Spherical Protein Research Articles

Prolonged self-assembly of H. pylori ferritin globules at physiological conditions.

One of the promising drug delivery tools is ferritin, which features high stability at a wide range of conditions and protects cargo by its spherical protein shell. We studied the self-assembly into homoglobules of ferritin from H. pylori and a chimeric protein ferritin-SUMO. We exposed the globules to pH-driven dis/reassembly and in both cases we observed two fractions during size exclusion chromatography (SEC) procedure. The higher molecular weight fraction contained fully assembled globules of ferritin and ferritin-SUMO that well coincides with literature. Interestingly, the lower molecular weight fraction contained intermediate subglobular oligomers that also formed globules, but on a time scale of hours, while being under physiological conditions. We performed biochemical characterization of this fraction and found that, in the case of ferritin, it contained almost the whole range of intermediate oligomers with different stoichiometry. In contrast, the ferritin-SUMO fraction contained only two distinct states: dimers and globules, without any other ferritin-SUMO intermediate oligomers. We built AlphaFold-derived schemes of ferritin and ferritin-SUMO self-assembly which also indicated differences in their assembly pathways. Our results could potentially open the possibility of cargo loading into ferritins at physiological conditions and improved purification of ferritin-based products if using a ferritin-SUMO modification with following cleavage of the SUMO-tag by the SUMO protease.

Freeze-Induced Protein Assembly of α-Synuclein into Stable Microspheres to Fabricate Light-Induced Cargo Release Systems.

Stable hollow-type microspheres (MSs) have been fabricated using α-synuclein (αS), an amyloidogenic protein, via freeze-induced protein self-assembly. This assembly process involves three steps: rapid freezing to form spherical protein condensates from αS oligomers, frozen annealing to form a crust on the condensate and freeze-drying to create an interior lumen via the three-dimensional (3D) coffee-stain effect. The crust produced during the frozen-annealing step is a β-sheet-mediated protein structure that is presumed to be created at the quasi-liquid layer of the protein-ice interface and thus contributes to the stability of MSs in aqueous solutions at room temperature without any additional surface stabilization. MSs transform into amyloid fibril condensates when heated to 70 °C, and the drug is loaded via centrifugal membrane filtration. Additionally, the MSs were shielded with an iron-alginate layer embedded with gold nanoparticles (AuNPs) to prevent premature leakage and to control drug release. This takes advantage of the photothermal effect of AuNPs, resulting in combined cytotoxicity between the drug and heat. Therefore, drug-loaded MSs comprising αS and AuNPs can be suggested as light-controllable drug delivery systems that exhibit chemical and physical anticancer therapeutic effects.

Structural Analysis of Variants of the Ferritin Light Chain Protein and Its Relationship with Neuroferritinopathy.

Ferritin is a highly conserved spherical protein that stores iron and possesses triple and quadruple symmetry input ports. Additionally, it is composed of light chains that can be affected by post-translational mutations, reducing the iron storage capacity in the brain and leading to neuroferritinopathy, which is a rare disease with limited bioinformatics data. In this study, we analyzed the biochemical mechanism of different ferritin mutations reported in the literature, through the characterization and determination of the in silico structural model by searching databases, implementing bioinformatics programs such as Jalview, NetNGlyc 1.0, NetOGlyc 3.1, and three-dimensional structure predictors with machine learning such as Alphafold, demonstrating the generation of hairpin and steric hindrances that hinder the aggregation of subunits and changes in the size and arrangement of quadruple and triple entry holes of the A96T mutation compared to the wild-type protein, since in the quadruple entry hole, a decrease in area is observed compared to the wild-type protein and the triple entry hole has a decrease in distance measurements of 6.504 Å. This possibly affects the functionality of the protein, thus releasing high concentrations of iron in the brain and causing neurodegeneration.

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.

