Bacterial biopolymers: from pathogenesis to advanced materials

Abstract

Bacteria are prime cell factories that can efficiently convert carbon and nitrogen sources into a large diversity of intracellular and extracellular biopolymers, such as polysaccharides, polyamides, polyesters, polyphosphates, extracellular DNA and proteinaceous components. Bacterial polymers have important roles in pathogenicity, and their varied chemical and material properties make them suitable for medical and industrial applications. The same biopolymers when produced by pathogenic bacteria function as major virulence factors, whereas when they are produced by non-pathogenic bacteria, they become food ingredients or biomaterials. Interdisciplinary research has shed light on the molecular mechanisms of bacterial polymer synthesis, identified new targets for antibacterial drugs and informed synthetic biology approaches to design and manufacture innovative materials. This Review summarizes the role of bacterial polymers in pathogenesis, their synthesis and their material properties as well as approaches to design cell factories for production of tailor-made bio-based materials suitable for high-value applications.

Fig. 1. Bacterial biopolymers and their functions.

Bacteria can survive in diverse ecosystems and infect a variety of living organisms. When produced by bacterial pathogens, secreted biopolymers can function as virulence factors, whereas intracellular polymers are mainly reserve materials that increase survival during starvation. The switch from motility to sessility of bacterial pathogens is a strategic decision that is often connected with the production of exopolysaccharides. Pathogens benefit from the production of high molecular weight polysaccharides as they are an integral part of the biofilm matrix and interact with counterions and other polymers to form a hydrogel-like niche^,. Furthermore, they protect embedded bacterial cells from environmental stresses, the immune systems and antimicrobial treatment. This lifestyle transition underlies the establishment of many chronic and hard to eradicate infections. Capsular polysaccharides are attached to the cell surface and protect the pathogen from phagocytosis and antimicrobial drugs. Glycogen is an intracellular storage polysaccharide that promotes the survival of some pathogens during the intracellular phase of infection. Polyhydroxyalkanoates (PHAs) are highly reduced biopolyesters that function as storage compounds that increase bacterial fitness and potentially function as an electron sink in anaerobic zones of biofilms^,. PHA-metabolizing enzymes are produced under specific nutritional and environmental stresses to enhance bacterial survival. Polyamides function as bacterial capsules or slimes to protect cells or as intracellular storage material. Bacillus anthracis, which can cause lethal infections, produces such a capsule. Polyphosphates (polyPs) are chains of condensed phosphates that function as a storage material with high energy-rich bonds. The metabolism of polyP is positively correlated with the production of virulence factors^,. Extracellular DNA (eDNA) mediates the surface adhesion of cells and stabilizes the biofilm matrix through interaction with other secreted polymers and cations. Proteinaceous components such as fimbriae, pili and flagella are extracellular self-assembling nanostructures that contribute to surface attachment, the formation of the biofilm matrix and/or bacterial motility.

Fig. 2. Bacterial polysaccharides as biomaterials and their properties.

High molecular weight exopolysaccharides, such as alginate, cellulose and hyaluronate, are well-known virulence factors constituting the biofilm matrix. The interaction of polysaccharides and other polymeric substances can determine the properties of the biofilm matrix. Bacterial polysaccharides are very diverse, and their diversity and material properties are determined by the constituent sugars or sugar acids, the type of glycosidic linkages and whether they are unbranched or branched, the length of the polymer (and thus the molecular weight), the type of side group (for example, acetyl, pyruvate or succinate) and the degree of substitution^,. Bacterial polysaccharides are important biomaterials due to their unique material properties, including solubility, rheological characteristics, viscoelastic properties, interaction with cations, ionic strength, crosslinking, gelation, water retention, extendibility and stability under different conditions. Hence, polysaccharides have been applied as natural viscosifiers, thickeners, stabilizers, gel and film formers, and additives or have been processed into nanostructures (for example, nanoparticles and nanotubes), microspheres, microcapsules, sponges, hydrogels, foams, elastomers and fibres^,,. Besides the desired material properties, high purity and the purification process are crucial for the use of bacterial polysaccharides as high-value biomaterials^,. d-Glc, d-glucose; d-GlcA, d-glucuronic acid; GlcNAc, N-acetylglucosamine; l-GulA, l-guluronic acid; d-ManA, d-mannuronic acid.

