Lipopolysaccharide Structure Research Articles

Effect of bacterial dissociation on lipopolysaccharide structure: A study of O-polysaccharide from the marine bacterium Pseudoalteromonas agarivorans KMM 232 (O-form)

The lipopolysaccharide (LPS) was obtained from a bacterium Pseudoalteromonas agarivorans KMM 232 (O-form) isolated from a seawater sample collected at a depth of 500 m. The O-polysaccharide (OPS) was isolated by mild acid degradation of the LPS and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY and 1H,13C HSQC, and 1H,13C HMBC experiments. The following new structure of the OPS from P. agarivorans KMM 232 (O-form) containing 2-acetamido-2-deoxy-d-glucose (D-GlcNAc), d-glucose (D-Glc), d-glucuronic acid (D-GlcA), 4,6-O-[(R)-1-carboxyethylidene]-d-galactose [D-Galp4,6 (R-Pyr)] and two residues of d-galactose (D-Gal) was established:

Exopolysaccharide is detrimental for the symbiotic performance of Sinorhizobium fredii HH103 mutants with a truncated lipopolysaccharide core.

The nitrogen-fixing rhizobia-legume symbiosis relies on a complex interchange of molecular signals between the two partners during the whole interaction. On the bacterial side, different surface polysaccharides, such as lipopolysaccharide (LPS) and exopolysaccharide (EPS), might play important roles for the success of the interaction. In a previous work we studied two Sinorhizobium fredii HH103 mutants affected in the rkpK and lpsL genes, which are responsible for the production of glucuronic acid and galacturonic acid, respectively. Both mutants produced an altered LPS, and the rkpK mutant, in addition, lacked EPS. These mutants were differently affected in symbiosis with Glycine max and Vigna unguiculata, with the lpsL mutant showing a stronger impairment than the rkpK mutant. In the present work we have further investigated the LPS structure and the symbiotic abilities of the HH103 lpsL and rkpK mutants. We demonstrate that both strains produce the same LPS, with a truncated core oligosaccharide devoid of uronic acids. We show that the symbiotic performance of the lpsL mutant with Macroptilium atropurpureum and Glycyrrhiza uralensis is worse than that of the rkpK mutant. Introduction of an exoA mutation (which avoids EPS production) in HH103 lpsL improved its symbiotic performance with G. max, M. atropurpureum, and G. uralensis to the level exhibited by HH103 rkpK, suggesting that the presence of EPS might hide the truncated LPS produced by the former mutant.

The human gut Bacteroides eggerthii expresses a new galactofuranose-containing lipooligosaccharide with weak immunostimulatory properties

Lipopolysaccharides (LPS) decorating the cell surface of Gram-negative bacteria exhibit nuanced functionalities linked to their precise structural composition. However, despite their critical role in health and disease, information on the structure and function of LPS from members of the human gut microbiota is still limited. Here, we deciphered the complete structure of the LPS isolated from the human gut bacterium Bacteroides eggerthii 1_2_48FAA. We showed that B. eggerthii 1_2_48FAA produces an R-type LPS (or lipooligosaccharide, LOS) composed of a heterogeneous mixture of tetra- and penta-acylated lipid A species with different degree of phosphorylation, and a compact galactofuranose-containing core oligosaccharide. Using in vitro human cell lines, we showed that B. eggerthii 1_2_48FAA LOS acts as a weak activator of TLR4-mediated signaling. Moreover, we observed that expression of maturation markers CD40, CD80 and CD86 on monocytes-derived dendritic cells upon B. eggerthii 1_2_48FAA LOS exposure was significantly lower compared to pro-inflammatory Escherichia coli LPS. Taken together, these data provide new structural and biological insights into LPS from gut bacteria, underscoring the importance of structural features in modulating host immunity.

Colanic acid and lipopolysaccharide in Pectobacterium carotovorum Pcc21 serve as receptors for the bacteriophage phiPccP-2

