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Sample records for aggregatibacter actinomycetemcomitans lipopolysaccharide

  1. Evolutionary Divergence of Aggregatibacter actinomycetemcomitans.

    PubMed

    Kittichotirat, W; Bumgarner, R E; Chen, C

    2016-01-01

    Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e' (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e' isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved

  2. Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

    PubMed

    Im, Jintaek; Baik, Jung Eun; Kim, Kyoung Whun; Kang, Seok-Seong; Jeon, Jun Ho; Park, Ok-Jin; Kim, Hyun Young; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

    2015-08-01

    Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M. PMID:25840438

  3. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    PubMed Central

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  4. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  5. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  6. Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.

    PubMed

    Zijnge, Vincent; Kieselbach, Thomas; Oscarsson, Jan

    2012-01-01

    The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth. Further to confirming the release of established virulence factors (e.g. cytolethal distending toxin [CDT], and leukotoxin [LtxA]), we identified additional putative virulence determinants in the secretome. These included DegQ, fHbp, LppC, Macrophage infectivity protein (MIP), NlpB, Pcp, PotD, TolB, and TolC. This finding indicates that the number of extracellular virulence-related proteins is much larger than previously demonstrated, which was also supported by in silico analysis of the strain D7S genome. Moreover, our LC-MS/MS and in silico data revealed that at least Type I, II, and V secretion are actively used to excrete proteins directly into the extracellular space, or via two-step pathways involving the Sec/Tat systems for transport across the inner membrane, and outer membrane factors, secretins and auto-transporters, respectively for delivery across the outer membrane. Taken together, our results provide a molecular basis for further elucidating the role of A. actinomycetemcomitans in periodontal and systemic diseases. PMID:22848560

  7. Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis.

    PubMed

    Herbert, B A; Novince, C M; Kirkwood, K L

    2016-06-01

    Aggregatibacter actinomycetemcomitans is a perio-pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra-physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non-hematopoietic and hematopoietic. Non-hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid-derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo-immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast-osteoblast-mediated bone remodeling, which subsequently leads to net alveolar bone loss. PMID:26197893

  8. Leukotoxic activity of Aggregatibacter actinomycetemcomitans and periodontal attachment loss.

    PubMed

    Höglund Åberg, Carola; Haubek, Dorte; Kwamin, Francis; Johansson, Anders; Claesson, Rolf

    2014-01-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. This leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a well-characterized specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been isolated. In particular, the presence of the JP2 genotype significantly increases the risk for the progression of periodontal attachment loss (AL). Based on these findings we hypothesized that variations in the leukotoxicity are linked to disease progression in infected individuals. In the present study, the leukotoxicity of 239 clinical isolates of A. actinomycetemcomitans was analysed with different bioassays, and the genetic peculiarities of the isolates were related to their leukotoxicity based on examination with molecular techniques. The periodontal status of the individuals sampled for the presence of A. actinomycetemcomitans was examined longitudinally, and the importance of the observed variations in leukotoxicity was evaluated in relation to disease progression. Our data show that high leukotoxicity correlates with an enhanced risk for the progression of AL. The JP2 genotype isolates were all highly leukotoxic, while the isolates with an intact leukotoxin promoter (non-JP2 genotypes) showed substantial variation in leukotoxicity. Genetic characterization of the non-JP2 genotype isolates indicated the presence of highly leukotoxic genotypes of serotype b with similarities to the JP2 genotype. Based on these results, we conclude that A. actinomycetemcomitans harbours other highly virulent genotypes besides the previously described JP2 genotype. In addition, the results from the present study further highlight the importance of the leukotoxin as a key virulence factor in aggressive forms of periodontitis. PMID:25093857

  9. Neutrophil-derived resistin release induced by Aggregatibacter actinomycetemcomitans.

    PubMed

    Furugen, Reiko; Hayashida, Hideaki; Yoshii, Yumiko; Saito, Toshiyuki

    2011-08-01

    Resistin is an adipokine that induces insulin resistance in mice. In humans, resistin is not produced in adipocytes, but in various leukocytes instead, and it acts as a proinflammatory molecule. The present investigation demonstrated high levels of resistin in culture supernatants of neutrophils that are stimulated by a highly leukotoxic strain of Aggregatibacter actinomycetemcomitans. In contrast, the level of resistin was remarkably low when neutrophils were exposed to two other strains that produce minimal levels of leukotoxin and a further isogenic mutant strain incapable of producing leukotoxin. Pretreatment of neutrophils with a monoclonal antibody to CD18, β chain of lymphocyte function-associated molecule 1 (LFA-1), or an Src family tyrosine kinase inhibitor before incubation with the highly leukotoxic strain inhibited the release of resistin. These results show that A. actinomycetemcomitans-expressed leukotoxin induces extracellular release of human neutrophil-derived resistin by interacting with LFA-1 on the surface of neutrophils and, consequently, activating Src family tyrosine kinases. PMID:21658109

