Search Results (109)

Class IId bacteriocins are linear, unmodified antimicrobial peptides produced by Gram-positive bacteria, and often display potent, narrow-spectrum inhibition spectra. Garvicin Q (GarQ) is a class IId bacteriocin produced by the lactic acid bacterium Lactococcus garvieae. It stands out for its unusual broad-spectrum antimicrobial activity against various bacterial species, including Listeria monocytogenes, Pediococcus pentosaceus, Carnobacterium maltaromaticum, Enterococcus faecalis, and Lactococcus spp. Its protein target is the mannose phosphotransferase system (Man-PTS) of susceptible bacterial strains, though little is known about the precise molecular mechanism behind GarQ’s unusual broad spectrum of activity. In this work, ¹³C- and ¹⁵N-labelled GarQ was recombinantly produced using our previously described “sandwiched” protein expression system in Escherichia coli. We also developed a protocol to purify a uniformly labelled sample of the small ubiquitin-like modifier His₆-SUMO, which is produced as a byproduct of the expression procedure. We demonstrated its use as a “free” protein standard for 3D NMR experiment calibrations. The GarQ solution structure was solved using triple-resonance nuclear magnetic resonance (NMR) spectroscopy and was compared with the structures of other Man-PTS-targeting bacteriocins. GarQ adopts a helix–hinge–helix fold, which is contrary to its structural predictions according to AlphaFold 3. Full article

The emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Type IV pili (T4Ps) are multifunctional surface fibers essential for bacterial motility, adhesion, and virulence, found across Gram-negative and Gram-positive bacteria and archaea. Detailed descriptions of T4P structural biology are allowing progress in understanding T4P biogenesis. Secretins, large outer membrane channels, are crucial for T4P extrusion in Gram-negative bacteria. Using cryo-EM and AlphaFold, we modeled the structure of BfpB, the secretin of the Bundle-Forming Pilus (BFP) of enteropathogenic Escherichia coli. BfpB exhibits a unique 17-fold symmetry, correlating with the thicker BFP filaments, and diverging from the 12–15 subunits typical of T4P, type 2 secretion (T2S), and type 3 secretion (T3S) systems. Additionally, we identified an extended β-hairpin loop in the N3 domain, resembling features of distantly related T3SS secretins, and an N-terminal helix where a C-terminal S-domain is seen in some T2S and T3S secretins. These findings reveal evolutionary parallels and structural adaptations in secretins, highlighting the link between oligomerization and pilus structure. This work advances our understanding of T4P biogenesis, secretin evolution, and bacterial secretion systems, offering insights into pathogenic diversity and future research directions. Full article

The human CHCHD4 protein, which is a prototypical family member, carries a coiled–coil–helix–coiled–coil–helix motif that is stabilized by two disulfide bonds. Using its CPC sequence motif, CHCHD4 plays a key role in mitochondrial metabolism, cell survival, and response to stress conditions, controlling the mitochondrial import of diversified protein substrates that are specifically recognized through an interplay between covalent and non-covalent interactions. In the present review, we provide an updated and comprehensive analysis of CHCHD4 substrates controlled by its redox activities. A particular emphasis has been placed on the molecular and structural aspects of these partnerships. The literature survey has been integrated with the mining of structural databases reporting either experimental structures (Protein Data Bank) or structures predicted by AlphaFold, which provide protein three-dimensional models using machine learning-based approaches. In providing an updated view of the thirty-four CHCHD4 substrates that have been experimentally validated, our analyses highlight the notion that this protein can operate on a variety of structurally diversified substrates. Although in most cases, CHCHD4 plays a crucial role in the formation of disulfide bridges that stabilize helix–coil–helix motifs of its substrates, significant variations on this common theme are observed, especially for substrates that have been more recently identified. Full article