Bacteriophages (phages) are viruses that specifically bind to and infect target bacteria. The phage phiPccP-2, belonging to the Myoviridae family, efficiently controls Pectobacterium spp. In the present study, we aimed to elucidate the mechanism of recognition of P. carotovorum Pcc21 by phiPccP-2. The EZ-Tn5 transposon mutant library of Pcc21 was used to screen for phage-resistant mutants. Among 4,072 mutants screened, 12 harbored disruptions in genes associated with the biosynthesis of either colanic acid (CA) or lipopolysaccharide (LPS) showed resistance to phiPccP-2. Complementation of 4 representative phage-resistant mutants with the corresponding genes fully restored the binding ability and lytic activity of PhiPccP-2. The amounts of CA or LPS structure in these mutants were significantly altered compared with those in the wild-type strain. Adsorption competition assays between CA and LPS extracted from Pcc21 and the natural receptors in Pcc21 showed that unbound phages were significantly increased, indicating that both CA and LPS are associated with the adsorption of the phiPccP-2 to Pcc21. In contrast, the adsorption of phiPccP-2 to extracted CA or LPS did not inactivate the lytic activity of phiPccP-2, indicating that the adsorption to the extracted CA or LPS is not sufficient for DNA injection. Treatment with polymyxin B, which disrupts LPS, interfered with phiPccP-2 adsorption to Pcc21. Furthermore, phage-resistant mutants showed reduced virulence in the host plant, suggesting a trade-off between phage resistance and bacterial virulence. Overall, our results indicate that both CA and LPS serve as receptors for the binding of phiPccP-2 to P. carotovorum Pcc21.

Determination of Initial Rates of Lipopolysaccharide Transport.

Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min-1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.

Heterogeneity of Salmonella enterica lipopolysaccharide counteracts macrophage and antimicrobial peptide defenses.

Salmonella enterica is comprised of over 2,500 serovars, in which non-typhoidal serovars (NTS), Enteritidis (SE), and Typhimurium (STM) are the most clinically associated with human infections. Although NTS have similar genetic elements to cause disease, phenotypic variation including differences in lipopolysaccharide (LPS) composition may control immune evasion. Here, we demonstrate that macrophage host defenses and LL-37 antimicrobial efficacy against SE and STM are substantially altered by LPS heterogeneity. We found that SE evades macrophage killing by inhibiting phagocytosis while STM survives better intracellularly post-phagocytosis. SE-infected macrophages failed to activate the inflammasomes and subsequently produced less interleukin-1β (IL-1β), IL-18, and interferon λ. Inactivation of LPS biosynthesis genes altered LPS composition, and the SE LPS-altered mutants could no longer inhibit phagocytosis, inflammasome activation, and type II interferon signaling. In addition, SE and STM showed differential susceptibility to the antimicrobials LL-37 and colistin, and alteration of LPS structure substantially increased susceptibility to these molecules. Collectively, our findings highlight that modification of LPS composition by Salmonella increases resistance to host defenses and antibiotics.

Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability.

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.

Immunobiological effects of lipopolysaccharide derived from Helicobacter pylori and influence of a proton pump inhibitor lansoprazole on human polymorphonuclear leukocytes.

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs.

Influence of chitosanon on the ability of LPS to interact with cells of the immune system

Complexes of lipopolysaccharide (LPS) from the bacterium Escherichia coli and chitosan (CN) with a molecular weight of 5 kDa were obtained and their supramolecular organization was studied. Using atomic force microscopy, it was shown that during the formation of complexes there is a transition from the micellar structure of the original LPS to linear network structures uniformly distributed over the surface of mica. The stability of LPS-CN complexes of various stoichiometries in biological media in the presence of serum proteins was investigated. It was shown that complexes with an LPS:CN ratio of 1:1 in the presence of serum proteins lost their surface charge and tended to aggregate; while complexes with maximum saturation of CN (1:5) did not aggregate under these conditions and maintained their surface charge. The effect of CNs of different molecular weights on the ability of LPS to interact with neutrophils in human whole blood was studied. It was observed that LPS-CN complexes were capable of binding to neutrophils and entering the cell, and this ability was enhanced in the presence of serum proteins. Chitosan exhibited the ability to suppress the synthesis of the proinflammatory cytokine TNF-α, induced by LPS, not only as part of the complex but also when cells were pretreated with a polycation.

