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PubMed2026

wbp-encoded LPS O-antigen architecture as a prognostic and therapeutic target in Pseudomonas aeruginosa keratitis.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) keratitis can progress rapidly to vision-threatening disease, even with intensive therapy. Virulence-associated genes are key determinants of ocular-surface pathogenesis. We therefore sought to develop a composite wbp-exo genotyping framework for risk stratification and to guide wbp-dependent, LPS-directed, levofloxacin-polymyxin B (LVX-POL) combination therapy for high-risk corneal infections. METHODS: A well-characterised clinical P. aeruginosa keratitis cohort was integrated with whole-genome sequencing. Based on comprehensive virulence-gene identification and annotation, the relationship between strain-level genetic features and clinical prognosis was analysed. The differences between WBP1 strains and WBP2 strains in adhesion, invasion, and biofilm formation in corneal epithelial cells were further evaluated. To establish biological plausibility, wbp genotypes were correlated with LPS O-antigen electrophoretic profiles and in vivo corneal inflammatory phenotypes in murine infection, including the observation of leucocyte recruitment and cytokine responses. To further confirm the key role of wbp gene status and LPS O-antigen in pathogenicity, wbpL knockout and reconstitution strains were constructed. Their appearances in vitro and in vivo were evaluated. Finally, a mechanistic rationale for an LPS-directed LVX-POL regimen was tested in a high-risk WBP1 P. aeruginosa murine keratitis. FINDINGS: Whole-genome sequencing was performed on 46 clinical P. aeruginosa isolates and identified an average of 332 virulence- and fitness-associated genes per strain. The exo and wbp gene families were significantly associated with patient prognosis. A fusion model (AUC = 0.86) outperformed single-gene-family models (EXO: 0.66; WBP: 0.72) for predicting clinical outcomes. Intact wbp cassettes were enriched in poor-outcome isolates, and electrophoretic LPS profiles indicated that WBP1 strains produce highly polymerised O-antigen associated with sustained neutrophil recruitment and cytokine production. Murine experiments further implicated wbp genes in clinical pathogenesis, showing stronger immune responses and higher expression of TLR4, MyD88, TRAF6, p65, p-p65, IL-6, TNF-α, and IL-1β throughout the inflammatory course. After knocking out wbpL gene, the WBP1 strain got stronger in biofilm formation and adhesion but weaker in inflammation and ocular surface survival. In the high-risk WBP1 P. aeruginosa keratitis model, LVX-POL combinations achieved complete ulcer resolution and markedly improved stromal infiltration and hypopyon, outperforming LVX monotherapy. INTERPRETATION: The wbp gene family was identified as a key genetic factor that contributes to the LPS O-antigen structure, inflammatory intensity, bacterial ocular surface survival and poor prognosis in P. aeruginosa keratitis. A WBP1-targeted, LPS-directed LVX-POL regimen was proposed as a mechanistically informed option for high-risk strains. FUNDING: This research was supported by Beijing Public Health High-level Talent Training Program (Phase III-03-14), Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2026ZD01909300) and Beijing Natural Science Foundation "QiYan" Undergraduate Research Fund (QY26496).

Pseudomonas aeruginosa↗

PubMed2025

Characterization of gut microbiota signatures in Indian preterm infants with necrotizing enterocolitis: a shotgun metagenomic approach.

INTRODUCTION: Necrotizing enterocolitis (NEC) is an inflammatory bowel disease that primarily affects preterm infants. Predisposing risk factors for NEC include prematurity, formula feeding, anemia, and sepsis. To date, no studies have investigated the gut microbiota of preterm infants with NEC in India. METHOD: In the current study, shotgun metagenomic sequencing was performed on fecal samples from premature infants with NEC and healthy preterm infants (n = 24). Sequencing was conducted using the NovaSeq X Plus platform, generating 2 × 150 bp paired-end reads. The infants were matched based on gestational age and postnatal age. RESULT: The median time to NEC diagnosis was 9 days (range: 1-30 days). Taxonomic analysis revealed a high prevalence of Enterobacteriaceae at the family level, with the genera Klebsiella and Escherichia particularly prominent in neonates with NEC. No statistically significant differences in alpha or beta diversity were observed between stool samples from infants with and without NEC. Linear regression analysis demonstrated that Enterobacteriaceae were significantly more abundant in stool samples from infants with NEC than without NEC (q < 0.05). Differential abundance analysis using Linear Discriminant Analysis Effect Size (LEfSe) identified Klebsiella pneumoniae and Escherichia coli as enriched in the gut microbiota of preterm infants with NEC. Functional analysis revealed an increase in genes associated with lipopolysaccharide (LPS) O-antigen, the type IV secretion system (T4SS), the L-rhamnose pathway, quorum sensing, and iron transporters, including ABC transporters, in stool samples from infants with NEC. CONCLUSION: The high prevalence of Enterobacteriaceae and enrichment of LPS O-antigen and T4SS genes may be associated with NEC in Indian preterm infants.