Since 2017, an infectious disease characterized by gosling gout and caused by goose astrovirus (GAstV) has affected geese in most major goose-producing regions of China. In this study, a total of 385 geese displaying gout symptoms were sampled from 12 cities in Guangdong Province, China, between 2019 and 2021. RT-PCR analysis revealed that all samples were positive for GAstV (385/385), with GAstV-II being the predominant subtype, accounting for 90.4% (348/385) of the cases. Co-infection with GAstV-I and GAstV-II was detected in 50.4% (194/385) of the samples. Additionally, different GAstV subtypes were successfully isolated using goose embryos, namely GDYJ-21-01 (GAstV-I) and GDZJ-21-01 (GAstV-II). Analysis of viral copy numbers in major pathological tissues following infection of goslings and goose embryos revealed that GDZJ strain exhibited broader tissue tropism than GDYJ strain. Compared to other tissues, GDYJ strain displayed tissue tropism exclusively in the cecal tonsils of goslings and the allantoic fluid of embryos. Structural prediction and alignment using AlphaFold 2.0 identified an α-helix in the S223-A226 region of the GDZJ VP34 protein, while a loop structure was observed in the Q235-Q237 region of the corresponding GDYJ VP34 protein. Furthermore, although the VP27 protein regions of both subtypes contained five β-sheet structures, the overall sequence similarity was relatively low, at 37.1%. This study broadens our understanding of the prevalence differences among GAstV subtypes and provides valuable insights into the development of reagents for preventing these viral infections. Full article

The antimicrobial peptide P6.2 was previously de novo designed as an alpha helix cationic amphipathic molecule. In previous work, we have shown that this peptide displayed significant antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. However, while P6.2 lacked biofilm-inhibiting properties against the P. aeruginosa strain PA01, it displayed anti-inflammatory effects in a murine acute lung infection model challenged with this pathogen. In this work, the peptide P6.2 antimicrobial activity and its possible synergy with meropenem were evaluated both in vitro and in vivo using a Galleria mellonella infection model against a carbapenem-resistant KPC-producing clinical isolate of P. aeruginosa. Firstly, the cytotoxic effect of the peptide on A549 and RAW264.7 cell lines was assayed, showing no cytotoxicity at 64 µg/mL and below. Then, the MIC (minimal inhibitory concentration) and bactericidal effect against the carbapenemase-producing strain P. aeruginosa M13513 strain were determined. P6.2 showed a MIC between 32 and 64 µg/mL, and a rapid bactericidal activity against this strain (less than 45 min). The peptide stability at different temperatures and in bovine serum at 37 °C was also analyzed, showing good stability and almost no degradation after 15 min of incubation at 100 °C or 24 h at 37 °C in serum, respectively. The antibiofilm activity was also evaluated, and although the peptide did not show biofilm inhibitory activity, it did demonstrate biofilm disruptive activity, together with bactericidal activity inside the pre-formed biofilm. The possible synergistic effect with the carbapenem meropenem was then analyzed in vitro by killing kinetics, revealing a synergistic interaction between P6.2 and the antibiotic against this strain. Finally, P6.2 was evaluated in vivo in the Galleria mellonella larvae infection model. Interestingly, in G. mellonella, P6.2 alone did not completely clear the infection caused by P. aeruginosa M13513. However, when combined with meropenem, P6.2 demonstrated a synergistic effect, leading to increased survival rates in infected larvae. The results presented here highlight the potential that this peptide displays when used in combination with carbapenems against a clinically relevant KPC-producing P. aeruginosa. Full article