The lipopolysaccharide structure affects the detoxifying ability of intestinal alkaline phosphatases

Lipopolysaccharide (LPS) is one of the most potent mediators of inflammation. In swine husbandry, weaning is associated with LPS-induced intestinal inflammation, resulting in decreased growth rates due to malabsorption of nutrients by the inflamed gut. A potential strategy to treat LPS-mediated disease is administering intestinal alkaline phosphatase (IAP). The latter can detoxify lipid A, the toxic component of LPS, by removal of phosphate groups. Currently, 183 LPS O-serotypes from E. coli have been described, however, comparative experiments to elucidate functional differences between LPS serotypes are scarce. In addition, these functional differences might affect the efficacy of LPS detoxifying enzymes. Here, we evaluated the ability of four LPS serotypes (O26:B6, O55:B5, O111:B4 and O127:B8) derived from Escherichia coli to trigger the secretion of pro-inflammatory cytokines by porcine PBMCs. We also tested the ability of three commercially available IAPs to detoxify these LPS serotypes. The results show that LPS serotypes differ in their ability to trigger cytokine secretion by immune cells, especially at lower concentrations. Moreover, IAPs displayed a different detoxification efficiency of the tested serotypes. Together, this study sheds light on the impact of LPS structure on the detoxification by IAPs. Further research is however needed to elucidate the LPS serotype-specific effects and their implications for the development of novel treatment options to alleviate LPS-induced gut inflammation in weaned piglets.

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Investigating the mechanism of rough phenotype in a naturally attenuated Brucella strain: insights from whole genome sequencing.

Brucellosis, a significant zoonotic disease, not only impacts animal health but also profoundly influences the host immune responses through gut microbiome. Our research focuses on whole genome sequencing and comparative genomic analysis of these Brucella strains to understand the mechanisms of their virulence changes that may deepen our comprehension of the host immune dysregulation. The Brucella melitensis strain CMCC55210 and its naturally attenuated variant CMCC55210a were used as models. Biochemical identification tests and in vivo experiments in mice verified the characteristics of the strain. To understand the mechanism of attenuation, we then performed de novo sequencing of these two strains. We discovered notable genomic differences between the two strains, with a key single nucleotide polymorphism (SNP) mutation in the manB gene potentially altering lipopolysaccharide (LPS) structure and influencing host immunity to the pathogen. This mutation might contribute to the attenuated strain's altered impact on the host's macrophage immune response, overing insights into the mechanisms of immune dysregulation linked to intracellular survival. Furthermore, we explore that manipulating the Type I restriction-modification system in Brucella can significantly impact its genome stability with the DNA damage response, consequently affecting the host's immune system. This study not only contributes to understanding the complex relationship between pathogens, and the immune system but also opens avenues for innovative therapeutic interventions in inflammatory diseases driven by microbial and immune dysregulation.

Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-β-d-Fucf-(1→3)-β-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-β-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

There is no association between serum endotoxin levels and inflammation in asthma

Backround Endotoxin, in lipopolysaccharide structure (LPS), is the main component of the outer membrane of gram negative bacteria. LPS levels were associated with inflammatory disease. Asthma is a chronic inflammatory disease involving many different cell types and cellular elements. The association between LPS serum levels and the asthma is not well known. The aim of this study was to investigate the association between the LPS serum levels and the severity of asthma, demographic data and laboratory parameters. Methodology The study included 67 patients aged >18 years with a diagnosis of asthma, and 15 healthy volunteers with no history of chronic disease as a control group. The Asthma Control Test (ACT), Respiratory Function Tests (RFTs), fractional exhaled nitric oxide (FeNO), and endotoxin levels were measured and compared between the groups. The endotoxin measurements were performed using the ELISA method. Results The mild-moderate asthma group included 33 patients and the severe asthma group, 34 patients. The endotoxin level was measured as 17.78 (range 3.59 to 304.55) EU/ml in the patient group and 15 (range 4.01 to 74.06) EU/ml in the control group with no statistically significant difference determined between the groups. In the subgroups, the endotoxin level was measured as 15.21 (range 3.69 to 304.55) EU/ml in the mild-moderate group and 14.46 (range 3.59 to 278.86) EU/ml in the severe asthma group with no statistically significant difference determined between the groups. Conclusion The results of this study showed no relationship between serum endotoxin level and asthma or asthma severity.

Synthesis and biological evaluation of lipid A derived from commensal Bacteroides.