Humans↗

PubMed1975

Lipophilic O-antigens containing D-glycero-D-mannoheptose as the sole neutral sugar in Rhodopseudomonas gelatinosa.

Lipopolysaccharides (LPS, O-antigens) of 12 strains of the photosynthetic bacterium Rhodopseudomonas gelatinosa were obtained by the phenol/chloroform/petroleum ether method, recommended for extracting lipophilic glycolipids of enterobacterial R-mutants. All R. gelatinosa LPS have essentially the same chemical composition. Similar to LPS of Salmonella R-mutants of chemotypes Rd1 and Rd2, the sole neutral sugar constituent is an aldoheptose. The heptose of R. gelatinosa LPS has the D-glycero-D-manno- configuration, in contrast to the L-glycero-D-mannoheptose of enterobacterial LPS. 2-Keto-3-deoxyoctonate forms the acid-labile linkage between the lipid moiety (lipid A) and the oligosaccharide moiety of R. gelatinosa LPS. Like enterobacterial lipid A, lipid A of this species contains phosphate and D-glucosamine as the sole amino sugar. The fatty acid spectrum conprises beta-hydroxycapric, lauric, and myristic acids. Beta-Hydroxymyristic acid, the typical fatty acid of enterobacterial LPS, is lacking. The R. gelatinosa LPS show O-antigenic acitivity; passive hemagglutinations with untreated or heat-treated (not well alkali-treated) LPS and antisera prepared against heat-killed cells yield high titers. According to the serological cross-reactions observed, the LPS of the 12 strains could be arranged into two different serotypes: serotype I comprising strains 29/1, 29/2, 25/2, and serotype II comprising strains 44/K/6, 3/1, IS/10, 39/2, Dr2, 2150, P8P9, K32, P18f3.1. No serological cross-reactions were observed between LPS of these two different serotypes in passive hemagglutinations.

Caprylates↗

PubMed2026

Loss of O-antigen due to wbbL mutations is common and associated with increased mortality in Escherichia coli bloodstream infections.

Escherichia coli bloodstream infections are common and associated with high mortality. A key feature of E. coli is the lipopolysaccharide (LPS) O-antigen, which contributes to immune evasion during invasive infection. We analyzed serial isolates from patients with relapsed E. coli bacteremia and identified frequent disruption of O-antigen synthesis due to mutations in wbbL, resulting in a rough LPS phenotype. Rough LPS isolates were more serum sensitive and less pathogenic in mice. Despite this apparent attenuation, 11 of 61 (18%) E. coli sequence type 131 bloodstream isolates in our cohort harbored disruptive wbbL mutations and were associated with significantly worse clinical outcomes, including septic shock and mortality. Using a murine model of recurrent bacteremia, we show that rough LPS isolates partially evade protective immunity generated against smooth LPS E. coli, highlighting the importance of host immune context in invasive disease.

O Antigens↗

PubMed1978

Composition of the fractions separated by polyacrylamide gel electrophoresis of the lipopolysaccharide of a marine bacterium.

The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.

Cell Wall↗

PubMed2026

Surface architecture of the bacterial envelope determines phage adsorption route in pathogenic Escherichia coli O157:H7.

