Precise Structural Analysis of Neutral Glycans Using Aerolysin Mutant T240R Nanopore.

Glycans play vital roles in nearly all life processes of multicellular organisms, and understanding these activities is inseparable from elucidating the biological significance of glycans. However, glycan research has lagged behind that of DNA and protein due to the challenges posed by structural heterogeneity and isomerism (i.e., structures with equal molecular weights) the lack of high-efficiency structural analysis techniques. Nanopore technology has emerged as a sensitive single-molecule biosensor, shining a light on glycan analysis. However, a significant number of glycans are small and uncharged, making it challenging to elicit identifiable nanopore signals. Here we introduce a R-binaphthyl tag into glycans, which enhances the cation-π interaction between the derivatized glycan molecules and the nanopore interface, enabling the detection of neutral glycans with an aerolysin nanopore. This approach allows for the distinction of di-, tri-, and tetrasaccharides with monosaccharide resolution and has the potential for group discrimination, the monitoring of enzymatic transglycosylation reactions. Notably, the aerolysin mutant T240R achieves unambiguous identification of six disaccharide isomers, trisaccharide and tetrasaccharide linkage isomers. Molecular docking simulations reveal that multiple noncovalent interactions occur between residues R282, K238, and R240 and the glycans and R-binaphthyl tag, significantly slowing down their translocation across the nanopore. Importantly, we provide a demonstration of the kinetic translocation process of neutral glycan isomers, establishing a solid theoretical foundation for glycan nanopore analysis. The development of our technology could promote the analysis of glycan structural isomers and has the potential for nanopore-based glycan structural determination and sequencing.

Profiling the chemistry- and confinement-controlled sensing capability of an octameric aerolysin-like protein.

Octameric Aep1 was employed, for the first time to the best of our knowledge, as a nanopore to expand applications. After investigating the optimized conditions of Aep1 for single-channel recording, the sensing features were characterized. Cyclic and linear molecules of varying sizes and charges were employed to probe the radius and chemical environment of the pore, providing deep insights for expected future endeavors at predicting the structure of octameric Aep1. γ-CD showed unique suitability as an 8-subunit adapter in octameric Aep1, enabling the discrimination of ß-nicotinamide mononucleotide.

Structural basis for GSDMB pore formation and its targeting by IpaH7.8.

Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis1,2. Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential3-7. Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity4. Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB4. Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB-IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity.

Structural mechanisms for regulation of GSDMB pore-forming activity.

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein1,2, to trigger target cell pyroptosis3. GSDMB and the charter gasdermin family member GSDMD4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently6,8. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins9,10. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies6,8. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins.

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies.

Bacterial pore-forming toxins.

Pore-forming toxins (PFTs) are widely distributed in both Gram-negative and Gram-positive bacteria. PFTs can act as virulence factors that bacteria utilise in dissemination and host colonisation or, alternatively, they can be employed to compete with rival microbes in polymicrobial niches. PFTs transition from a soluble form to become membrane-embedded by undergoing large conformational changes. Once inserted, they perforate the membrane, causing uncontrolled efflux of ions and/or nutrients and dissipating the protonmotive force (PMF). In some instances, target cells intoxicated by PFTs display additional effects as part of the cellular response to pore formation. Significant progress has been made in the mechanistic description of pore formation for the different PFTs families, but in several cases a complete understanding of pore structure remains lacking. PFTs have evolved recognition mechanisms to bind specific receptors that define their host tropism, although this can be remarkably diverse even within the same family. Here we summarise the salient features of PFTs and highlight where additional research is necessary to fully understand the mechanism of pore formation by members of this diverse group of protein toxins.

Membrane Dynamics and Remodelling in Response to the Action of the Membrane-Damaging Pore-Forming Toxins.

Pore-forming protein toxins (PFTs) represent a diverse class of membrane-damaging proteins that are produced by a wide variety of organisms. PFT-mediated membrane perforation is largely governed by the chemical composition and the physical properties of the plasma membranes. The interaction between the PFTs with the target membranes is critical for the initiation of the pore-formation process, and can lead to discrete membrane reorganization events that further aids in the process of pore-formation. Punching holes on the plasma membranes by the PFTs interferes with the cellular homeostasis by disrupting the ion-balance inside the cells that in turn can turn on multiple signalling cascades required to restore membrane integrity and cellular homeostasis. In this review, we discuss the physicochemical attributes of the plasma membranes associated with the pore-formation processes by the PFTs, and the subsequent membrane remodelling events that may start off the membrane-repair mechanisms.