The inflammation-inducing properties of lipopolysaccharides (LPS) of Gram-negative bacteria reside in their lipid A moiety. Bacillus fragilis, which is a commensal Gram-negative bacterium, biosynthesises lipid A that is structurally distinct from that of E. coli and other enteric bacteria. It is composed of a β1,6-linked glucosamine (GlcN) disaccharide that is only phosphorylated at the anomeric center. The major species of B. fragilis has five fatty acids and the amine of the distal GlcN moiety carries the unusual (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid. A recent study indicates that the LPS of B. fragilis has anti-viral activity by selective induction of interferon (IFN)-β and is protective in mouse models of vesicular stomatitis virus (VSV) and influenza A. Heterogeneity in the structures of LPS and lipid A and possible contamination with other inflammatory components make it difficult to unambiguously define the immune-modulatory properties of LPS or lipid A. Therefore, we developed a synthetic approach for the preparation of the unusual major lipid A species derived from B. fragilis, which includes a synthetic approach for (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid by the Wittig olefination to install the terminal isopropyl moiety. The proinflammatory and antiviral responses of synthetic B. fragilis lipid A were investigated in several cell lines and primary human monocytes by examining the production of interleukin (IL)-6 and IFN-β. It was found that B. fragilis does not induce the production of IL-6 and IFN-β but can partially antagonize the production of pro-inflammatory cytokines induced by E. coli LPS and lipid A.

Bucking the trend: understanding lipopolysaccharide structure and outer membrane dynamics in cold-adapted Pseudomonas isolated from Enigma Lake, Antarctica.

Cold environments are predominant over the Earth and are inhabited by bacteria able to cope with a series of simultaneous environmental pressures. Gram-negative species of the Pseudomonas genus are the predominant ones isolated from cold habitats, making them an excellent model for studying the mechanisms of bacterial adaptation to the most extreme habitats on our planet. Here we focused on the lipopolysaccharide (LPS) structure and the outer membrane dynamics of Pseudomonas sp. EW#7, a strain isolated from Enigma Lake in Antarctica where, among other extreme characteristics, water temperature can reach 0.4 °C. We show that near-zero growth temperature mostly affects the LPS lipid A component. An uncommon tendency of decreasing lipid A secondary hydroxylation while increasing its phosphorylation degree was observed. This resulted in a faster lateral diffusion of lipid chains in the membrane and therefore in an enhancement of its fluctuations that guarantee membrane integrity and flexibility.

Insights into colistin-mediated fluorescence labelling of bacterial LPS.

Gram-negative bacterial infections are becoming untreatable due to their ability to mutate, and the gradual development of their resistance to the available antimicrobials. In recent times colistin, a drug of last resort, started losing its efficacy towards multidrug-resistant bacterial infections. Colistin targets bacterial endotoxin lipopolysaccharides (LPS) and destabilises the cytoplasmic membrane by disrupting the outer LPS membrane. In this study, we have tried to label the bacterial LPS, the main constituent of the cytoplasmic membrane of bacterial cells, to try to understand the interaction mechanism of LPS with colistin. The chemosensor, naphthaldehyde appended furfural (NAF) that selectively recognises colistin can label LPS, by showing its fluorescence signals. The computationally derived three-dimensional structure of LPS has been introduced to speculate on the possible binding mode of colistin with LPS, and this was also thoroughly studied with the help of quantum mechanics and molecular dynamics energy minimisation. Fluorescence microscopy and FE-SEM microscopic studies were also used to observe the change in the structural morphology of colistin-sensitive and resistant Salmonella typhi in different experimental conditions.

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation.

Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory effects on epithelial and immune cells in vitro

Cyanobacterial harmful blooms (CyanoHABs) pose a global ecological problem, and their lipopolysaccharides (LPS) are among the bioactive compounds they release. Previous studies on CyanoHAB-LPS from single cyanobacterial species have shown varying bioactivities in different in vitro cell models. In this study, we isolated LPS from 19 CyanoHAB samples collected at 18 water bodies in the Czech Republic over two consecutive seasons. The proportions of cyanobacteria, Gram-negative bacteria (G-), and other bacteria in the biomass were determined by qPCR, while the cyanobacterial genera were identified using light microscopy. In vitro models of keratinocytes (HaCaT), the intestinal epithelium (co-culture of differentiated Caco-2 cells and peripheral blood mononuclear cells — PBMC), and PBMC alone were treated with isolated LPS at concentrations of 50, 100, and 1 µg/ml, respectively. The endotoxin activities of these concentrations were within the range measured in the aquatic environment. Approximately 85–90% of the samples displayed biological activity. However, the potency of individual LPS effects and response patterns varied across the different in vitro models. Furthermore, the observed activities did not exhibit a clear correlation with the taxonomic composition of the phytoplankton community, the relative share of microbial groups in the biomass, endotoxin activity of the LPS, or LPS migration and staining pattern in SDS-PAGE. These findings suggest that the effects of CyanoHAB-LPS depend on the specific composition and abundance of various LPS structures within the complex environmental sample and their interactions with cellular receptors.