UNLABELLED: The outermost surface layers of Gram-negative bacteria determine phage access to terminal receptors, yet their genetic basis has been mapped almost exclusively in laboratory strains that lack them. Here we apply genome-wide RB-TnSeq fitness profiling to four Escherichia coli O157:H7 strains from distinct phylogenetic clades sharing the O157 O-antigen, using 38 phages with terminal receptors previously mapped in E. coli K-12 strain. RB-TnSeq fitness landscapes across all four pathogenic backgrounds were mostly similar, and dominated by surface-associated loci, including the gfc-etk group 4 capsule operon, O-antigen biosynthesis genes, LPS core assembly genes and outer membrane proteins. Disruption of gfc-etk abolished infection in 11 genetically diverse myoviruses, establishing the O-antigen capsule as a widespread required primary recognition substrate. O-antigen loci generated two classes of fitness score patterns. For 10 phages, disruption increased infectivity, indicating it is a barrier to receptor access; for 3 others, disruption abolished infectivity, demonstrating it can also be a primary recognition substrate. Outer membrane protein receptor identity was conserved across laboratory and pathogenic backgrounds, with the same proteins recognized in both K-12 and O157:H7, while glycan layer state determines whether these receptors are reached. These results demonstrate that outer surface glycan layers can act as primary and optional recognition substrates for phage infection, or as physical barriers preventing terminal receptor access. Extending the ability to probe phage-targeted receptors beyond outer membrane proteins provides a framework for incorporating glycan layer state into predictive models of phage-host interactions. IMPORTANCE: Bacteriophage-based interventions for controlling Escherichia coli O157:H7, a major foodborne pathogen responsible for tens of thousands of illnesses annually in the United States, require a mechanistic understanding of the factors governing strain-level susceptibility. Predictive frameworks developed in laboratory model strains lacking O-antigen and capsular polysaccharides can map the terminal protein receptors that phages bind, but are currently limited in their ability to determine whether those receptors are accessible in pathogenic isolates carrying full outer surface complexity. This study provides the first genome-scale, functional genetic map of phage susceptibility determinants in O157:H7 and demonstrates that the state of the outer surface layers, specifically the O-antigen and the gfc-etk capsule, determines whether phages can reach conserved terminal receptors. This finding explains differences in phage susceptibility between strains sharing nearly identical gene content, and identifies the molecular layers that must be characterized to predict phage host interaction in pathogenic E. coli backgrounds.

Journal Article↗

PubMed1975

Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid--or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.

Absorption↗

PubMed1979

Endotoxins of anaerobic gram-negative rods.

Endotoxic lipopolysaccharides (LPS) isolated from Fusobacterium are similar to those of Salmonella with respect to chemical composition, O-antigenic specificity and endotoxic activity. Bacteroides LPS are highly atypical with respect to the chemical composition, and their endotoxic activity is low. O-Antigenic specificity is present. B. fragilis LPS is chemotactic for polymorphonuclear leukocytes in vitro and in vivo. The chemotactic activity is brought about by activation of the alternative complement pathway.

Animals↗

PubMed1979

O-antigenic cross-reactivity in Fusobacterium nucleatum.

Lipopolysaccharides (LPS) of the same chemotype isolated from seven strains of Fusobacterium nucleatum were examined by indirect haemagglutination and inhibition of haemagglutination in unabsorbed and absorbed antisera. Four common major antigenic specificities were detected, and two of the LPS examined contained antigenic specificities not shared with any of the other LPS. In addition, a few weak cross-reactions were observed, also with some LPS of other chemotypes.

Antibodies, Bacterial↗

PubMed2025

Advances in Helicobacter pylori lipopolysaccharide structure and function.

Helicobacter pylori is a widespread pathogen responsible for chronic gastritis, peptic ulcers, and an elevated risk of gastric cancer. Lipopolysaccharide (LPS), localized exclusively in the outer leaflet of the outer membrane, is essential for maintaining bacterial integrity. Recent advances have deepened our understanding of H. pylori LPS structure, particularly lipid A modifications and the redefinition of the core oligosaccharide and O-antigen regions. The complete set of enzymes involved in LPS biosynthesis has been identified in the reference strain G27, and comparative genomics has revealed a notable regional difference (the absence of the heptan domain in East Asian strains). Here, we summarize recent insights into the structure and function of H. pylori LPS, emphasizing its role in bacterial persistence and its promise as a target for LPS-based glycoconjugate vaccine development.

Helicobacter pylori↗

PubMed1975

O-antigenic specificity of lipopolysaccharides from Bacteroides fragilis ss. fragilis.

Lipopolysaccharides (LPS) isolated from three strains of Bacteroides fragilis ss. fragilis were examined by indirect haemagglutination and inhibition of haemagglutination in unabsorbed and absorbed rabbit antisera. Six different antigenic specificities were detected. All serological activity was destroyed by oxidation of LPS with periodate.

Animals↗

PubMed1978

[Monosaccharide composition of the lipopolysaccharides of bacteria of the genus Citrobacter].