Correlation study between structural parameters of droplets and rheological properties of O/W high internal phase Pickering emulsions above the closest packing density

The correlation between the structural parameters of droplets and rheological properties of O/W high internal phase Pickering emulsions (HIPPEs) stabilized by soy protein isolate (SPI) nanoparticles was elucidated, with a volume fraction of dispersed phase (ϕ) above the closest packing density (74%). The structural basis of SPI nanoparticles was confirmed to be spherical heat-induced protein aggregates, which had moderate wettability and excellent interfacial activity. The structural parameters of emulsion droplets (contact length L, diameter D, length d1, width d2, the number of adjacent contact droplets Z), and the stability and rheological properties of HIPPEs were characterized by the confocal laser scanning microscopy (CLSM), diffusing wave spectroscopy (DWS), and mechanical rheometer respectively. The results of CLSM indicated the transition of droplets from spherical to polyhedral shapes as increased ϕ, accompanied by an increased L and Z. The rheological and stability analysis confirmed the widened linear viscoelastic range, elevated gel point (εc), and the extended half−life of the intensity autocorrelation function of HIPPEs as ϕ increased. Moreover, the loss factor (tan δ) remained constant at 0.1 within the ϕ of 74%–86%, revealing the solid−like nature of HIPPEs. The correlation between ϕ, structural parameters of droplets, and rheological properties of HIPPEs was further analyzed. The nonlinear regression fitting between the L/D and G' (elastic modulus) yielded a better fitting effect than d1/d2 and Z, suggesting an intrinsic correlation between emulsion structure and rheology. In summary, this study elucidated the intricate relationship between ϕ, droplet structure, and rheological properties of HIPPEs, offering a research foundation and theoretical basis for the development and application of protein particle−stabilized HIPPEs.

Fabrication of stable spherical soybean lipophilic protein nanogel for curcumin delivery: Carbony oxygen and aromatic ring flexibility binding to the beta−barrel

Soybean lipophilic protein (SLP), which comprises approximately 10 % polar lipids (especially phosphatidylcholine (PC)) in glycinin (11S) and β-conglycinin (7S), is a potentially effective delivery for nutraceuticals. Herein, we successfully engineered a stable spherical soybean lipophilic protein nanogel through a pH−shift treatment at its isoelectric point, designed for the efficient delivery of curcumin (Cur) to enhance its bioavailability. The analysis of the physicochemical and structural properties of the SLP−nanogel revealed its exceptional encapsulation efficiency, achieving 93.52 %, surpassing that of soybean isolated protein at 83.67 %. Moreover, the loading ability experienced a remarkable 8.7−fold increase, reaching 13.02 μg/mg. Fluorescence kinetics analysis reveals that Cur selectively binds to a single site on 7SPC/11SPC, predominantly through van der Waals forces and hydrogen bonds. Molecular dynamics results revealed that the pH−shifted treatment expanded the beta−barrel volume of 7SPC/11SPC, enhancing the binding affinity between 7SPC/11SPC and Cur by forming H−bonds and pi−H interaction in the beta−barrel regions of K232−Y388 (7SPC) and D300−I410 (11SPC). Our findings provided a novel and effective way to prepare stable spherical nanogels for delivering Cur and a novel insight into the binding mechanism between Cur and 11SPC/7SPC.

Analysis of serum proteomic profiles of endangered Siamese and Burmese Eld's deer infected with subclinical Babesia bovis in Thailand

The endangered Eld's deer is a conserved species in Thailand, where tropical parasitic infections are endemic. Although Eld's deer with babesiosis are generally asymptomatic, they can still harbor the parasite and serve as reservoirs for ticks, spreading the infection to healthy animals within the herd. The present study aimed to investigate potential serum proteome biomarkers of Eld's deer with subclinical Babesia bovis infection. A total of 67 blood samples were collected from captive Siamese and Burmese Eld's deer showing no signs of parasitic infection. The nested polymerase chain reaction (nPCR) of a conserved spherical body protein 2 (sbp-2) gene of B. bovis was utilized to classify Eld's deer groups, with 25.37 % (17/67) testing positive for B. bovis. Additionally, the application of proteomic studies showed that six B. bovis proteins, such as Obg-like ATPase 1 (OLA1) and heat shock protein 90 (HSP90), were significantly upregulated by more than a two-fold change compared with the PCR-negative samples. Of the 55 overexpressed serum proteins in the PCR-positives, alpha 2-HS glycoprotein (AHSG) and immunoglobulin lambda variable 2–8 (IGLV2–8) were notably among the top 10 proteins with the highest area under curve (AUC) values. Hence, they were proposed as potential biomarkers for subclinical B. bovis infection in Eld's deer. Analysis of the protein interaction network revealed interactions between Eld's deer AHSG and B. bovis OLA1 and HSP90, alongside associations with other proteins such as erb-b2 receptor tyrosine kinase 2 (ERBB2) and epidermal growth factor receptor (EGFR). These interactions were involved in the immune system pathway and inflammatory responses. Our findings shed light on subclinical infection of B. bovis in Eld's deer and identify potential biomarkers, contributing to the further effective detection and monitoring of B. bovis infection in this endangered species.

