanced synthesis methods using a handful of starting materials. Thus, there is a need to explore more generic avenues to expand the feasibility to the next generation 2D materials beyond atomic and molecular-level covalent networks. In this context, self-assembly of atomically precise noble nanoclusters can, in principle, suggest modular approaches for new generation 2D materials, provided that the ligand engineering allows symmetry breaking and directional internanoparticle interactions. Here the self-assembly of silver nanoclusters (NCs) capped with p-mercaptobenzoic acid ligands (Na4 Ag44 -pMBA30 ) into large-area freestanding membranes by trapping the NCs in a transient solvent layer at air-solvent interfaces is demonstrated. The patchy distribution of ligand bundles facilitates symmetry breaking and preferential intralayer hydrogen bondings resulting in strong and elastic membranes. The membranes with Young's modulus of 14.5 ± 0.2 GPa can readily be transferred to different substrates. The assemblies allow detection of Raman active antibiotic molecules with high reproducibility without any need for substrate pretreatment.

Fusarium species isolated from post-hatchling loggerhead sea turtles (Caretta caretta) in South Africa.

Species in the Fusarium solani species complex are fast growing, environmental saprophytic fungi. Members of this genus are filamentous fungi with a wide geographical distribution. Fusarium keratoplasticum and F. falciforme have previously been isolated from sea turtle nests and have been associated with high egg mortality rates. Skin lesions were observed in a number of stranded, post-hatchling loggerhead sea turtles (Caretta caretta) in a rehabilitation facility in South Africa. Fungal hyphae were observed in epidermal scrapes of affected turtles and were isolated. The aim of this study was to characterise the Fusarium species that were isolated from these post-hatchling loggerhead sea turtles (Caretta caretta) that washed up on beaches along the South African coastline. Three gene regions were amplified and sequenced, namely the internal transcribed spacer region (ITS), a part of the nuclear large subunit (LSU), and part of the translation elongation factor 1 α (tef1) gene region. Molecular characteristics of strains isolated during this study showed high similarity with Fusarium isolates, which have previously been associated with high egg mortality rates in loggerhead sea turtles. This is the first record of F. keratoplasticum, F. falciforme and F. crassum isolated from stranded post-hatchling loggerhead sea turtles in South Africa.

Hydroxyapatite Pellets as Versatile Model Surfaces for Systematic Adhesion Studies on Enamel: A Force Spectroscopy Case Study.

Research into materials for medical application draws inspiration from naturally occurring or synthesized surfaces, just like many other research directions. For medical application of materials, particular attention has to be paid to biocompatibility, osseointegration, and bacterial adhesion behavior. To understand their properties and behavior, experimental studies with natural materials such as teeth are strongly required. The results, however, may be highly case-dependent because natural surfaces have the disadvantage of being subject to wide variations, for instance in their chemical composition, structure, morphology, roughness, and porosity. A synthetic surface which mimics enamel in its performance with respect to bacterial adhesion and biocompatibility would, therefore, facilitate systematic studies much better. In this study, we discuss the possibility of using hydroxyapatite (HAp) pellets to simulate the surfaces of teeth and show the possibility and limitations of using a model surface. We performed single-cell force spectroscopy with single Staphylococcus aureus cells to measure adhesion-related parameters such as adhesion force and rupture length of cell wall proteins binding to HAp and enamel. We also examine the influence of blood plasma and saliva on the adhesion properties of S. aureus. The results of these measurements are matched to water wettability, elemental composition of the samples, and the change in the macromolecules adsorbed over time on the surface. We found that the adhesion properties of S. aureus were similar on HAp and enamel samples under all conditions: Significant decreases in adhesion strength were found equally in the presence of saliva or blood plasma on both surfaces. We therefore conclude that HAp pellets are a good alternative for natural dental material. This is especially true when slight variations in the physicochemical properties of the natural materials may affect the experimental series.

Quantification of the Adhesion Strength of Candida albicans to Tooth Enamel.

Caries is one of the most prevalent diseases worldwide, which is caused by the degradation of the tooth enamel surface. In earlier research the opportunistic pathogen Candida albicans has been associated with the formation of caries in children. Colonization of teeth by C. albicans starts with the initial adhesion of individual yeast cells to the tooth enamel surface. In this study, we visualized the initial colonization of C. albicans yeast cells on pellicle-covered enamel by scanning electron microscopy. To quantitatively unravel the initial adhesion strength, we applied fluidic force microscopy-based single-cell force spectroscopy to examine the key adhesion parameters adhesion force, rupture length and de-adhesion work. We analyzed single saliva-treated or untreated yeast cells on tooth enamel specimens with or without salivary pellicle. Under all tested conditions, adhesion forces in the lower nanonewton range were determined. Furthermore, we have found that all adhesion parameters were enhanced on the pellicle-covered compared to the uncovered enamel. Our data suggest that initial adhesion occurs through a strong interaction between yeast cell wall-associated adhesins and the salivary pellicle. Future SCFS studies may show whether specific management of the salivary pellicle reduces the adhesion of C. albicans on teeth and thus contributes to caries prophylaxis.

Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces.

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties.

Distribution patterns of Acidobacteriota in different fynbos soils.

The Acidobacteriota is ubiquitous and is considered as one of the major bacterial phyla in soils. The current taxonomic classifications of this phylum are divided into 15 class-level subdivisions (SDs), with only 5 of these SDs containing cultured and fully described species. Within the fynbos biome, the Acidobacteriota has been reported as one of the dominant bacterial phyla, with relative abundances ranging between 4-26%. However, none of these studies reported on the specific distribution and diversity of the Acidobacteriota within these soils. Therefore, in this study we aimed to first determine the relative abundance and diversity of the Acidobacteriota in three pristine fynbos nature reserve soils, and secondly, whether differences in the acidobacterial composition can be attributed to environmental factors, such as soil abiotic properties. A total of 27 soil samples were collected at three nature reserves, namely Jonkershoek, Hottentots Holland, and Kogelberg. The variable V4-V5 region of the 16S rRNA gene was sequenced using the Ion Torrent S5 platform. The mean relative abundance of the Acidobacteriota were 9.02% for Jonkershoek, 14.91% for Kogelberg, and most significantly (p<0.05), 18.42% for Hottentots Holland. A total of 33 acidobacterial operational taxonomic units (OTUs) were identified. The dominant subdivisions identified in all samples included SDs 1, 2, and 3. Significant differences were observed in the distribution and composition of these OTUs between nature reserves. The SD1 were negatively correlated to soil pH, hydrogen (H+), potassium (K+) and carbon (C). In contrast, SD2, was positively correlated to soil pH, phosphorus (P), and K+, and unclassified members of SD3 was positively correlated to H+, K, and C. This study is the first to report on the specific acidobacterial distribution in pristine fynbos soils in South Africa.

Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa.

Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.

Impact of cigarette butts on bacterial community structure in soil.

Cigarette butts contribute significantly to global pollution present on the planet. The filters found in cigarette butts contain a microplastic, cellulose acetate, as well as toxic metals and metalloids which are responsible for pollution in the environment. Although cigarette butt litter is prevalent in many soils, research on the effects of these cigarette butts is limited. In this study, we used Automated Ribosomal Intergenic Spacer Analysis (ARISA) to generate DNA fingerprints of bacterial communities in soil before and after the addition of cigarette butt leachate treatments. An ICP-MS analysis of the biodegradable and non-biodegradable cigarette butts revealed the presence of various elements: Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, V, and Zn. The analysis also specified which metals were present at the highest concentrations in the biodegradable and non-biodegradable cigarette butts, and these were, respectively, Al (1,31 g/kg and 2,35 g/kg), Fe (2,03 g/kg and 1,11 g/kg), and Zn (3,18 mg/kg and 15,70 mg/kg). Our results show that biodegradable cigarette butts had a significant effect on bacterial community composition (beta diversity), unlike the non-biodegradable butts. This effect can be attributed to higher concentrations of certain metals and metalloids in the leachate of biodegradable cigarette butts compared to the non-biodegradable ones. Our findings suggest that biodegradable and non-biodegradable cigarette butts can significantly affect bacterial communities in soil as a result of the leaching of significant quantities of certain elements into the surrounding soils.

Molecular Origin of Electrical Conductivity in Gold-Polythiophene Hybrid Particle Films.

Hybrid electronic materials combine inorganic metals and semiconductors with π-conjugated polymers. The orientation of the polymer molecules in relation to the inorganic components is crucial for electrical material properties and device performance, but little is known of the configuration of π-conjugated polymers that bind to inorganic surfaces. Highly curved surfaces are common when using nanoscale components, for example, metal nanocrystal cores covered with conductive polymers. It is important to understand their effect on molecular arrangement. Here, we compare the molecular structures and electrical conductivities of well-defined nanoscale gold spheres and rods with shells of the covalently bound polythiophene PTEBS (poly[2-(3-thienyl)-ethyloxy-4-butylsulfonate]). We prepared aqueous sinter-free inks from the particles and printed them. The particles formed highly conductive films immediately after drying. Films with spherical metal cores consistently had 40% lower conductivities than films based on nanorods. Raman and X-ray photoelectron spectroscopy revealed differences in the gold-sulfur bonds of PTEBS on rods and spheres. The fractions of bond sulfur groups implied differences in the alignment of PTEBS with the surface. More polymer molecules were bound in an edge-on configuration on spheres than on rods, where almost all polymers aligned "face-on" with the metal surface. This leads to different interface resistances: gold-polythiophene-gold interfaces between rods with π-π-tacked face-on PTEBS apparently foster electron transport along the surface-normal direction, while edge-on PTEBS does not. Molecular confinement thus increases the conductivity of hybrid inks based on highly curved nanostructures.