The authors studied antigens obtained by Grasset's method from 13 strains of Citrobacter of the International collection. The strains possessed O- and H-antigens whose behaviur in the electric field differed. All the strains under study were divided into two groups (by the number of serologically-active components of their O-antigens); representatives of the second group had no cathode O-antigen component. Chemical composition of specific lipopolysaccharides (LPS) obtained by Westphal's method was determined. Fourteen different sugars were revealed. The strains under study were referred to the known chemotypes. Strain 16/52 (8a, 8c) was for the first time studied in respect to the monosaccharide composition of specific LPS, and was referred to chemotype designated as CC-L.

Agglutination Tests↗

PubMed2025

A rhamnose-rich O-antigen of Paraburkholderia phymatum MP20 is required for symbiosis with Mimosa pudica.

Paraburkholderia phymatum, a β-proteobacterium, forms a nitrogen-fixing symbiosis with many species of the large legume genus Mimosa as well as with common bean (Phaseolus vulgaris L.). Paraburkholderia are considered to have evolved nodulation independently from the well-studied α-proteobacteria symbionts of legumes. However, the detailed mechanisms important for β-rhizobia-legume symbiosis have not yet been determined. In this manuscript, we have sequenced the genome of P. phymatum MP20, a strain isolated from Mimosa pudica nodules, and utilized transposon mutagenesis to identify a mutant that showed delayed and ineffective nodulation of M. pudica. Further analysis revealed that the mutant strain produced an altered lipopolysaccharide lacking rhamnose containing O-antigen. Complementation with the wild-type gene restored the symbiosis. Microscopic analysis of the ineffective nodules showed that the mutant strain did not infect the cortical cells but was restricted to the endodermis. The results suggest that the O-antigen of P. phymatum is important for the bacterial infection of cortical cells and for nodule maturation. Further research will unveil the specific involvement of the glycosyltransferase gene in LPS biosynthesis and its impact on successful nodule formation by P. phymatum.IMPORTANCEThe nitrogen-fixing symbiosis between legumes and rhizobia is important for agricultural and environmental sustainability. The mechanisms of the symbiotic interactions are extensively studied using α-rhizobia. In contrast, mechanisms of symbiotic interactions important for β-rhizobia and their Caesalpinioid (mimosoid) legume hosts are not well known. Here, we describe the genome sequence of P. phymatum MP20, a β-rhizobia isolated from the nodules of M. pudica, and isolation and characterization of a transposon mutant defective in symbiosis. We demonstrate that the O-antigen of the LPS is required for nodulation and symbiotic nitrogen fixation. This study broadens our knowledge of symbiotic interactions in β-rhizobia and will lead to a better understanding of the wider rhizobial-legume symbiosis apart from the α-rhizobia.

Symbiosis↗

PubMed1978

Effect on particle size of solubilization of wild-type and Re chemotype lipopolysaccharides solubilized with bovine serum albumin and triethylamine.

Disaggregation of wild-type and Re chemotype lipopolysaccharides (LPS) has been accomplished by solubilization with triethylamine followed by stabilization with bovine serum albumin in order to determine the degree of aggregation. Solubilization with 1.5% triethylamine was found to be more effective than with lower concentrations, as judged by permeation chromatography. Chromatographic analysis of triethylamine-bovine serum albumin-disaggregated LPS showed a range of particle sizes. Wild-type LPS yielded size classes of 1 X 10(6) to 4 X 10(6) daltons, 250,000 daltons, and 20,000 daltons. Re LPS yielded size classes of 1 X 10(6) to 4 X 10(6) daltons and 20,000 daltons. Untreated LPS was always greater than 4 X 10(6) daltons. Chemical characterization of the size classes revealed 3-keto-2-deoxyoctonate in all size classes and suggests that the O-antigenic side chain length may determine the aggregate size in solubilized LPS.

Carbohydrates↗

PubMed1977

Lipid A as the biologically active moiety in bacterial endotoxin (LPS)-initiated generation of procoagulant activity by peripheral blood leukocytes.