Adipogenesis is regulated by the coordinated activity of adipogenic transcription factors including PPAR-gamma and C/EBP alpha, while dysregulated adipogenesis can predispose adipose tissues to adipocyte hypertrophy and hyperplasia. We have previously reported that Cthrc1-null mice have increased adiposity compared to wildtype mice, supporting the notion that CTHRC1 regulates body composition. Herein, we derived conditioned medium from 3T3-L1 cells expressing human CTHRC1 and investigated its anti-adipogenic activity. This constituent significantly reduced 3T3-L1 cell adipogenic differentiation commensurate to the marked suppression of Cebpa and Pparg gene expression. It also increased the expression of the anti-adipogenic transcription factor SOX9 and promoted its nuclear translocation. Importantly, Sox9 gene knockdown demonstrated that the anti-adipogenic effect produced by this conditioned medium is dependent on SOX9 expression, while its ability to positively regulate SOX9 was attenuated by the application of Rho and Rac1 signaling pathway inhibitors. We also identified the selective expression of CTHRC1 in PDGFRA-expressing cell populations in human white adipose tissue, but not brown or perivascular adipose tissues. Congruently, flow cytometry revealed CTHRC1 expression in PDGFR-alpha⁺ stromal cells of mouse white adipose tissue, thus defining a novel stromal cell population that could underpin the ability of CTHRC1 to regulate adiposity. Full article

The rapid induction of drug resistance is considered a fatal drawback of conventional antibiotics and requires the continuous development of new antibiotics. Accordingly, antibacterial peptides (AMPs) have attracted interest as next-generation antibiotics and many studies have been conducted. However, much remains unknown regarding the mechanism of AMPs and the effects of amino acid sequence changes. We compared the structures and antifungal effects of HnMc-W (F1W substitution, straight alpha-helical structure), HnMc-WP1 (S9P substitution, bending alpha-helical structure), and HnMc-WP2 (addition of the PXXP motif, helix-to-helix structure) to those of a parent hybrid AMP (HnMc) regarding their mechanism of action. The most active was HnMc-WP2, which exhibited an antifungal effect via membranolytic action on the fungal cell membrane. The others inhibited fungal growth by inducing apoptosis through reactive oxygen species production caused by mitochondrial damage. This study proposes the addition of the ‘PXXP’ motif in the design of AMPs acting on cell membranes. Full article

Several mutations of the uppermost arginine, R219, in the voltage-sensing sliding helix S4_I of cardiac sodium channel Nav1.5 are reported in the ClinVar databases, but the clinical significance of the respective variants is unknown (VUSs). AlphaFold 3 models predicted a significant downshift of S4_I in the R219C VUS. Analogous downshift S4_I, upon its in silico deactivation, resulted in a salt bridge between R219 and the uppermost glutamate, E161, in helix S2_I. To understand how salt bridge elimination affects biophysical characteristics, we generated mutant channel R219E, expressed it in the HEK293-T cells, and employed the patch-clamp method in a whole-cell configuration. Mutation R219E did not change the peak current density but shortened time to the peak current at several potentials, significantly enhanced activation, enhanced steady-state inactivation and steady-state fast inactivation, and slowed recovery from inactivation. Taken together, these data suggest that mutation R219E destabilized the resting state of Nav1.5. Cardiac syndromes associated with mutations R219P/H/C/P or E161Q/K are consistent with the observed changes of biophysical characteristics of mutant channel R219E suggesting pathogenicity of the respective VUSs, as well as ClinVar-reported VUSs involving arginine or glutamate in homologous positions of several Nav1.5 paralogs. Full article

