Against the rules: a marine bacterium, Loktanella rosea, possesses a unique lipopolysaccharide.

Bacteria are an inimitable source of new glyco-structures potentially useful in medicinal and environmental chemistry. Lipopolysaccharides (LPS; endotoxins) are the major components of the outer membrane of Gram-negative bacteria; being exposed toward the external environment they can undergo structural changes and thus, they often possess peculiar chemical features that allow them to thrive in harsh chemical and physical environments. Marine bacteria have evolved and adapted over millions of years in order to succeed in different environments, finding a niche for their survival characterized by severe physical or chemical parameters. The present work focuses on the structural investigation of the LPS from Loktanella rosea, a marine Gram-negative bacterium. Through chemical analysis, 2D nuclear magnetic resonance and matrix-assisted laser desorption ionization mass spectrometry investigations, a unique LPS carbohydrate backbone has been defined. The lipid A skeleton consists of a trisaccharide backbone lacking the typical phosphate groups and is characterized by two beta-glucosamines and an alpha-galacturonic acid. The core region is built up of three ulosonic acids, with two 3-deoxy-d-manno-oct-2-ulopyranosonic acid residues, one of which is carrying a neuraminic acid. This carbohydrate structure is an exceptional variation from the typical architectural skeleton of endotoxins which consequently implies a very different biosynthesis.

The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model.

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular, lipid A is the endotoxic portion of LPS responsible for eliciting host innate immune response. In this work, the complete primary structure of the lipid A from B. multivorans C1576 has been defined and, further, its pro-inflammatory activity in a cystic fibrosis airways model is shown. The structure of B. multivorans lipid A was attained by chemical, mass spectrometry and nuclear magnetic resonance analyses whereas its biological activity was assessed on the intestinal epithelial cell line CACO-2 cells, on the airway epithelial IB3-1 cells, carrying the ΔF508/W1282X CFTR mutation and on an ex vivo model of culture explants of nasal polyps.

Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection.

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1beta cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.

First structural characterization of Burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains.

This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis. We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF-alpha secretion in the post-transplantation strain.

Structural study and conformational behavior of the two different lipopolysaccharide O-antigens produced by the cystic fibrosis pathogen Burkholderia multivorans.

Lipopolysaccharides (LPSs) are virulence factors expressed by gram-negative bacteria; they are among those mainly responsible for bacterial virulence. In this work we define the primary structure and the conformational features of the O-chain from the LPS produced by the highly virulent clinical isolate Burkholderia multivorans strain C1576, an opportunistic human pathogen isolated in a cystic fibrosis center and causative of an outbreak with lethal outcome. We demonstrate that the LPS from this clinical isolate consists of two O-polysaccharide chains present in different amounts and made up of repeating units, both containing deoxy sugar. Additionally, conformational studies have been performed to establish and compare the spatial arrangements of the two polysaccharides and differences in their shape have been highlighted. The comprehension of the structural and conformational features of the two repeating units may help to explain their biological significance, the molecular shape of the bacterial external surface, and the comprehension at the molecular level of the recognition mechanisms of the antibodies.

The structure and proinflammatory activity of the lipopolysaccharide from Burkholderia multivorans and the differences between clonal strains colonizing pre and posttransplanted lungs.

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens for humans especially in cystic fibrosis patients. Lipopolysaccharide (LPS) molecules are potent virulence factors of Gram-negative bacteria organisms essential for bacterial survival. A complete analysis of the bacterial lipopolysaccharide structure to function relationship is required to understand the chemical basis of the inflammatory process. We have therefore investigated the structures of lipopolysaccharides from clonally identical Burkholderia multivorans strains (genomovar II) isolated pre- and post-lung transplantation through compositional analysis, mass spectrometry, and 2D NMR spectroscopy. We tested the LPS proinflammatory activity as a stimulant of human myelomonocytic U937 cell cytokine induction and assessed TLR4/MD2 signaling. Marked changes between the paired strains were found in the lipid A-inner core region. Such structural variations can contribute to the bacterial survival and persistence of infections despite the loss of a CF milieu following lung transplantation.

Bacterial polysaccharides suppress induced innate immunity by calcium chelation.

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.

Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli.

In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (<32 degrees C), but not at 37 degrees C. We show that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.

The complete structure and pro-inflammatory activity of the lipooligosaccharide of the highly epidemic and virulent gram-negative bacterium Burkholderia cenocepacia ET-12 (strain J2315).

Members of genus Burkholderia include opportunistic Gram-negative bacteria that are responsible for serious infections in immunocompromised and cystic fibrosis (CF) patients. The Burkholderia cepacia complex is a group of microorganisms composed of at least nine closely related genomovars. Among these, B. cenocepacia is widely recognized to cause epidemics associated with excessive mortality. Species that belong to this strain are problematic CF pathogens because of their high resistance to antibiotics, which makes respiratory infections difficult to treat and impossible to eradicate. Infection by these bacteria is associated with higher mortality in CF and poor outcomes following lung transplantation. One virulence factor contributing to this is the pro-inflammatory lipopolysaccharide (LPS) molecules. Thus, the knowledge of the lipopolysaccharide structure is an essential prerequisite to the understanding of the molecular mechanisms involved in the inflammatory process. Such data are instrumental in aiding the design of antimicrobial compounds and for developing therapeutic strategies against the inflammatory cascade. In particular, defining the structure of the LPS from B. cenocepacia ET-12 clone LMG 16656 (also known as J2315) is extremely important given the recent completion of the sequencing project at the Sanger Centre using this specific strain. In this paper we address this issue by defining the pro-inflammatory activity of the pure lipopolysaccharide, and by describing its full primary structure. The activity of the lipopolysaccharide was tested as a stimulant in human myelomonocytic U937 cells. The structural analysis was carried out by compositional analysis, mass spectrometry and 2D NMR spectroscopy on the intact lipooligosacchride (LOS) and its fragments, which were obtained by selective chemical degradations.