Chemistry and Biology of the Potent Endotoxin from a Burkholderia dolosa Clinical Isolate from a Cystic Fibrosis Patient

Jang

Journal of Biological Chemistry

et al. 2017

Self Cite

Lipopolysaccharide, the outer cell-wall component of Gram-negative bacteria, has been shown to be important for symbiotic associations. We recently reported that the lipopolysaccharide O-antigen of enhances the initial colonization of the midgut of the bean bug, However, the midgut-colonizing symbionts lack the O-antigen but display the core oligosaccharide on the cell surface. In this study, we investigated the role of the core oligosaccharide, which directly interacts with the host midgut, in the symbiosis. To this end, we generated the core oligosaccharide mutant strains, Δ, Δ, Δ and Δ and determined the chemical structures of their oligosaccharides, which exhibited different compositions. The symbiotic properties of these mutant strains were compared with those of the wild-type and O-antigen-deficient Δ strains. Upon introduction into via the oral route, the core oligosaccharide mutant strains exhibited different rates of colonization of the insect midgut. The symbiont titers in fifth-instar insects revealed significantly reduced population sizes of the inner core oligosaccharide mutant strains Δ and Δ These two strains also negatively affected host growth rate and fitness. Furthermore, individuals colonized with the Δ and Δ strains were vulnerable to septic bacterial challenge, similar to insects without a symbiont. Taken together, these results suggest that the core oligosaccharide from symbionts plays a critical role in maintaining a proper symbiont population and in supporting the beneficial effects of the symbiont on its host in the symbiosis.

supporting

confidence: 89%

The lipopolysaccharide core oligosaccharide of Burkholderia plays a critical role in maintaining a proper gut symbiosis with the bean bug Riptortus pedestris

Jang

Journal of Biological Chemistry

et al. 2017

Self Cite

“…Furthermore, dampened pro-inflammatory signaling induced by pentaacylated Ara4N-Lipid A 1 compared with hexaacyl E. coli LPS/Re-LPS is presumably related to its underacylated structure. High E. coli LPS-like activity of the BCC bacterial LOS isolates could be also associated with the presence of the core-sugar region attached at position 6′ of Lipid A,[ 7 ] leading to enhanced TLR4 activation, which has been already shown for the underacylated LOS variants of other bacterial species. [ 60 ] Moreover, glycosylation of Lipid A by negatively charged 3-deoxy- D - manno -octulosonic acid (Kdo) residues (as in LOS isolates) can also result in the boosted pro-inflammatory signaling.…”

Section: Resultsmentioning

confidence: 99%

“…[ 2 ] The pro-inflammatory activity of lipooligosaccharides (LOS) from BCC isolates has been extensively studied. Heterogeneous tetra- and pentaacylated LOS/Lipid A isolates from B. mallei ,[ 3 ] B. multivorans ,[ 4 ] B. cenocepacia ,[ 5 , 6 ] B. cepacia [ 2 ] and B. dolosa [ 7 ] were reported to be very potent activators of human (h) LPS-sensing innate immune receptor, Toll-like Receptor 4 (TLR4)–myeloid differentiation-2 (MD-2) complex. Furthermore, it has been previously established that the terminal membrane-bound portion of LPS, glycophospholipid Lipid A, is primarily responsible for induction of pro-inflammatory signalling, which was shown both with isolated[ 8 ] and synthetic Lipid A derivatives.…”

Section: Introductionmentioning

confidence: 99%

Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Hollaus

Ittig

Hofinger

et al. 2015

Chemistry A European J

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-β-l-arabinose (β-l-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β-l-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the β-l-Ara4N H-phosphonate to α-lactol of β(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1′-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β-l-Ara4N-substituted Burkholderia Lipid A. The β-l-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A.

“…MALDI preparations were performed as previously reported. 42 HEK-Blue hTLR4 Cells. HEK-Blue hTLR4 cells (Inviv-oGen) were cultured according to manufacturer's instructions.…”

Section: Bioconjugate Chemistrymentioning

confidence: 99%

Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies

et al. 2019

Endotoxin (lipooligosaccharide, LOS, and lipopolysaccharide, LPS) is the major molecular component of Gram-negative bacteria outer membrane, and very potent proinflammatory substance. Visualizing and tracking the distribution of the circulating endotoxin is one of the fundamental approaches to understand the molecular aspects of infection with subsequent inflammatory and immune responses, LPS also being a key player in the molecular dialogue between microbiota and host. While fluorescently labeled LPS has previously been used to track its subcellular localization and colocalization with TLR4 receptor and downstream effectors, our knowledge on lipopolysaccharide (LOS) localization and cellular activity remains almost unexplored. In this study, LOS was labeled with a novel fluorophore, Cy7N, featuring a large Stokes-shifted emission in the deep-red spectrum resulting in lower light scattering and better imaging contrast. The LOS-Cy7N chemical identity was determined by mass spectrometry, and immunoreactivity of the conjugate was evaluated. Interestingly, its application to microscopic imaging showed a faster cell internalization compared to LPS-Alexa488, despite that it is also CD14-dependent and undergoes the same endocytic pathway as LPS toward lysosomal detoxification. Our results suggest the use of the new infrared fluorophore Cy7N for cell imaging of labeled LOS by confocal fluorescence microscopy, and propose that LOS is imported in the cells by mechanisms different from those responsible for LPS uptake.