Porcine blood, a significant byproduct of the pork industry, represents a potential source of antimicrobial peptides (AMPs). AMPs offer a promising alternative to chemical antimicrobials, which can be used as natural preservatives in the food industry. AMPs can exhibit both antibacterial and/or antifungal properties, thus improving food safety and addressing the growing concern of antibiotic and antifungal resistance. The objective of this study was to evaluate the antimicrobial activity of potential AMPs previously identified from porcine cruor hydrolysates. To this end, a total of sixteen peptides were chemically synthesized and their antimicrobial activities (antibacterial, anti-mold, and anti-yeast) were evaluated using microtitration and agar well diffusion methods against a wide range of microorganisms. Five new peptide sequences demonstrated antifungal activity, with Pep5 (FQKVVAGVANALAHKYH), an alpha-helix peptide, exhibiting the most promising results. Pep5 demonstrated efficacy against nine of the eleven fungal isolates, exhibiting low minimum inhibitory concentrations (MICs) and a fungicidal effect against key spoilage fungi (Rhodotorula mucilaginosa, Debaryomyces hansenii, Candida guilliermondii, Paecilomyces spp., Eurotium rubrum, Mucor racemosus, Aspergillus versicolor, Penicillium commune, and P. chrysogenum). These findings illustrate the potential of porcine blood hydrolysates as a source of AMPs, particularly antifungal peptides, which are less known and less studied than the antibacterial ones. Among the tested sequences, Pep5 exhibited the most promising characteristics, including broad-spectrum activity, low MICs, and a fungicidal effect. It is, therefore, a promising candidate for further research and for potential applications in the porcine industry and beyond. Full article

Background: The consumption of nutraceuticals or food supplements has increased crucially, aiming to address nutrient deficits and enhance immune system function. To develop safe food products with unique nutritional and functional benefits, new production methods of these nutraceuticals such as the fermentative process have been gaining prominence for industrial applications. Bionutri-AR1^® is a nutraceutical produced via this bioprocess, featuring a complex composition, that has been used to improve the immune systems of debilitated people. Objectives: Considering the various biological properties attributed to glucans, one of its main components, this study aims to structurally characterize and evaluate, in vitro, the antioxidant and immunomodulatory potential of the polymers from this nutraceutical to assess whether these polymers contribute to the product’s reported biological effects. Methods/Results: Unlike previous reports, this study characterized by NMR, GC-MS, and Congo Red assay techniques two main glucans: a water-insoluble linear α-D-glucan with glycosidic bonds (1→4) and a soluble branched (1→3)- and (1→6)-linked β-glucan with a triple helix. Both glucans showed significant antioxidant activity, measured by their capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. They were also capable of inducing the secretion of cytokines such as tumoral necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and interleukin 6 (IL-6), determined through capture enzyme-linked immunosorbent assay (ELISA), especially when co-stimulated with lipopolysaccharide (LPS). Conclusions: This suggests a dual action of these glucans in both proinflammatory and regulatory pathways. Future studies will describe the mechanisms by which these glucans, especially the insoluble ones, enhance immune system function, highlighting their potential use in immunotherapy. Full article

This study identified a salt-tolerant GH11 xylanase, Xyn^st, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn^st and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn^st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (K_m = 0.30 mg mL⁻¹ for Xyn^st and 0.18 mg mL⁻¹ for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (K_cat/K_m = 15483.33 mL mg⁻¹ s⁻¹). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn^st did. The conversion efficiencies of Xyn^st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries. Full article

Polyphenol oxidase (PPO) plays a key role in the enzymatic browning process, and this study employed Gaussian-accelerated molecular dynamics (GaMD) simulations to investigate the catalytic efficiency mechanisms of lotus root PPO with different substrates, including catechin, epicatechin, and chlorogenic acid, as well as the inhibitor oxalic acid. Key findings reveal significant conformational changes in PPO that correlate with its enzymatic activity. Upon substrate binding, the alpha-helix in the Q53-D63 region near the copper ion extends, likely stabilizing the active site and enhancing catalysis. In contrast, this helix is disrupted in the presence of the inhibitor, resulting in a decrease in enzymatic efficiency. Additionally, the F350-V378 region, which covers the substrate-binding site, forms an alpha-helix upon substrate binding, further stabilizing the substrate and promoting catalytic function. However, this alpha-helix does not form when the inhibitor is bound, destabilizing the binding site and contributing to inhibition. These findings offer new insights into the substrate-specific and inhibitor-induced structural dynamics of lotus root PPO, providing valuable information for enhancing food processing and preservation techniques. Full article

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271–276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration. Full article