Preparations of rabbit or human leukocytes, when incubated with bacterial endotoxins (lipopolysaccharides, LPS) are stimulated to generate a procoagulant-tissue factor activity (TFa). As LPS has been shown to consist of specific repeating oligosaccharide side chains (O-antigen) linked to a central polysaccharide core region that is, in turn, linked to the lipid region of the molecule (lipid A), we have examined the biochemical requirement of the LPS necessary for generation of TFa. Using preparations of LPS from mutant strains of bacteria, which contain varying amounts of polysaccharide in relation to lipid A, we have demonstrated that activity is associated with the lipid A region of the LPS molecule. These observations have been confirmed using isolated lipid A, which is a potent stimulator of TFa, as well as a native protoplasmic polysaccharide that is both devoid of lipid A and without detectable TFa stimulatory activity. Modification of LPS by treatment with mild alkali abrogated its capacity to stimulate TFa generation. In addition, such altered preparations of LPS partially inhibit the stimulatory effect of native LPS. Similarly, treatment of LPS (or lipid A) with the antibiotic polymyxin B substantially inhibited the stimulatory effect of LPS.

Animals↗

PubMed1977

Chemical composition, serological reactivity and endotoxicity of lipopolysaccharides extracted in different ways from Bacteroides fragilis, Bacteroides melaninogenicus and Bacteroides oralis.

Lipopolysaccharides (LPS) extracted from strains of Bacteroides fragilis, Bacteroides melaninogenicus and Bacteroides oralis with phenol-water, trichloroacetic acid, EDTA or liquid phenol-chloroform-petroleum ether (PCP) and isolated by ultracentrifugation, varied considerably in their quantitative chemical composition. Negligible yields of LPS were obtained by PCP-extraction. All preparations were more or less serologically active. All methods (except PCP) extracted the same O-antigenic determinants from B. fragilis. Endotoxic activity, as measured by primary skin inflammations in rabbits, was low but was present in all preparations. Proteins (and/or lipoproteins) co-precipitated with LPS in the ultracentrifuge.

Bacteroides↗

PubMed1976

Cell wall lipopolysaccharide response to the ColIb plasmid mutants.

Mutants of ColIb plasmid affected the synthesis of O-side chains of lipopolysaccharides (LPS) in Salmonella. The plasmid srd 25 (defective in colicin synthesis) caused a significant decline of rhamnose and mannose content and lack of abequose in LPS of S. typhimurium. The number of repeating units in O-side chains was decreased after the indroduction of srd 25. Cultures of S. typhimurium and S. enteritidis harboring drd2 (derepressed in colicin production) polymerised dideoxyhexose-defective O-side chains i.e. deprived of abequose and tyvelose, respectively. In dideoxyhexoseless S. meleagridis the content of rhamnose and mannose were reduced. The information for the alterations of Salmonella LPS was contained in the plasmid genome. In the wild-type plasmids the genes controlling the O-antigen changes were not expressed.

Cell Wall↗

PubMed2025

Polysaccharide synthesis operon modulates Rickettsia-endothelial cell interactions.

Pathogenic Rickettsia species target vascular endothelial cells and cause systemic vasculitis. As obligate intracellular bacterial pathogens, Rickettsia must secure nutritional resources within the cytoplasm of endothelial cells while simultaneously subverting the innate immune defense system. With advances in rickettsial and host genetics, recent studies have identified novel molecular mechanisms involved in the complex interactions between Rickettsia and endothelial cells. However, it remains unclear how Rickettsia shields pathogen-derived immune stimulants, such as lipopolysaccharides (LPS) and peptidoglycan fragments, from immune recognition during intracellular replication. Prior work described two Rickettsia conorii variants with kkaebi transposon insertions in the polysaccharide synthesis operon (pso). Biochemical and immunological analyses revealed that pso is responsible for the biosynthesis of O-antigen (O-Ag) and the proper assembly of surface proteins. In the present work, we document that pso variant HK2 exhibits reduced capacities to adhere to and invade microvascular endothelial cells. Despite the low intracellular abundance, HK2 induced significantly higher levels of proinflammatory cytokines and chemokines, leading to premature cell death. Notably, HK2 exhibited defective intracellular survival in bone marrow-derived macrophages. This inability to dampen endothelial cell-mediated immune stimulation and resist macrophage-induced bactericidal activities resulted in the rapid elimination of viable Rickettsia in the mouse model of spotted fever. Further, when tested as a live-attenuated vaccine, HK2 elicited robust protective immunity against lethal spotted fever pathogenesis. Our work highlights the crucial role of pso in enabling Rickettsia to evade immune surveillance during intracellular replication within endothelial cells, ultimately delaying pathogen-induced programmed cell death and escaping immune defense mechanisms.

Operon↗