Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

Recent advances on biomedical applications of gellan gum: A review.

Gellan gum (GG) has attracted considerable attention as a versatile biopolymer with numerous potential biological applications, especially in the fields of tissue engineering, wound healing, and cargo delivery. Due to its distinctive characteristics like biocompatibility, biodegradability, nontoxicity, and gel-forming ability, GG is well-suited for these applications. This review focuses on recent research on GG-based hydrogels and biocomposites and their biomedical applications. It discusses the incorporation of GG into hydrogels for controlled drug release, its role in promoting wound healing processes, and its potential in tissue engineering for various tissues including bone, retina, cartilage, vascular, adipose, and cardiac tissue. It provides an in-depth analysis of the latest findings and advancements in these areas, making it a valuable resource for researchers and professionals in these fields.

Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Biosynthesis of Cytidine Diphosphate-6-d-Glucitol for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C. jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C. jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C. jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Depolymerisation of the Klebsiella pneumoniae Capsular Polysaccharide K21 by Klebsiella Phage K5.

Klebsiella pneumoniae is a pathogen associated with various infection types, which often exhibits multiple antibiotic resistance. Phages, or bacterial viruses, have an ability to specifically target and destroy K. pneumoniae, offering a potential means of combatting multidrug-resistant infections. Phage enzymes are another promising therapeutic agent that can break down bacterial capsular polysaccharide, which shields K. pneumoniae from the immune response and external factors. In this study, Klebsiella phage K5 was isolated; this phage is active against Klebsiella pneumoniae with the capsular type K21. It was demonstrated that the phage can effectively lyse the host culture. The adsorption apparatus of the phage has revealed two receptor-binding proteins (RBPs) with predicted polysaccharide depolymerising activity. A recombinant form of both RBPs was obtained and experiments showed that one of them depolymerised the capsular polysaccharide K21. The structure of this polysaccharide and its degradation fragments were analysed. The second receptor-binding protein showed no activity on capsular polysaccharide of any of the 31 capsule types tested, so the substrate for this enzyme remains to be determined in the future. Klebsiella phage K5 may be considered a useful agent against Klebsiella infections.

Cell wall polysaccharides of streptococci: A genetic and structural perspective.

The Streptococcus genus comprises both commensal and pathogenic species. Additionally, Streptococcus thermophilus is exploited in fermented foods and in probiotic preparations. The ecological and metabolic diversity of members of this genus is matched by the complex range of cell wall polysaccharides that they present on their cell surfaces. These glycopolymers facilitate their interactions and environmental adaptation. Here, current knowledge on the genetic and compositional diversity of streptococcal cell wall polysaccharides including rhamnose-glucose polysaccharides, exopolysaccharides and teichoic acids is discussed. Furthermore, the species-specific cell wall polysaccharide combinations and specifically highlighting the presence of rhamnose-glucose polysaccharides in certain species, which are replaced by teichoic acids in other species. This review highlights model pathogenic and non-pathogenic species for which there is considerable information regarding cell wall polysaccharide composition, structure and genetic information. These serve as foundations to predict and focus research efforts in other streptococcal species for which such data currently does not exist.

The anticancer mechanisms of exopolysaccharide from Weissella cibaria D-2 on colorectal cancer via apoptosis induction.

Exopolysaccharide (EPS) from Weissella cibaria has been devoted to the study of food industry. However, the anticancer activity of W. cibaria derived EPS has not yet been investigated. In this study, we obtained the EPS from W. cibaria D-2 isolated from the feces of healthy infants and found that D-2-EPS, a homopolysaccharide with porous web like structure, could effectively inhibit the proliferation, migration, invasion and induce cell cycle arrest in G0/G1 phase of colorectal cancer (CRC) cells. In HT-29 tumor xenografts, D-2-EPS significantly retarded tumor growth without obvious cytotoxicity to normal organs. Furthermore, we revealed that D-2-EPS promoted the apoptosis of CRC cells by increasing the levels of Fas, FasL and activating Caspase-8/Caspase-3, indicating that D-2-EPS might induce apoptosis through the extrinsic Fas/FasL pathway. Taken together, the D-2-EPS has the potential to be developed as a nutraceutical or drug to prevent and treat colorectal cancer.

Pseudomonas aeruginosa biofilm exopolysaccharides: assembly, function, and degradation.

