Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Direct Identification of Complex Glycans via a Highly Sensitive Engineered Nanopore.

The crucial roles that glycans play in biological systems are determined by their structures. However, the analysis of glycan structures still has numerous bottlenecks due to their inherent complexities. The nanopore technology has emerged as a powerful sensor for DNA sequencing and peptide detection. This has a significant impact on the development of a related research area. Currently, nanopores are beginning to be applied for the detection of simple glycans, but the analysis of complex glycans by this technology is still challenging. Here, we designed an engineered α-hemolysin nanopore M113R/T115A to achieve the sensing of complex glycans at micromolar concentrations and under label-free conditions. By extracting characteristic features to depict a three-dimensional (3D) scatter plot, glycans with different numbers of functional groups, various chain lengths ranging from disaccharide to decasaccharide, and distinct glycosidic linkages could be distinguished. Molecular dynamics (MD) simulations show different behaviors of glycans with ß1,3- or ß1,4-glycosidic bonds in nanopores. More importantly, the designed nanopore system permitted the discrimination of each glycan isomer with different lengths in a mixture with a separation ratio of over 0.9. This work represents a proof-of-concept demonstration that complex glycans can be analyzed using nanopore sequencing technology.

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity.

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.

Structural Determinants of Chirally Selective Transport of Amino Acids through the α-Hemolysin Protein Nanopores of Free-Standing Planar Lipid Membranes.

Despite the importance of the enantioselective transport of amino acids through transmembrane protein nanopores from fundamental and practical perspectives, little has been explored to date. Here, we study the transport of amino acids through α-hemolysin (αHL) protein pores incorporated into a free-standing lipid membrane. By measuring the transport of 13 different amino acids through the αHL pores, we discover that the molecular size of the amino acids and their capability to form hydrogen bonds with the pore surface determine the chiral selectivity. Molecular dynamics simulations corroborate our findings by revealing the enantioselective molecular-level interactions between the amino acid enantiomers and the αHL pore. Our work is the first to present the determinants for chiral selectivity using αHL protein as a molecular filter.

Narrowing Signal Distribution by Adamantane Derivatization for Amino Acid Identification Using an α-Hemolysin Nanopore.

The rapid progress in nanopore sensing has sparked interest in protein sequencing. Despite recent notable advancements in amino acid recognition using nanopores, chemical modifications usually employed in this process still need further refinements. One of the challenges is to enhance the chemical specificity to avoid downstream misidentification of amino acids. By employing adamantane to label proteinogenic amino acids, we developed an approach to fingerprint individual amino acids using the wild-type α-hemolysin nanopore. The unique structure of adamantane-labeled amino acids (ALAAs) improved the spatial resolution, resulting in distinctive current signals. Various nanopore parameters were explored using a machine-learning algorithm and achieved a validation accuracy of 81.3% for distinguishing nine selected amino acids. Our results not only advance the effort in single-molecule protein characterization using nanopores but also offer a potential platform for studying intrinsic and variant structures of individual molecules.

Peptide sequencing based on host-guest interaction-assisted nanopore sensing.

Direct protein sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose an alternative method for peptide sequencing based on enzymatic cleavage and host-guest interaction-assisted nanopore sensing. We serendipitously discovered that the identity of any proteinogenic amino acid in a particular position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through α-hemolysin nanopores in the presence of cucurbit[7]uril. Building upon this, we further present a proof-of-concept demonstration of peptide sequencing by sequentially cleaving off amino acids from C terminus of a peptide with carboxypeptidases, and then determining their identities and sequence with a peptide probe in nanopore. With future optimization, our results point to a different way of nanopore-based protein sequencing.

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis.

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

Mapping the Acetylamino and Carboxyl Groups on Glycans by Engineered α-Hemolysin Nanopores.

Glycan is a crucial class of biological macromolecules with important biological functions. Functional groups determine the chemical properties of glycans, which further affect their biological activities. However, the structural complexity of glycans has set a technical hurdle for their direct identification. Nanopores have emerged as highly sensitive biosensors that are capable of detecting and characterizing various analytes. Here, we identified the functional groups on glycans with a designed α-hemolysin nanopore containing arginine mutations (M113R), which is specifically sensitive to glycans with acetamido and carboxyl groups. Molecular dynamics simulations indicated that the acetamido and carboxyl groups of the glycans produce unique electrical signatures by forming polar and electrostatic interactions with the M113R nanopores. Using these electrical features as the fingerprints, we mapped the length of the glycans containing acetamido and carboxyl groups at the monosaccharide, disaccharide, and trisaccharide levels. This proof-of-concept study provides a promising foundation for developing single-molecule glycan fingerprinting libraries and demonstrates the capability of biological nanopores in glycan sequencing.