  10. Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype g Strain NUM4039 (JCM 30399)

    PubMed Central

    Saito, Masanori; Hirasawa, Masaaki; Kuwahara, Noriko; Okada, Tamami; Umezawa, Koji; Kobayashi, Taira; Okamoto, Masaaki; Naito, Mariko; Hirasawa, Masatomo

    2016-01-01

    Aggregatibacter actinomycetemcomitans is considered to be a major etiological agent of aggressive periodontitis and includes serotype a to g strains. We herein report the first complete genome sequence of A. actinomycetemcomitans serotype g strain NUM4039. The genome is 2,382,853 bp in length with a G+C content of 44.34%. PMID:26988057

  11. Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

    PubMed Central

    Brown, Angela C.; Boesze-Battaglia, Kathleen; Du, Yurong; Stefano, Frank P.; Kieba, Irene R.; Epand, Raquel F.; Kakalis, Lazaros; Yeagle, Philip L.; Epand, Richard M.; Lally, Edward T.

    2012-01-01

    Summary The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization. PMID:22309134

  12. Influence of Aae Autotransporter Protein on Adhesion and Biofilm Formation by Aggregatibacter actinomycetemcomitans.

    PubMed

    Nunes, Ana Carla Robatto; Longo, Priscila Larcher; Mayer, Marcia Pinto Alves

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins. PMID:27224556

  13. Mature Biofilm Degradation by Potential Probiotics: Aggregatibacter actinomycetemcomitans versus Lactobacillus spp.

    PubMed Central

    Mizuno, Kouhei; Okinaga, Toshinori

    2016-01-01

    The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics. PMID:27438340

  14. Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Rompikuntal, Pramod Kumar; Thay, Bernard; Khan, Muhammad Khanzeb; Alanko, Jonna; Penttinen, Anna-Maija; Asikainen, Sirkka; Wai, Sun Nyunt

    2012-01-01

    Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. PMID:22025516

  15. Effect of mouthrinses on Aggregatibacter actinomycetemcomitans biofilms in a hydrodynamic model.

    PubMed

    Sliepen, Isabelle; Van Essche, Mark; Quirynen, Marc; Teughels, Wim

    2010-06-01

    The aim of the study was to evaluate the effects of Listerine, Meridol, and Perioaid on the viability and total number of bacteria in established biofilms using an in vitro model under hydrodynamic conditions. Biofilms of Aggregatibacter actinomycetemcomitans were placed in a modified Robbins device and rinsed twice daily during 4 days. Bacteria were quantified by culture and quantitative polymerase chain reaction. Visualization of the samples was performed by scanning electron and confocal laser scanning microscopy, combined with a fluorescent vital staining. All three mouthrinses caused a significant reduction in the number of cultivable A. actinomycetemcomitans in a biofilm. Perioaid was significantly the most powerful in killing the biofilm-protected bacteria and also in counteracting the development of thick dense microbial communities. The total amount of bacteria was not significantly affected by Listerine and Meridol. PMID:19462186

  16. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  17. Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β

    PubMed Central

    Paino, Annamari; Tuominen, Heidi; Jääskeläinen, Mari; Alanko, Jonna; Nuutila, Jari; Asikainen, Sirkka E.; Pelliniemi, Lauri J.; Pöllänen, Marja T.; Chen, Casey; Ihalin, Riikka

    2011-01-01

    Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation. PMID:21533109

  18. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  19. Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite

    PubMed Central

    Longo, PL; Ota-Tsuzuki, C; Nunes, ACR; Fernandes, BL; Mintz, K; Fives-Taylor, P; Mayer, MPA

    2009-01-01

    The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces. PMID:24031399

  20. Interactions of extracts from selected chewing stick sources with Aggregatibacter actinomycetemcomitans

    PubMed Central

    2012-01-01

    Background Aggregatibacter actinomycetemcomitans produces a leukotoxin that activates a pro-inflammatory death of human monocytes/macrophages. A specific clone of this bacterium (JP2) has a 530-base pair deletion in the leukotoxin promoter gene and significantly enhanced expression of leukotoxin. This specific clone of A. actinomycetemcomitans is common in some African populations and has a strong association with periodontal attachment loss in adolescents in these populations. Chewing sticks of plant origin are commonly used as oral hygiene tool in Africa, but their role as a therapeutic agent in periodontal disease is poorly investigated. Results Ethanol extracts were made from 7 common plants used as chewing sticks in West-Africa. None of the tested extracts inhibited growth of A. actinomycetemcomitans. However, extracts from Psidium guajava (Guava) completely neutralized the cell death and pro-inflammatory response of human leukocytes induced by the leukotoxin. None of the six other tested chewing stick extracts showed this effect. Conclusions The discovery that extracts from Guava efficiently neutralizes A. actinomycetemcomitans leukotoxicity might lead to novel therapeutic agents and strategies for prevention and treatment of aggressive forms of periodontitis induced by infections with the highly leukotoxic JP2 clone of this bacterium. PMID:22537711

