A pleiotropic role of sialidase in the pathogenicity of Porphyromonas gingivalis.

As one of the keystone pathogens of periodontitis, the oral bacterium Porphyromonas gingivalis produces an array of virulence factors, including a recently identified sialidase (PG0352). Our previous report involving loss-of-function studies indicated that PG0352 plays an important role in the pathophysiology of P. gingivalis. However, this report had not been corroborated by gain-of-function studies or substantiated in different P. gingivalis strains. To fill these gaps, herein we first confirm the role of PG0352 in cell surface structures (e.g., capsule) and serum resistance using P. gingivalis W83 strain through genetic complementation and then recapitulate these studies using P. gingivalis ATCC33277 strain. We further investigate the role of PG0352 and its counterpart (PGN1608) in ATCC33277 in cell growth, biofilm formation, neutrophil killing, cell invasion, and P. gingivalis-induced inflammation. Our results indicate that PG0352 and PGN1608 are implicated in P. gingivalis cell surface structures, hydrophobicity, biofilm formation, resistance to complement and neutrophil killing, and host immune responses. Possible molecular mechanisms involved are also discussed. In summary, this report underscores the importance of sialidases in the pathophysiology of P. gingivalis and opens an avenue to elucidate their underlying molecular mechanisms.

Adhesin domains responsible for binding bacteria to surfaces they colonize project outwards from companion split domains.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly ß-sandwich extender modules, but other domains like a ß-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

Genome-wide identification of B-box zinc finger (BBX) gene family in Medicago sativa and their roles in abiotic stress responses.

BACKGROUND: B-box (BBX) family is a class of zinc finger transcription factors (TFs) that play essential roles in regulating plant growth, development, as well as abiotic stress. However, no systematic analysis of BBX genes has yet been conducted in alfalfa (Medica go sativa L.), and their functions have not been elucidated up to now. RESULTS: In this study, 28 MsBBX genes were identified from the alfalfa genome, which were clustered into 4 subfamilies according to an evolutionary tree of BBX proteins. Exon-intron structure and conserved motif analysis reflected the evolutionary conservation of MsBBXs in alfalfa. Collinearity analysis showed that segmental duplication promoted the expansion of the MsBBX family. Analysis of cis-regulatory elements suggested that the MsBBX genes possessed many growth/development-, light-, phytohormone-, and abiotic stress-related elements. MsBBX genes were differentially expressed in leaves, flowers, pre-elongated stems, elongated stems, roots and nodules, and most MsBBXs were remarkably induced by drought, salt and various plant growth regulators (ABA, JA, and SA). Further functional verification demonstrated that overexpressing of the MsBBX11 gene clearly promoted salt tolerance in transgenic Arabidopsis by regulating growth and physiological processes of seedlings. CONCLUSIONS: This research provides insights into further functional research and regulatory mechanisms of MsBBX family genes under abiotic stress of alfalfa.

Sugar-binding and split domain combinations in repeats-in-toxin adhesins from Vibrio cholerae and Aeromonas veronii mediate cell-surface recognition and hemolytic activities.

Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.

The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures.

Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple "mix-and-match" method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non-phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be effectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy-aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics.

Multiple free-radical-trapping and hydrogen-bonding-enhanced polyurethane foams with long-lasting flame retardancy, aging resistance, and toughness.

Flexible polyurethane foam (FPUF) is a ubiquitous material utilized in furniture cushions, mattresses, and various technical applications. Despite the widespread use, FPUF faces challenges in maintaining long-lasting flame retardancy and aging resistance, particularly in harsh environments, while retaining mechanical robustness. Here, we present a novel approach to address these issues by enhancing FPUF through multiple free-radical-trapping and hydrogen-bonding mechanisms. A hindered amine phosphorus-containing polyol (DTAP) was designed and chemically introduced into FPUF. The distinctive synergy between hindered amine and phosphorus-containing structures enables the formation of multiple hydrogen bonds with urethane, while also effectively capturing free radicals across a broad temperature spectrum. As a result, incorporating only 5.1 wt% of DTAP led to the material successfully passing vertical burning tests and witnessing notable enhancements in tensile strength, elongation at break, and tear strength. Even after enduring accelerated thermal aging for 168 hours, the foam maintained exceptional flame retardancy and mechanical properties. This study offers novel insights into material enhancement, simultaneously achieving outstanding long-lasting flame retardancy, toughness, and anti-aging performance.

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Self-evolutionary recycling of flame-retardant polyurethane foam enabled by controllable catalytic cleavage.

Polyurethane (PU) foams, pivotal in modern life, face challenges suh as fire hazards and environmental waste burdens. The current reliance of PU on potentially ecotoxic halogen-/phosphorus-based flame retardants impedes large-scale material recycling. Here, our demonstrated controllable catalytic cracking strategy, using cesium salts, enables self-evolving recycling of flame-retardant PU. The incorporation of cesium citrates facilitates efficient urethane bond cleavage at low temperatures (160 °C), promoting effective recycling, while encouraging pyrolytic rearrangement of isocyanates into char at high temperatures (300 °C) for enhanced PU fire safety. Even in the absence of halogen/phosphorus components, this foam exhibits a substantial increase in ignition time (+258.8%) and a significant reduction in total smoke release (-79%). This flame-retardant foam can be easily recycled into high-quality polyol under mild conditions, 60 °C lower than that for the pure foam. Notably, the trace amounts of cesium gathered in recycled polyols stimulate the regenerated PU to undergo self-evolution, improving both flame-retardancy and mechanical properties. Our controllable catalytic cracking strategy paves the way for the self-evolutionary recycling of high-performance firefighting materials.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins (INPs) of common environmental bacteria like Pseudomonas syringae and Pseudomonas borealis. However, individually, these 100 kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an INP gene into Escherichia coli led to efficient ice nucleation. Here, we demonstrate that a positively charged subdomain at the C-terminal end of the central ß-solenoid of the INP is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the INP multimers form fibres that are ~5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.