Distribution and Genetic Diversity of Genes from Brazilian Bacillus thuringiensis Strains Toxic to Agricultural Insect Pests Revealed by Real-Time PCR.

Bacillus thuringiensis is a Gram-positive aerobic bacterium and the most used biopesticide worldwide. Given the importance of B. thuringiensis strain characterization for the development of new bioinsecticides or transgenic events and the identification and classification of new B. thuringiensis genes and strains to understand its distribution and diversity, this work is aimed at creating a gene identification system based on qPCR reactions utilizing core B. thuringiensis genes cry1, cry2, cry3, cry4, cry5, app6, cry7, cry8, cry9, cry10, cry11, vpb1, vpa2, vip3, cyt1, and cyt2 for the characterization of 257 strains of B. thuringiensis. This system was based on the Invertebrate Bacteria Collection from Embrapa Genetic Resources and Biotechnology and analyzed (a) the degree of correlation between the distribution of these strains and the origin of the substrate from which the strain was isolated and (b) between its distribution and geoclimatic conditions. This study made it possible to observe that the cry1, cry2, and vip3A/B genes occur homogeneously in the Brazilian territory, and some genes are found in specific regions. The biggest reservoir of variability is within B. thuringiensis strains in each region, and it is suggested that both geoclimatic conditions and regional crops interfere with the genetic diversity of the B. thuringiensis strains present in the region, and B. thuringiensis strains can constantly exchange genetic information.

Electron beam irradiation degrades the toxicity and alters the protein structure of Staphylococcus aureus alpha-hemolysin.

α-Hemolysin (Hla) is a potent pore-forming toxin (PFT) produced by Staphylococcus aureus that exacerbates the pathogenesis of S. aureus enterotoxicity and plays a role in population food poisoning. Hla lyses cells by binding to host cell membranes and oligomerizing to form heptameric structures, thereby disrupting the cell barrier. Although the broad bactericidal effect of electron beam irradiation (EBI) has been demonstrated whether it has a damaging or degrading effect on Hla's remains unknown. In this study, EBI was found to have the effect of altering the secondary structure of Hla proteins, verifying that the damaging effect of EBI-treated Hla on intestinal and skin epithelial cell barriers was significantly reduced. It was noted by hemolysis and protein interactions that EBI treatment significantly disrupted the binding of Hla to its high-affinity receptor, but did not affect the binding between Hla monomers to form heptamers. Thus, EBI can effectively reduce the threat of Hla to food safety.

Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography.

Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L-weight filter to enhance the structural features. By L-weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three-dimensional structures determined by x-ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

The role of glycoconjugates as receptors for insecticidal proteins.

Bacillus thuringiensis (Bt) proteins are an environmentally safe and effective alternative to chemical pesticides and have been used as biopesticides, with great commercial success, for over 50 years. Global agricultural production is predicted to require a 70% increase until 2050 to provide for an increasing population. In addition to agriculture, Bt proteins are utilized to control human vectors of disease-namely mosquitoes-which account for >700 000 deaths annually. The evolution of resistance to Bt pesticial toxins threatens the progression of sustainable agriculture. Whilst Bt protein toxins are heavily utilized, the exact mechanisms behind receptor binding and toxicity are unknown. It is critical to gain a better understanding of these mechanisms in order to engineer novel toxin variants and to predict, and prevent, future resistance evolution. This review focuses on the role of carbohydrate binding in the toxicity of the most utilized group of Bt pesticidal proteins-three domain Cry (3D-Cry) toxins.

Assembly dynamics and structure of an aegerolysin, ostreolysin A6.

Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.

Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations.

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches.

Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from Bacillus thuringiensis subsp. israelensis ISPC-12.

Cry11Aa is the most potent mosquito larvicidal protein of Bacillus thuringiensis subsp. israelensis (Bti). Development of resistance against insecticidal proteins including Cry11Aa is known but no field resistance was observed with Bti. The phenomenon of increasing resistance in insect pests necessitates the development of new strategies and techniques to enhance efficacy of insecticidal proteins. Recombinant technology offers better control over the molecule and allows modification of protein to achieve maximal effect against target pests. In this study, we standardised protocol for recombinant purification of Cry11Aa. Recombinant Cry11Aa found active against larvae of Aedes and Culex mosquito species and LC50 were estimated. Detailed biophysical characterization provides crucial insights into stability and in-vitro behaviour of the recombinant Cry11Aa. Moreover, trypsin hydrolysis doesn't improve overall toxicity of recombinant Cry11Aa. Proteolytic processing suggests domain I and II are more prone to proteolysis in comparison to domain III. Significance of structural features for proteolysis of Cry11Aa was observed after performing molecular dynamics simulations. Findings reported here are contributing significantly in method for purification, understanding in-vitro behaviour and proteolytic processing of Cry11Aa which could facilitate in efficient utilisation of Bti for insect pests and vectors control.