A deep learning model to detect novel pore-forming proteins.

Many pore-forming proteins originating from pathogenic bacteria are toxic against agricultural pests. They are the key ingredients in several pesticidal products for agricultural use, including transgenic crops. There is an urgent need to identify novel pore-forming proteins to combat development of resistance in pests to existing products, and to develop products that are effective against a broader range of pests. Existing computational methodologies to search for these proteins rely on sequence homology-based approaches. These approaches are based on similarities between protein sequences, and thus are limited in their usefulness for discovering novel proteins. In this paper, we outline a novel deep learning model trained on pore-forming proteins from the public domain. We compare different ways of encoding protein information during training, and contrast it with traditional approaches. We show that our model is capable of identifying known pore formers with no sequence similarity to the proteins used to train the model, and therefore holds promise for identifying novel pore formers.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Enhanced identification of Tau acetylation and phosphorylation with an engineered aerolysin nanopore.

Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep-P was decelerated by up to 46 folds compared to the wild-type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep-Ac, monophosphorylated Pep-P, di-modified Pep-P-Ac and non-modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non-covalent interactions between nanopores and single analytes.

Production and characterization of polyclonal and monoclonal antibodies of lamprey pore-forming protein.

In the most primitive jawless vertebrate lamprey, the complement-dependent cytotoxicity regulated by variable lymphocyte receptors (VLRs) plays an important role in the adaptive immunity. Our previous studies have shown that the lamprey pore-forming protein (LPFP) acted as the terminal effector of VLR to lyse and kill the target cells. Here, the recombinant GST-LPFP protein was expressed and purified in prokaryotic expression system, and then used as the immunogen to produce mouse monoclonal antibody and rabbit polyclonal antibody. With these antibodies, we proved that LPFP existed as homodimers in the lamprey serum, and could be recruited to the membrane of target cells after stimulation. In conclusion, the antibodies we produced could specifically recognize the LPFP protein, which could be the useful tools to further study the pore-forming mechanism of LPFP.

Molecular and structural aspects of gasdermin family pores and insights into gasdermin-elicited programmed cell death.

Pyroptosis is a highly inflammatory and lytic type of programmed cell death (PCD) commenced by inflammasomes, which sense perturbations in the cytosolic environment. Recently, several ground-breaking studies have linked a family of pore-forming proteins known as gasdermins (GSDMs) to pyroptosis. The human genome encodes six GSDM proteins which have a characteristic feature of forming pores in the plasma membrane resulting in the disruption of cellular homeostasis and subsequent induction of cell death. GSDMs have an N-terminal cytotoxic domain and an auto-inhibitory C-terminal domain linked together through a flexible hinge region whose proteolytic cleavage by various enzymes releases the N-terminal fragment that can insert itself into the inner leaflet of the plasma membrane by binding to acidic lipids leading to pore formation. Emerging studies have disclosed the involvement of GSDMs in various modalities of PCD highlighting their role in diverse cellular and pathological processes. Recently, the cryo-EM structures of the GSDMA3 and GSDMD pores were resolved which have provided valuable insights into the pore formation process of GSDMs. Here, we discuss the current knowledge regarding the role of GSDMs in PCD, structural and molecular aspects of autoinhibition, and pore formation mechanism followed by a summary of functional consequences of gasdermin-induced membrane permeabilization.

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMß) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.

Delivery of Ultrasmall Nanoparticles to the Cytosolic Compartment of Pyroptotic J774A.1 Macrophages via GSDMDNterm Membrane Pores.

Endosome capture is a major physiological barrier to the successful delivery of nanomedicine. Here, we found a strategy to deliver ultrasmall nanoparticles (<10 nm) to the cytosolic compartment of pyroptotic cells with spontaneous endosomal escape. To mimic pathological pyroptotic cells, J774A.1 macrophages were stimulated with lipopolysaccharide (LPS) plus nigericin (Nig) or adenosine triphosphate (ATP) to form specific gasdermin D protein-driven membrane pores at an N-terminal domain (GSDMDNterm). Through GSDMDNterm membrane pores, both anionic and cationic nanoparticles (NPs) with diameters less than 10 nm were accessed into the cytosolic compartment of pyroptotic cells in an energy- and receptor-independent manner, while NPs larger than the size of GSDMDNterm membrane pores failed to enter pyroptotic cells. NPs pass through GSDMDNterm membrane pores via free diffusion and then access into the cytoplasm of pyroptotic cells in a microtubule-independent manner. Interestingly, we found that LPS-primed NPs may act as Trojan horse, deliver extracellular LPS into normal cells through endocytosis, and in turn induce GSDMDNterm membrane pores, which facilitate further internalization of NPs. This study presented a straightforward method of distinguishing normal and pyroptotic cells through GSDMD membrane pores, implicating their potential application in monitoring the delivery of desired nanomedicines in pyroptosis-related diseases and conditions.