Molecular genotyping of Babesia caballi

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

Does particle size matter for soy protein nanoparticles in the fabrication and lipolysis of pickering-like emulsions?

Enzyme-mediated nanostructures have gained increasing interests and shown promising applications in functional delivery for food and pharmaceuticals. In our previous study, we fabricated three types of spherical soy protein nanoparticles (SPNP) by flavorzyme to different degree of hydrolysis (DH, 3%, 7% and 11%). These SPNPs exhibited similar subunit composition, secondary structure and surface properties, but differ in size, which is 89 nm, 106 nm and 151 nm, respectively. This study further applied them in emulsion systems for functional delivery. We systematically studied the emulsifying activities, interfacial properties including wettability, interfacial packing pattern, interfacial adsorption and dilatational rheological behavior of SPNPs, and their performance in modulating lipolysis of the emulsions under the influence of lipase, bile salts, pepsin and trypsin. All SPNPs behaved as Pickering-like stabilizers with moderate wettability at oil-water interface with three phase contact angle of 92–101°, and the SPNP with the smallest size has better emulsifying activities (D3,2 = 3.22 ± 0.00 μm in SDS solution) than native soy protein isolate (SPI) (D3,2 = 4.04 ± 0.03 μm in SDS solution). These SPNPs formed similar viscoelastic oil-water interfaces (Ed' = 20.2–21.5 mN/m), which were more glassy-like with weak in-plane interactions compared to SPI (Ed' = 28.6 ± 0.5 mN/m). All SPNPs can delay lipolysis of emulsions by lowering the access/replacement of lipase and bile salts and depressing the proteolysis by trypsin at interface in similar ways. Our work elucidated the role of particle size of enzyme-mediated plant protein-based particle in emulsion stabilization and lipolysis control, and is expected to give the theoretical support for the design of functional food emulsions with controllable energy intake.

Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

Quantifying Protein Shape to Elucidate Its Influence on Solution Viscosity in High-Concentration Electrolyte Solutions.

Therapeutic proteins with a high concentration and low viscosity are highly desirable for subcutaneous and certain local injections. The shape of a protein is known to influence solution viscosity; however, the precise quantification of protein shape and its relative impact compared to other factors like charge-charge interactions remains unclear. In this study, we utilized seven model proteins of varying shapes and experimentally determined their shape factors (v) based on Einstein's viscosity theory, which correlate strongly with the ratios of the proteins' surface area to the 2/3 power of their respective volumes, based on protein crystal structures resolved experimentally or predicted by AlphaFold. This finding confirms the feasibility of computationally estimating protein shape factors from amino acid sequences alone. Furthermore, our results demonstrated that, in high-concentration electrolyte solutions, a more spherical protein shape increases the protein's critical concentration (C*), the transition concentration beyond which protein viscosity increases exponentially relative to concentration increases. In summary, our work elucidates protein shape as a key determinant of solution viscosity through quantitative analysis and comparison with other contributing factors. This provides insights into molecular engineering strategies to optimize the molecular design of therapeutic proteins, thus optimizing their viscosity.

Polarization-sensitive Two-photon Microscopy for a Label-free Amyloid Structural Characterization.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

