Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification.

Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/ß-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-ß-stranded outer-membrane (OM) WzpX, WzpS, and WzpB ß-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium Myxococcus xanthus DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the M. xanthus WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each ß-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the ß-barrels and inhibiting polysaccharide secretion.

Recycling solid waste to produce eco-friendly ultra-high performance concrete: A review of durability, microstructure and environment characteristics.

Recycling waste materials (WMs) is a cost-effective method for saving natural resources, protecting the environment, and reducing the use of high-carbon raw materials. This review aims to illustrate the impact of solid waste on the durability and microstructure of ultra-high performance concrete (UHPC) and to provide guidance for the research of eco-friendly UHPC. The results show that the proper use of solid waste to replace part of the binder or aggregate has a positive effect on the performance development of UHPC, but further enhancement techniques should be developed. When solid waste is prepared as a binder, the durability of waste based UHPC can be effectively improved by grinding and activation. When solid waste is used as an aggregate, its rough surface, potential reactivity and internal curing effect are also beneficial to the improvement of UHPC performance. Since UHPC has a dense microstructure, it can effectively prevent the leaching of harmful elements (heavy metal ions) in solid waste. However, the effect of waste modification on the reaction products of UHPC needs to be further studied, and design methods and testing standards suitable for eco-friendly UHPCs should be developed. The use of solid waste in UHPC effectively reduces the carbon footprint of the mixture, which is beneficial to the development of cleaner production technologies.

Unmasking of the von Willebrand A-domain surface adhesin CglB at bacterial focal adhesions mediates myxobacterial gliding motility.

The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling adhesin of the gliding transducer (Glt) machinery at bFAs. Biochemical and genetic analyses reveal that CglB localizes to the cell surface independently of the Glt apparatus; once there, it is recruited by the OM module of the gliding machinery, a heteroligomeric complex containing the integral OM ß barrels GltA, GltB, and GltH, as well as the OM protein GltC and OM lipoprotein GltK. This Glt OM platform mediates the cell-surface accessibility and retention of CglB by the Glt apparatus. Together, these data suggest that the gliding complex promotes regulated surface exposure of CglB at bFAs, thus explaining the manner by which contractile forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective ß-Barrel Porin Requirements for Polymer Secretion.

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM ß-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 ß-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize ß-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning ß-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.

Evaluation of Azido 3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Analogues for Click Chemistry-Mediated Metabolic Labeling of Myxococcus xanthus DZ2 Lipopolysaccharide.

Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N3) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N3-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N3-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N3-Kdo and 7-N3-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N3-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N3-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N3-Kdo is indeed a substrate of the enzyme, 7-N3-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N3-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N3-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

Harmonizing Prokaryotic Nomenclature: Fixing the Fuss over Phylum Name Flipping.

Lloyd and Tahon recently criticized proposed bacterial phylum nomenclature changes (K.G. Lloyd, G. Tahon, Nat Rev Microbiol 20:123-124, 2022, https://doi.org/10.1038/s41579-022-00684-2) precipitated by the International Committee on Systematics of Prokaryotes (ICSP)'s official recognition of phylum nomenclature rules. Here, we extend the critique. While we applaud bringing consistency to phylum names, we prognosticate what this minute but momentous change entails for the future of microbial nomenclature and how this will sow confusion among researchers. Several pitfalls of the proposed ICSP framework-based nomenclature are also detailed, including (i) improper type genus name and suffix usage, (ii) loss of Bacteria/Archaea distinctions, (iii) disruption of major phylum name prefixes, and (iv) absence of organism name prevalidation. Finally, we suggest new names for the key bacterial phyla Proteobacteria (Proteobacteriota), Firmicutes (Firmicuteota), Actinobacteria (Actinobacteriota), and Tenericutes (Tenericuteota), while keeping the archaeal phylum names Crenarchaeota, Thaumarchaeota, and Euryarchaeota. Together, these changes will help researchers attain chaos-free uniform nomenclature.

Bacterial Glycocalyx Integrity Impacts Tolerance of Myxococcus xanthus to Antibiotics and Oxidative-Stress Agents.

The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a "glycocalyx", confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative stress-inducing compounds of various mutant strains of the social predatory Gram-negative soil bacterium Myxococcus xanthus; the selected mutants were those that manifest different physical states of their respective EPS glycocalyces. While the overall presence of an EPS layer was indeed beneficial for tolerance, the integrity of this layer was also found to affect the susceptibility of the bacterium to killing; however, this finding was not universal, and instead was dependent on the specific compound tested. Thus, the integrity of the cell-surface EPS glycocalyx plays an important role in the tolerance of M. xanthus to harmful compounds.

The possible association between neurodegenerative/demyelinating neurological disorders in achalasia patients.

The precise pathogenesis of achalasia is still unclear. Neurodegenerative and/or demyelinating disorders (NDD) appear to share some common pathophysiological pathways described in achalasia such as inflammation, autoimmune, mitochondrial dysfunction, and neurodegeneration. Jerie et al. have published on the October issue a prospective study assessing the prevalence of several NDD in achalasia patients. In this commentary, we shed some light on the possible link between achalasia and NDD as well as comment on the study by Jerie et al.