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity.

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

In vitro and in silico approach for characterization of antimicrobial peptides from potential probiotic cultures against Staphylococcus aureus and Escherichia coli.

The focus of the present study was to characterize antimicrobial peptide produced by potential probiotic cultures of Enterococcus durans DB-1aa (MCC4243), Lactiplantibacillus plantarum Cu2-PM7 (MCC4246) and Limosilactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus MTCC 96 and Escherichia coli MTCC118. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound of all the three selected cultures after ion-exchange chromatography was found to be thermoresistant and stable under a wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. Comparatively, bacteriocins from L. fermentum Cu3-PM8 and L. plantarum Cu2-PM7 showed higher stability under studied parameter, hence was taken up for further investigation. The apparent molecular weight of bacteriocin from L. fermentum MCC4233 and L. plantarum MCC4246 was found to be 3.5 kDa. Further, plantaricin gene from MCC4246 was characterized in silico. The translated partial amino acid sequence of the plnA gene in MCC4246 displayed 48 amino acids showing 100 % similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 ß sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The predicted properties of the peptide included an isoelectric point of 10.82 and a hydrophobicity of 48.6 %. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts "KSSAYSLQMGATAIKQVKKLFKKWGW" to be a peptide responsible for antimicrobial activity. The study provides information about a broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as a biopreservative agent.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Key Physicochemical Determinants in the Antimicrobial Peptide RiLK1 Promote Amphipathic Structures.

Antimicrobial peptides (AMPs) represent a skilled class of new antibiotics, due to their broad range of activity, rapid killing, and low bacterial resistance. Many efforts have been made to discover AMPs with improved performances, i.e., high antimicrobial activity, low cytotoxicity against human cells, stability against proteolytic degradation, and low costs of production. In the design of new AMPs, several physicochemical features, such as hydrophobicity, net positive charge, propensity to assume amphipathic conformation, and self-assembling properties, must be considered. Starting from the sequence of the dodecapeptide 1018-K6, we designed a new 10-aminoacid peptide, namely RiLK1, which is highly effective against both fungi and Gram-positive and -negative bacteria at low micromolar concentrations without causing human cell cytotoxicity. In order to find the structural reasons explaining the improved performance of RiLK1 versus 1018-K6, a comparative analysis of the two peptides was carried out with a combination of CD, NMR, and fluorescence spectroscopies, while their self-assembling properties were analyzed by optical and atomic force microscopies. Interestingly, the different spectroscopic and microscopic profiles exhibited by the two peptides, including the propensity of RiLK1 to adopt helix arrangements in contrast to 1018-K6, could explain the improved bactericidal, antifungal, and anti-biofilm activities shown by the new peptide against a panel of food pathogens.

Membrane Interactions of Latarcins: Antimicrobial Peptides from Spider Venom.

A group of seven peptides from spider venom with diverse sequences constitute the latarcin family. They have been described as membrane-active antibiotics, but their lipid interactions have not yet been addressed. Using circular dichroism and solid-state 15N-NMR, we systematically characterized and compared the conformation and helix alignment of all seven peptides in their membrane-bound state. These structural results could be correlated with activity assays (antimicrobial, hemolysis, fluorescence vesicle leakage). Functional synergy was not observed amongst any of the latarcins. In the presence of lipids, all peptides fold into amphiphilic α-helices as expected, the helices being either surface-bound or tilted in the bilayer. The most tilted peptide, Ltc2a, possesses a novel kind of amphiphilic profile with a coiled-coil-like hydrophobic strip and is the most aggressive of all. It indiscriminately permeabilizes natural membranes (antimicrobial, hemolysis) as well as artificial lipid bilayers through the segregation of anionic lipids and possibly enhanced motional averaging. Ltc1, Ltc3a, Ltc4a, and Ltc5a are efficient and selective in killing bacteria but without causing significant bilayer disturbance. They act rather slowly or may even translocate towards intracellular targets, suggesting more subtle lipid interactions. Ltc6a and Ltc7, finally, do not show much antimicrobial action but can nonetheless perturb model bilayers.