The biofilm matrix is a fortress; sheltering bacteria in a protective and nourishing barrier that allows for growth and adaptation to various surroundings. A variety of different components are found within the matrix including water, lipids, proteins, extracellular DNA, RNA, membrane vesicles, phages, and exopolysaccharides. As part of its biofilm matrix, Pseudomonas aeruginosa is genetically capable of producing three chemically distinct exopolysaccharides - alginate, Pel, and Psl - each of which has a distinct role in biofilm formation and immune evasion during infection. The polymers are produced by highly conserved mechanisms of secretion, involving many proteins that span both the inner and outer bacterial membranes. Experimentally determined structures, predictive modelling of proteins whose structures are yet to be solved, and structural homology comparisons give us insight into the molecular mechanisms of these secretion systems, from polymer synthesis to modification and export. Here, we review recent advances that enhance our understanding of P. aeruginosa multiprotein exopolysaccharide biosynthetic complexes, and how the glycoside hydrolases/lyases within these systems have been commandeered for antimicrobial applications.

Extracellular DNA enhances biofilm integrity and mechanical properties of mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). P. aeruginosa becomes tolerant to antimicrobials in the biofilm state and is difficult to treat. Production of extracellular polymeric substances (EPS), such as alginate and extracellular DNA (eDNA), can allow adherence to abiotic and biotic surfaces, antimicrobial evasion, and resilience to environmental pressures. Alginate-producing mucoid variants of P. aeruginosa are frequently isolated from CF airway samples and are associated with worsening patient outcomes. While eDNA is a major structural component of nonmucoid P. aeruginosa biofilms, the potential role of eDNA in mucoid biofilms is unclear. Here, we investigate how eDNA contributes to clinical mucoid biofilm physiology and integrity. We predicted that eDNA plays a structural and mechanical role in mucoid biofilms. To test this, we quantified biofilm eDNA in mucoid biofilms and used microscopy and rheology to visualize eDNA and detect changes in biofilm structure and mechanics upon DNaseI treatment. We showed that biofilm eDNA abundance is diverse across clinical mucoid strains and observed a temporal increase in foci of eDNA within intact mucoid biofilms. Increased cell dispersal and reduced biomass were also observed following DNaseI treatment of mucoid biofilms. Degradation of eDNA also impacted the mechanical integrity of mucoid biofilms by increasing the stiffness and decreasing the cohesion of the biofilm. These findings advance our understanding of clinical mucoid P. aeruginosa biofilms and facilitate the development of new approaches to target biofilms by exploiting the functions of EPS components. IMPORTANCE Understanding the role of eDNA in mucoid Pseudomonas aeruginosa biofilms will lead to therapeutic strategies that combat the biophysical and structural function of EPS for the eradication of bacteria in mucoid biofilms during chronic infections. This knowledge can be used to further identify unknown matrix component interactions within pathogenic biofilm-forming clinical isolates.

Sources and methods of manufacturing xanthan by fermentation of various carbon sources.

Xanthan gum, an anionic polysaccharide with an exceptionally high molecular weight, is produced by the bacterium Xanthomonas sp. It is a versatile compound that has been utilized in various industries for decades. Xanthan gum was the second exopolysaccharide to be commercially produced, following dextran. In 1969, the US Food and Drug Administration (FDA) approved xanthan gum for use in the food and pharmaceutical industries. The food industry values xanthan gum for its exceptional rheological properties, which make it a popular thickening agent in many products. Meanwhile, the cosmetics industry capitalizes on xanthan gum's ability to form stable emulsions. The industrial production process of xanthan gum involves fermenting Xanthomonas in a medium that contains glucose, sucrose, starch, etc. as a substrate and other necessary nutrients to facilitate growth. This is achieved through batch fermentation under optimal conditions. However, the increasing costs of glucose in recent years have made the production of xanthan economically unviable. Therefore, many researchers have investigated alternative, cost-effective substrates for xanthan production, using various modified and unmodified raw materials. The objective of this analysis is to investigate how utilizing different raw materials can improve the cost-efficient production of xanthan gum.

Distinct types of multicellular aggregates in Pseudomonas aeruginosa liquid cultures.

Pseudomonas aeruginosa forms suspended multicellular aggregates when cultured in liquid media. These aggregates may be important in disease, and/or as a pathway to biofilm formation. The polysaccharide Psl and extracellular DNA (eDNA) have both been implicated in aggregation, but previous results depend strongly on the experimental conditions. Here we develop a quantitative microscopy-based method for assessing changes in the size distribution of suspended aggregates over time in growing cultures. For exponentially growing cultures of P. aeruginosa PAO1, we find that aggregation is mediated by cell-associated Psl, rather than by either eDNA or secreted Psl. These aggregates arise de novo within the culture via a growth process that involves both collisions and clonal growth, and Psl non-producing cells do not aggregate with producers. In contrast, we find that stationary phase (overnight) cultures contain a different type of multicellular aggregate, in which both eDNA and Psl mediate cohesion. Our findings suggest that the physical and biological properties of multicellular aggregates may be very different in early-stage vs late-stage bacterial cultures.

Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria.

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.

Lactic acid bacteria-derived exopolysaccharide: Formation, immunomodulatory ability, health effects, and structure-function relationship.

Exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) have implications for host health and act as food ingredients. Due to the variability of LAB-EPS (lactic acid bacteria-derived exopolysaccharide) gene clusters, especially the glycosyltransferase genes that determine monosaccharide composition, the structure of EPS is very rich. EPSs are synthesized by LAB through the extracellular synthesis pathway and the Wzx/Wzy-dependent pathway. LAB-EPS has a strong immunomodulatory ability. The EPSs produced by different genera of LAB, especially Lactobacillus, Leuconostoc, and Streptococcus, have different immunomodulatory abilities because of their specific structures. LAB-EPS possesses other health effects, including antitumor, antioxidant, intestinal barrier repair, antimicrobial, antiviral, and cholesterol-lowering activities. The bioactivities of LAB-EPS are tightly related to their structures such us monosaccharide composition, glycosidic bonds, and molecular weight (MW). For the excellent physicochemical property, LAB-EPS acts as product improvers in dairy, bakery food, and meat in terms of stability, emulsification, thickening, and gelling. We systematically summarize the detailed process of EPS from synthesis to application, with emphasis on physiological mechanisms of EPS, and specific structure-function relationship, which provides theoretical support for the potential commercial value in the pharmaceutical, chemical, food, and cosmetic industries.

Nisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2.

Glycosyltransferases synthesize a variety of exopolysaccharides (EPS) with different properties by altering the type of glycosidic linkage, degree of branching, length, mass, and conformation of the polymers. The genome analysis of an EPS-producing, Lactobacillus plantarum BR2 (Accession No: MN176402) showed twelve glycosyltransferase genes, and the gene BR2gtf (1116 bp), annotated as an EPS biosynthetic glycosyltransferase was cloned into the pNZ8148 vector. The recombinant pNZ8148 vector along with pNZ9530, a regulatory plasmid, were electroporated to L. plantarum BR2 for the over-expression of gtf gene under a nisin-controlled expression system and the glycosyltransferase activity of the recombinant and the wild-type strains were analysed. The recombinant strain showed 54.4% increase in EPS production with the maximum EPS production of 23.2 ± 0.5 g/L in a 5 L bioreactor study after 72 h of fermentation. This study shows an effective molecular strategy possibly to be adopted in lactic acid bacteria to enhance exopolysaccharide production.

Relationship of Gene-Structure-Antioxidant Ability of Exopolysaccharides Derived from Lactic Acid Bacteria: A Review.

Polysaccharides derived from lactic acid bacteria (LAB) have widespread industrial applications owing to their excellent safety profile and numerous biological properties. The antioxidant activity of exopolysaccharides (EPS) offers defense against disease conditions caused by oxidative stress. Several genes and gene clusters are involved in the biosynthesis of EPS and the determination of their structures, which play an important role in modulating their antioxidant ability. Under conditions of oxidative stress, EPS are involved in the activation of the nonenzyme (Keap1-Nrf2-ARE) response pathway and enzyme antioxidant system. The antioxidant activity of EPS is further enhanced by the targeted alteration of their structures, as well as by chemical methods. Enzymatic modification is the most commonly used method, though physical and biomolecular methods are also frequently used. A detailed summary of the biosynthetic processes, antioxidant mechanisms, and modifications of LAB-derived EPS is presented in this paper, and their gene-structure-function relationship has also been explored.

The divisome but not the elongasome organizes capsule synthesis in Streptococcus pneumoniae.

The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host.

Interlink between ExoD (Alr2882), exopolysaccharide synthesis and metal tolerance in Nostoc sp. strain PCC 7120: Insight into its role, paralogs and evolution.

Exopolysaccharides (EPS) produced by bacterial species are an important component of bacteria's survival strategy. Synthesis of EPS, principal component of extracellular polymeric substance, occurs through multiple pathways involving multitude of genes. While stress-induced concomitant increase in exoD transcript levels and EPS content have been shown earlier, experimental evidence for direct correlation is lacking. In the present study, role of ExoD in Nostoc sp. strain PCC 7120 was evaluated by generating a recombinant Nostoc strain AnexoD+, wherein the ExoD (Alr2882) protein was constitutively overexpressed. AnexoD+ exhibited higher EPS production, propensity for formation of biofilms and tolerance to Cd stress compared to vector control AnpAM cells. Both Alr2882 and its paralog All1787 exhibited 5 transmembrane domains, with only All1787 predicted to interact with several proteins in polysaccharide synthesis. Phylogenetic analysis of orthologs of these proteins across cyanobacteria indicated that the two paralogs Alr2882 and All1787 and their corresponding orthologs arose divergently during evolution, and could have distinct roles to perform in the biosynthesis of EPS. This study has thrown open the possibility of engineering overproduction of EPS and inducing biofilm formation through genetic manipulation of EPS biosynthesis genes in cyanobacteria, thus building a cost-effective green platform for large scale production of EPS.