scholarly journals Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Sineenart Sengyee ◽  
Sung Hwan Yoon ◽  
T. Eoin West ◽  
Robert K. Ernst ◽  
Narisara Chantratita
Keyword(s):  
Lipid A ◽  
Immunoblot Analysis ◽  
Immune Recognition ◽  
Pathogenic Species ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Content Type ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Sds Page

ABSTRACT Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

scholarly journals Hexa-Acylated Lipid A Is Required for Host Inflammatory Response to Neisseria gonorrhoeae in Experimental Gonorrhea

2013 ◽  
Vol 82 (1) ◽  
pp. 184-192 ◽  
Author(s):  
Xiyou Zhou ◽  
Xi Gao ◽  
Peter M. Broglie ◽  
Chahnaz Kebaier ◽  
James E. Anderson ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Lipid A ◽  
Innate Immune ◽  
Immune Recognition ◽  
Toll Like Receptor ◽  
Inflammatory Signaling ◽  
Toll Like Receptor 4 ◽  
Immune Signaling ◽  
Content Type ◽  
Innate Immune Signaling

ABSTRACTNeisseria gonorrhoeaecauses gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients withN. gonorrhoeaeremain asymptomatic, without evidence of localized inflammation. Inflammatory responses toN. gonorrhoeaeare generated by host innate immune recognition ofN. gonorrhoeaeby several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1β (IL-1β) processing complex known as the inflammasome. The lipooligosaccharide ofN. gonorrhoeaehas a hexa-acylated lipid A.N. gonorrhoeaestrains that carry an inactivatedmsbB(also known aslpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show thatmsbB-deficientN. gonorrhoeaeinduces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affectN. gonorrhoeae-mediated activation of the inflammasome. We demonstrate thatN. gonorrhoeaemsbBis dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection withmsbB-deficientN. gonorrhoeaeis associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition ofN. gonorrhoeaeLOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

scholarly journals Lack of Lipid A Pyrophosphorylation and FunctionallptAReduces Inflammation by Neisseria Commensals

2012 ◽  
Vol 80 (11) ◽  
pp. 4014-4026 ◽  
Author(s):  
Constance M. John ◽  
Mingfeng Liu ◽  
Nancy J. Phillips ◽  
Zhijie Yang ◽  
Courtney R. Funk ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Lipid A ◽  
Polymyxin B ◽  
Toll Like Receptor ◽  
Inflammatory Signaling ◽  
Toll Like Receptor 4 ◽  
Bioinformatics Analyses ◽  
Monocytic Cells ◽  
Content Type ◽  
High Level

ABSTRACTThe interaction of the immune system withNeisseriacommensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenicNeisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded bylptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensalNeisseriaspecies confirmed thatlptAwas absent in 15 of 17 strains examined but was present inN. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lackinglptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensalNeisseriastrains that lackedlptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of twoN. lactamicacommensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcallptAdeletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensalNeisseriastrains by reducing the inflammatory potential of LOS.

scholarly journals Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, inPorphyromonas gingivalis

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Sumita Jain ◽  
Ana M. Chang ◽  
Manjot Singh ◽  
Jeffrey S. McLean ◽  
Stephen R. Coats ◽  
...  
Keyword(s):  
Immune Response ◽  
Porphyromonas Gingivalis ◽  
Innate Immune Response ◽  
Lipid A ◽  
Innate Immune ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Gene Encoding ◽  
Content Type ◽  
Chronic Inflammatory Condition

ABSTRACTRemoval of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. InPorphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes theP. gingivalislipid A deacylase enzyme. This gene, PGN_1123 inP. gingivalis33277, is highly conserved withinP. gingivalis, and putative orthologs are phylogenetically restricted to theBacteroidetesphylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-typeP. gingivalis. Heterologous expression of PGN_1123 inBacteroides thetaiotaomicronpromoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCEPeriodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population.Porphyromonas gingivalisis a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection.P. gingivalisis particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

scholarly journals Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival ofBurkholderia pseudomallei

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
Michael H. Norris ◽  
Nawarat Somprasong ◽  
Herbert P. Schweizer ◽  
Apichai Tuanyok
Keyword(s):  
Lipid A ◽  
Severe Disease ◽  
Host Cells ◽  
Host Immune System ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

ABSTRACTBurkholderia pseudomalleicauses the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological “footprint.” The binding strength ofB. pseudomalleiLPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated fromB. pseudomalleiunder different temperatures were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes,lpxOandpagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A fromB. pseudomallei1026blpxOandpagLmutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication oflpxOandpagLmutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.