Fig. 3. Bacterial polymer granules as biomaterials.

a | Polyamides are composed of amino acids and are non-ribosomally synthesized by specific synthetases. They are found as intracellular granules without confining membranes and decorating proteins or as secreted extracellular capsules and slimes^,. Due to their biodegradability, non-toxicity and modifiability, bacterial polyamides have been considered as substitutes for chemically synthesized polymers that can be processed into formulations for industrial, biomedical, pharmaceutical and cosmetic applications. b | Polyhydroxyalkanoates (PHAs), such as polyhydroxybutyrate, are natural polyesters that are synthesized into hydrophobic spherical inclusions from (R)-3-hydroxybutyric acid. PHAs have been classified into short-chain-length PHAs (PHA_SCL; containing constituents with 3–5 carbon atoms) and medium-chain-length PHAs (PHA_MCL; containing constituents with 6–14 carbon atoms), which are primarily produced by pseudomonads. Synthases and other PHA-binding proteins decorate the surface of PHA inclusions. PHAs are unique bio-based materials processed as bioplastics or bioengineered functionalized nanoparticles for uses in medicine and industry. PHA nanobeads can function as effective platforms for enzyme immobilization, protein purification, bioseparation, drug or vaccine delivery, tissue engineering, diagnostics and imaging. c | Polyphosphates are composed of orthophosphates (inorganic phosphates, three to several hundred phosphates) linked by phosphoanhydride (P–O–P) bonds. They contribute to energy storage and can be processed into hydrogels or nanoparticles for various applications (Table 1). The phosphate and counterions such as Ca²⁺ and Sr²⁺ are released on hydrolysis and can be used for bone biomineralization, as smart bioinks for generating 3D scaffolds and for cell bioprinting of regeneratively active patient-specific osteoarticular implants^–,. Polyphosphate or collagen hydrogels were formulated for improving tissue integration of meshes to improve the outcome of surgical hernia repair.

Fig. 4. The main metabolic routes for the synthesis of bacterial biopolymers.

Intermediates of central metabolism are diverted towards the provision of precursors for polymer synthesis. Four general mechanisms for the production of polysaccharides in bacteria are shown. Synthesis of some secreted non-repeating polysaccharides, such as alginate and cellulose, is mediated by multiprotein complexes, usually consisting of a polymerase, a copolymerase, carbohydrate-modifying enzymes and secretion subunits. The genes encoding such functionally related protein subunits are co-clustered in large operons, such as the alg and bcs operons. Some polysaccharides, such as xanthan, are produced through the Wzy-dependent polysaccharide synthesis mechanism. In this pathway the repeating sugar units and their linked lipid carriers are assembled by several glycosyltransferases at the cytoplasmic membrane, followed by flipping across the cytoplasmic membrane, the final polymerization step in the periplasm and secretion. However, the synthesis of some polysaccharides, such as hyaluronate, dextrans and levans, is less complex and is mediated by a single enzyme. Dextrans and levans are synthesized outside the cell by sugar transferases that convert disaccharides into polysaccharides and use the energy that is released by hydrolysis of the glycosidic bond of the disaccharides. Modification of secreted polysaccharides (for example, acetylation, deacetylation, epimerization and phosphoethanolamine (pEtN) addition) can occur during translocation of nascent polymers across the cell envelope. Polyhydroxyalkanoates (for example, polyhydroxybutyrate (PHB)) are synthesized by a polyhydroxyalkanoate synthase that coverts hydroxyacyl-CoA derivatives of central metabolism into intracellular polyesters. Enzymatic processes independent of ribosomal protein biosynthesis synthesize polyamides. Dashed lines indicate multiple enzymatic steps, a circled plus sign indicates positive correlation and a circled minus sign indicates negative correlation. ABC, ATP-binding cassette; CPS, capsular polysaccharide; FA, fatty acid de novo biosynthesis; Fru-6-P, fructose 6-phosphate; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Glc-1-P, glucose 1-phosphate; Glc-6-P, glucose 6-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate pathway; LPS, lipopolysaccharide; ManA, mannuronic acid; polyP, polyphosphate; RBP, RNA-binding protein; SM, second messenger; sRNA small non-coding RNA; TCA, tricarboxylic acid; TCS, two-component system; TF, transcription factor.

Fig. 5. Production of novel enhanced biopolymers and biopolymer synthesis as a target for drug discovery.

Production of novel biopolymers can be achieved by synthetic biology for the development of cell factories. In vitro enzymatic synthesis or modification of biopolymers as well as chemical modifications can achieve novel biopolymers with altered material properties and functions. Molecular biology and biochemical or biophysical approaches have provided insight into biosynthesis pathways of bacterial biopolymers. Selective inhibition of biopolymers that function as virulence factors offers targets for antimicrobial drug discovery. Systems biology, synthetic biology and metabolic engineering tools have accelerated the construction of novel cell factories for the production of novel bio-based materials. PA, polyamide; pEtN, phosphoethanolamine; PHA, polyhydroxyalkanoate; polyP, polyphosphate.