New iron export pathways acting via holo-ferritin secretion

Ferritin is a spherical nanocage protein for iron storage, composed of 24 light- or heavy-polypeptide chain subunits. A single ferritin molecule can carry up to 4500 iron atoms in its core, which plays an important role in suppressing intracellular iron toxicity. Serum ferritin levels are used as a marker for the total amount of iron stored in the body. Most serum ferritin is iron-free (apo-ferritin) and it is unclear how ferritin is released from cells. Ferritin is secreted into serum via extracellular vesicles (EVs) or the secretory autophagy pathway but not via the classical endoplasmic reticulum (ER)-to-Golgi secretion pathway. We recently discovered that the level of tetraspanin CD63, a common EV marker, is post-transcriptionally regulated by the intracellular iron level and both CD63 and ferritin expression is induced by iron loading. Ferritin is incorporated into CD63(+)-EVs through the ferritin-specific autophagy adapter molecule, NCOA4, and then secreted from cells. EV production differs drastically depending on cell type and physiological conditions. Extracellular matrix detached cells express pentaspanin prominin 2 and prominin 2(+)-EVs secrete ferritin independently of NCOA4 trafficking. Ferritin is tightly bound to iron in EVs and functions as an iron-carrier protein in the extracellular environment. Cells can suppress ferroptosis by secreting holo-ferritin, which reduces intracellular iron concentration. However, this exposes the neighboring cells receiving the secreted holo-ferritin to a large excess of iron. This results in cellular toxicity through increased generation of reactive oxygen species (ROS). Here we review the machinery by which ferritin is incorporated into EVs and its role as an intercellular communication molecule.

An Overview of Heavy Chain Ferritin in Cancer.

As a spherical protein that acts as a repository for intracellular iron, Ferritin is the most important iron storage form and is known to influence tumor immunity. Unbound ferritin is composed of 24 subunits, made up of ferritin light chain (FTL) and ferritin heavy chain (FTH). Ferritin can be automatically put together to form hollow nanocages that measure 12 nm around the outside and 8 nm around the inside. Cancer causes the second-most deaths worldwide, effective elimination of tumor cells while protecting normal cells is the foundation of modern tumor therapy. To this end, the innate tumor-targeting activity of human FTH1, first identified ten years ago, is highly appealing. Unmodified human FTH1 binds to its receptor, transferrin receptor 1 (TfR1), which is frequently overexpressed in cancer cells. FTH1-TfR1 binding permits improved drug efficacy by promoting ferritin-mediated targeted delivery. In addition, FTH is also associated with the prognosis of multiple typies of cancer. The level of FTH1 is significantly and positively correlated with the infiltration of tumor-associated macrophages. FTH1 also plays an important role in regulating the tumor immunity of solid cancer. As such, FTH1 has been extensively applied in the targeted delivery of anticancer drugs, diagnostic molecules (e.g., radioisotopes and fluorophones), and inorganic nanoparticles (NPs) to tumors.This article reviews the role of FTH in cancer and its potential as a therapeutic target.

Structure of the HIV immature lattice allows for essential lattice remodeling within budded virions.

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<∆G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.

Paintable graphene oxide-hybridized soy protein-based biogels for skin radioprotection

Radiation-induced skin injury (RISI) represents a prevalent issue following radiotherapy. The main factors influencing RISI wound recovery include ROS and NLRP3 inflammasome activation as well as wound infection, amongst others. Herein, a biomaterial for the healing of radio-induced injury was designed using multiple strategies. A gallic acid (GA)-assisted isolation process was first applied to prepare gallic acid-grafted soy protein (SPI-GA) from spherical soy protein. The SPI-GA was then modified with simultaneously-grafted phenol and thiol cohorts (SH) (SPI-GA-SH). Due to the antibacterial properties of graphene oxide (GO), SPI-SH-GA@PGO biogels was finally self-assembled into an adhesive biogel in a binary solvent system (dopamine-glycerol/water). Characterization for adhesive and paintable SPI-based biogels indicated that the material was successfully synthesized. Cytological studies further highlighted the antibacterial properties of SPI-SH-GA@PGO, its non-toxicity to HaCaT cultures as well as its ability to promote vascularization and cell migrative while inhibiting apoptotic. Genomics assessment also demonstrated that the material could effectively improve the abnormal expression of skin cultures induced by radiation. Finally, a murine model suggested that SPI-SH-GA@PGO could reduce ROS and NLRP3 expression while promoting wound recovery. This work pioneered SPI-based biogel deployment as material for RISI recovery.