scholarly journals The Importance of Being an Antagonist as Well as Persistent

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Ann Progulske-Fox
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Current Work ◽  
Phenotypic Characterization ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Content Type ◽  
Link Type ◽  
Tlr4 Agonist

ABSTRACT The current work by Jain et al. (S. Jain, A. M. Chang, M. Singh, J. S. McLean, et al., J Bacteriol 201:e00683-18, 2019, https://doi.org/10.1128/JB.00683-18) reports the cloning of the lipid A deacylase gene of Porphyromonas gingivalis and the phenotypic characterization of the enzyme. Attempts to clone the gene and thus provide proof of the existence of this enzyme had gone on for 2 decades. The enzyme is central to the bacterium’s ability to modify and tailor the structure of its lipid A, changing a lipid A that is a moderate Toll-like receptor 4 (TLR4) agonist to an antagonist or silencer and thereby potentially changing the course of infection.

scholarly journals Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Jie Lin ◽  
Chunquan Xu ◽  
Renchi Fang ◽  
Jianming Cao ◽  
Xiucai Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Population Analysis ◽  
Colistin Resistance ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Expression Levels ◽  
Maldi Tof ◽  
Broth Microdilution Method ◽  
Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

scholarly journals Differential Induction of the Toll-Like Receptor 4-MyD88-Dependent and -Independent Signaling Pathways by Endotoxins

2005 ◽  
Vol 73 (5) ◽  
pp. 2940-2950 ◽  
Author(s):  
Susu M. Zughaier ◽  
Shanta M. Zimmer ◽  
Anup Datta ◽  
Russell W. Carlson ◽  
David S. Stephens
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Lipid A ◽  
Raw 264.7 ◽  
Receptor Complex ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Nitric Oxide Release ◽  
Tnf Α ◽  
Dependent Pathway

ABSTRACT The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), and the MyD88-independent molecules beta interferon (IFN-β), nitric oxide, and IFN-γ-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-α, IL-1β, and MIP-3α, but significantly less IFN-β, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-β, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-α and MIP-3α in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-α release but did not influence nitric oxide release. IFN-β polyclonal antibody and IFN-α/β receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

scholarly journals Immunomodulatory Effects of Yersinia pestis Lipopolysaccharides on Human Macrophages

2009 ◽  
Vol 17 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Motohiro Matsuura ◽  
Hideyuki Takahashi ◽  
Haruo Watanabe ◽  
Shinji Saito ◽  
Kazuyoshi Kawahara
Keyword(s):  
Inflammatory Response ◽  
Yersinia Pestis ◽  
Lipid A ◽  
Antagonistic Activity ◽  
Gram Negative Bacteria ◽  
Toll Like Receptor ◽  
Defense System ◽  
Toll Like Receptor 4 ◽  
Bacterial Binding ◽  
Binding Forms

ABSTRACTIn the current study, we investigated the activity of lipopolysaccharide (LPS) purified fromYersinia pestisgrown at either 27°C or 37°C (termed LPS-27 and LPS-37, respectively). LPS-27 containing hexa-acylated lipid A, similar to the LPS present in usual gram-negative bacteria, stimulated an inflammatory response in human U937 cells through Toll-like receptor 4 (TLR4). LPS-37, which did not contain hexa-acylated lipid A, exhibited strong antagonistic activity to the TLR4-mediated inflammatory response. The phagocytic activity in the cells was not affected by LPS-37. To estimate the activity of LPS in its bacterial binding form, formalin-killed bacteria (FKB) were prepared fromY. pestiscells grown at 27°C or 37°C (termed FKB-27 and FKB-37, respectively). FKB-27 strongly stimulated the inflammatory response. This activity was suppressed in the presence of an anti-TLR4 antibody but not an anti-TLR2 antibody. In addition, this activity was almost completely suppressed by LPS-37, indicating that the activity of FKB-27 is predominantly derived from the LPS-27 bacterial binding form. In contrast, FKB-37 showed no antagonistic activity. The results arising from the current study indicate thatY. pestiscauses infection in humans without stimulating the TLR4-based defense systemviabacterial binding of LPS-37, even when bacterial free LPS-37 is not released to suppress the defense system. This is in contrast to the findings for bacteria that possess agonistic LPS types, which are easily recognized by the defense systemviathe bacterial binding forms.

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