Metabolic Microenvironments Drive Microbial Differentiation and Antibiotic Resistance.

For bacteria, the transition from unicellular entities to multicellular biofilm communities generates distinct metabolic microenvironments. Dynamic and programmed metabolic responses allow the biofilms to react to local changes in nutrient levels. Moreover, metabolic adaptations contribute to phenotypic antibiotic resistance of the community, suggesting novel therapeutic approaches to target biofilms.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450 kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0 nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

UNLABELLED: The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. IMPORTANCE: Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibers and cellulose. Thus, EAEC, which adhere in a "stacked brick" biofilm mode, have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. In contrast, EHEC and UPEC, which use alternative adherence mechanisms, tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types of E. coli.

Bacterial epigenetics and its implication for agriculture, probiotics development, and biotechnology design.

In their natural habitats, organisms encounter numerous external stimuli and must be able to sense and adapt to those stimuli to survive. Unlike mutations, epigenetic changes do not alter the underlying DNA sequence. Instead, they create modifications that promote or silence gene expression. Bacillus subtilis has long been a model organism in studying genetics and development. It is beneficial for numerous biotechnological applications where it is included as a probiotic, in fermentation, or in bio-concrete design. This bacterium has also emerged recently as a model organism for studying bacterial epigenetic adaptation. In this review, we examine the evolving knowledge of epigenetic regulation (restriction-modification systems (RM), orphan methyltransferases, and chromosome condensation) in B. subtilis and related bacteria, and utilize it as a case study to test their potential roles and future applications in genetic engineering and microbial biotechnology. Finally, we suggest how the implementation of these fundamental findings promotes the design of synthetic epigenetic memory circuits and their future applications in agriculture, medicine, and biotechnology.

Redox-Responsive Hydrogels Loaded with an Antibacterial Peptide as Controlled Drug Delivery for Healing Infectious Wounds.

Infectious wounds occur when harmful microorganisms such as bacteria or viruses invade a wound site. Its problems associated include delayed healing, increased pain, swelling, and the potential for systemic infections. Therefore, developing new wound dressing materials with antibacterial effects is crucial for improving the healing process. Here a redox-degradable hydrogel loaded with an antibacterial peptide (vancomycin) in a straightforward gram-scale synthesis, is developed. The hydrogel structure consists of a disulfide bond-containing hyperbranched polyglycerol (SS-hPG) that is cross-linked by 4-arm polyethylene glycol-thiol (4-arm PEG-SH). The polymerization mechanism and full characterization of SS-hPG are described as this synthesis is reported for the first time. Rheology is used to ascertain the hydrogel's mechanical characteristics, such as stiffness, and self-healing, determining these properties for different ratios and concentrations of both gel components. The incorporation of disulfide bonds in the hydrogel is proved by conducting degradation experiments in reductive environments. Fluorescein isothiocyanate-albumin (FITC-BSA) and vancomycin both are loaded into the gel, and the guest release kinetics is assessed for both slow and on-demand releases. Finally, the in vitro and in vivo experiments prove that the vancomycin-loaded hydrogel acts as an antibacterial barrier for wound dressing and accelerates the healing of infectious wounds in a mouse model.

Supramolecular Architectures of Dendritic Polymers Provide Irreversible Inhibitor to Block Viral Infection.

In Nature, most known objects can perform their functions only when in supramolecular self-assembled from, e.g. protein complexes and cell membranes. Here, a dendritic polymer is presented that inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an irreversible (virucidal) mechanism only when self-assembled into a Two-dimmensional supramolecular polymer (2D-SupraPol). Monomeric analogs of the dendritic polymer can only inhibit SARS-CoV-2 reversibly, thus allowing for the virus to regain infectivity after dilution. Upon assembly, 2D-SupraPol shows a remarkable half-inhibitory concentration (IC50 30 nM) in vitro and in vivo in a Syrian Hamster model has a good efficacy. Using cryo-TEM, it is shown that the 2D-SupraPol has a controllable lateral size that can be tuned by adjusting the pH and use small angle X-ray and neutron scattering to unveil the architecture of the supramolecular assembly. This functional 2D-SupraPol, and its supramolecular architecture are proposed, as a prophylaxis nasal spray to inhibit the virus interaction with the respiratory tract.

Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.

In nature, bacteria frequently reside in differentiated communities or biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.

Pathways of transport protein evolution: recent advances.

We herein report recent advances in our understanding of transport protein evolution. Numerous families of complex transmembrane transport proteins are believed to have arisen from short channel-forming amphipathic or hydrophobic peptides by various types of intragenic duplication events. Distinct pathways distinguish families, demonstrating independent origins for some, and allowing assignment of others to superfamilies. Some families have diversified in topology, whereas others have remained uniform. An example of 'retroevolution' was discovered where a more complex carrier gave rise to a structurally and functionally simpler channel. The results described in this review article expand our understanding of protein evolution.

Emerging Roles of Functional Bacterial Amyloids in Gene Regulation, Toxicity, and Immunomodulation.

Bacteria often reside in multicellular communities, called biofilms, held together by an extracellular matrix. In many bacteria, the major proteinaceous component of the biofilm are amyloid fibers. Amyloids are highly stable and structured protein aggregates which were known mostly to be associated with neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases. In recent years, microbial amyloids were identified also in other species and shown to play major roles in microbial physiology and virulence. For example, amyloid fibers assemble on the bacterial cell surface as a part of the extracellular matrix and are extremely important to the scaffolding and structural integrity of biofilms, which contribute to microbial resilience and resistance. Furthermore, microbial amyloids play fundamental nonscaffold roles that contribute to the development of biofilms underlying numerous persistent infections. Here, we review several nonscaffold roles of bacterial amyloid proteins, including bridging cells during collective migration, acting as regulators of cell fate, as toxins against other bacteria or against host immune cells, and as modulators of the hosts' immune system. These overall points on the complexity of the amyloid fold in encoding numerous activities, which offer approaches for the development of a novel repertoire of antivirulence therapeutics.

Cyclic-di-GMP signalling and biofilm-related properties of the Shiga toxin-producing 2011 German outbreak Escherichia coli O104:H4.

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

'Life-style' control networks in Escherichia coli: signaling by the second messenger c-di-GMP.

Most bacteria can exist in either a planktonic-motile single-cell state or an adhesive multicellular state known as a biofilm. Biofilms cause medical problems and technical damage since they are resistant against antibiotics, disinfectants or the attacks of the immune system. In recent years it has become clear that most bacteria use cyclic diguanylate (c-di-GMP) as a biofilm-promoting second messenger molecule. C-di-GMP is produced by GGDEF-domain-containing diguanylate cyclases and is degraded by phosphodiesterases featuring EAL or HD-GYP domains. Many bacterial species possess multiple proteins with GGDEF and EAL domains, which actually belong to the most abundant protein families in genomic data bases. Via an unprecedented variety of effector components, which include c-di-GMP-binding proteins as well as RNAs, c-di-GMP controls a wide range of targets that down-regulate motility, stimulate adhesin and biofilm matrix formation or even control virulence gene expression. Moreover, local c-di-GMP signaling in macromolecular complexes seems to allow the independent and parallel control of different output reactions. In this review, we use Escherichia coli as a paradigm for c-di-GMP signaling. Despite the huge diversity of components and molecular processes involved in biofilm formation throughout the bacterial kingdom, c-di-GMP signaling represents a unifying principle, which suggests that the enzymes that make and break c-di-GMP may be promising targets for anti-biofilm drugs.