Farklı Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) popülasyonlarının yerel Bacillus thuringiensis suşlarına duyarlılığı

Tomato leafminer, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) is one of the most important tomato pests worldwide and causes 100% product loss if not controlled. Chemical insecticides, which have been overused for many years, have induced resistance in the pests and made it difficult to control their populations in the field. The use of biological agents that express insecticidal proteins, such as Bacillus thuringiensis, is an alternative to conventional insecticides to suppress pest populations. In this study, to recover novel B. thuringiensis strains from soil samples, a survey was conducted in Bilecik province in 2021. Thirteen local B. thuringiensis strains were isolated and the susceptibility of three different field populations (Samsun, İzmir, and Bilecik) of T. absoluta to these strains was evaluated. Bacillus thuringiensis B3 (Bt-B3) strain, which contains lepidopteran-active toxin genes, was more virulent for all T. absoluta populations tested. In addition, Samsun population was more sensitive to the B3 strain than İzmir and Bilecik. The LC50 values of Bt-B3 were determined to be 13.28, 26.06 and 24.24 ppm for Samsun, İzmir and Bilecik populations, respectively. Sequencing of the 16S rRNA gene region confirmed that the isolate was B. thuringiensis, while electron microscopy revealed that the isolate produced bipyramidal, cubic and spherical insecticidal proteins. The results of this study indicate that the isolate Bt-B3 appears to be a promising biocontrol agent for integrated pest management of T. absoluta in Türkiye.

Comparative genomics reveals unique features of two Babesia motasi subspecies: Babesia motasi lintanensis and Babesia motasi hebeiensis

Parasites of the Babesia genus are prevalent worldwide and infect a wide diversity of domestic animals and humans. Herein, using Oxford Nanopore Technology and Illumina sequencing technologies, we sequenced two Babesia subspecies, Babesia motasi lintanensis and Babesia motasi hebeiensis. We identified 3,815 one-to-one ortholog genes that are specific to ovine Babesia spp. Phylogenetic analysis reveals that the two B. motasi subspecies form a distinct clade from other piroplasmas. Consistent with their phylogenetic position, comparative genomic analysis reveals that these two ovine Babesia spp. share higher colinearity with Babesia bovis than with Babesia microti. Concerning the speciation date, B. m. lintanensis split from B. m. hebeiensis approximately 17 million years ago. Genes correlated to transcription, translation, protein modification and degradation, as well as differential/specialized gene family expansions in these two subspecies may favor adaptation to vertebrate and tick hosts. The close relationship between B. m. lintanensis and B. m. hebeiensis is underlined by a high degree of genomic synteny. Compositions of most invasion, virulence, development, and gene transcript regulation-related multigene families, including spherical body protein, variant erythrocyte surface antigen, glycosylphosphatidylinositol anchored proteins, and transcription factor Apetala 2 genes, is largely conserved, but in contrast to this conserved situation, we observe major differences in species-specific genes that may be involved in multiple functions in parasite biology. For the first time in Babesia spp., we find abundant fragments of long terminal repeat-retrotransposons in these two species. We provide fundamental information to characterize the genomes of B. m. lintanensis and B. m. hebeiensis, providing insights into the evolution of B. motasi group parasites.

Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Non-specific vs. specific DNA binding free energetics of a transcription factor domain protein between search and recognition.

Transcription factor (TF) proteins are key in genetic regulation by locating specific sites on the genome. It has been proposed that an optimized search can be achieved by protein alternating between 3D diffusion in the bulk and 1D diffusion along DNA in the facilitated diffusion model. While undergoing 1D diffusion, the protein can switch from a “search” mode for fast diffusion along non-specific DNA, to a “recognition” mode for protein stable binding to specific DNA. Though it was regarded that protein conformation transitions enable the search to recognition transitions, it was recently noticed that for a small TF domain protein, re-orientation of the protein on the DNA, without protein conformational changes, happens between the non-specific and specific DNA binding. We further conducted all-atom molecular dynamics simulations on the TF domain protein bound to a nonspecific or specific DNA site, to energetically confirm that the protein-DNA binding free energetics between the two systems is consistent with the “search” and “recognition”. Using both the Jarzynski equality and umbrella sampling methods pulling protein away from DNA, we obtained protein binding free energetics and a free energy difference of about 10 kBT between the two systems. However, the binding free energy difference estimated from experimental measurements was about 4 kBT on 15-bp DNA. We suggest that the discrepancy between the experimental and computational measurements can be due to DNA sequences flanking the 6-bp central specific and nonspecific binding sites. Such a proposal was also investigated through a highly simplified spherical protein model along with stochastic simulations, which well supported that flanking DNA sequences impact on overall protein dissociation kinetics and therefore on measuring binding affinity variations with specific or non-specific DNA in the central binding site.