  1. Withaferin A inhibits inflammatory responses induced by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

    PubMed

    Noh, Eui-Jeong; Kang, Ming-Jung; Jeong, Yu-Jin; Lee, Jun-Young; Park, Jung-Hwan; Choi, Hye-Jin; Oh, Sang-Muk; Lee, Kyung-Bok; Kim, Dong-Jae; Shin, Ji-Ae; Cho, Sung-Dae; Park, Jong-Hwan

    2016-07-01

    Periodontitis is a progressive chronic inflammatory disease and a major cause of tooth loss in humans. As a withanolides, withaferin A (WA) is known to exhibit strong anti‑inflammatory activity. The present study examined whether WA inhibited inflammatory responses in macrophages in response to two representative periodontal pathogens, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Murine bone marrow‑derived macrophages (BMDMs) were used in this study and cytokine production in culture supernatants was measured by enzyme‑linked immunosorbent assays. Western blot analysis was performed to determine the activation of nuclear factor‑κB and mitogen‑activated protein kinases (MAPKs) and the expression of inducible nitric oxide synthase (iNOS), toll‑like receptor (TLR) 2 and TLR4. The production of nitric oxide (NO) was determined by the Griess reaction. WA treatment was shown to decrease interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α production in BMDMs in response to F. nucleatum and A. actinomycetemcomitans in a dose‑dependent manner. The phosphorylation of IκB‑α and MAPKs (p38, extracellular signal‑regulated kinases and c‑Jun N‑terminal kinases) induced by F. nucleatum and A. actinomycetemcomitans was also inhibited by WA. F. nucleatum and A. actinomycetemcomitans induced iNOS expression and NO production in BMDMs, which was inhibited by WA in a dose‑dependent manner. WA also reduced endogenous and induced expression of TLR2 and TLR4 in these cells. These results suggest that WA may be a potential therapeutic agent or preventive additive for periodontitis control. PMID:27220676

  2. [Microbiological approach to a possible infective endocarditis case caused by Aggregatibacter actinomycetemcomitans].

    PubMed

    Gürcan, Şaban; Ünlü, Selahattin; Kuloğlu, Figen; Karadenizli, Aynur; Kuşkucu, Mert Ahmet

    2016-04-01

    Aggregatibacter (Actinobacillus) actinomycetemcomitans, a small, gram-negative coccobacillus that grows slow and fastidious, is generally colonized in the oral cavity. It is a rarely seen bacterium because of the difficulty of isolation but it can be a causative agent for dental infections and infective endocarditis (IE) particularly in the persons having prosthetic heart valves. In this report, a possible IE case caused by A.actinomycetemcomitans in a patient with aortic valve replacement has been presented. A 36-year-old man has admitted to Trakya University Hospital, Health Center for Medical Research and Practice, with the complaints of chills, malaise, intermittent fever, severe arthralgia and weight loss (20 kg). During his follow-up period, the blood cultures that were obtained three week intervals yielded the identical gram-negative coccobacilli morphology. The patient was then diagnosed as possible IE on the basis of having one major (growth of the typical microorganisms that may cause IE in two different blood cultures) and two minor (presence of prosthetic valve and high fever) criterias. The isolate could not be identified with conventional methods, while it was identified as Francisella tularensis with VITEK 2 (bioMerieux, France) system. Hence this identification was not confirmed by real-time Taqman polymerase chain reaction, so MALDI-TOF mass spectrometry was used to identify this bacteria. In the first run of the study, the isolate was named as Shigella dysenteriae initially, however when it was retested the next day it was identified as A.actinomycetemcomitans. In order to enlighten these conflicting results, 16S and 23S ribosomal DNA sequence analysis was performed, and consequently the bacterium was identified as A.actinomycetemcomitans. Doxycycline (2 x 100 mg po, 20 days) and streptomycin (2 x 10 mg/kg im, 10 days) therapy were initiated, considering the initial suspicious identification (F.tularensis), and on the fifth day of therapy the

  3. Aggregatibacter actinomycetemcomitans: Virulence of its leukotoxin and association with aggressive periodontitis

    PubMed Central

    Åberg, Carola Höglund; Kelk, Peyman; Johansson, Anders

    2015-01-01

    Periodontitis is an infection-induced inflammatory disease that causes loss of the tooth supporting tissues. Much focus has been put on comparison of the microbial biofilm in the healthy periodontium with the diseased one. The information arising from such studies is limited due to difficulties to compare the microbial composition in these two completely different ecological niches. A few longitudinal studies have contributed with information that makes it possible to predict which individuals who might have an increased risk of developing aggressive forms of periodontitis, and the predictors are either microbial or/and host-derived factors. The most conspicuous condition that is associated with disease risk is the presence of Aggregatibacter actinomycetemcomitans at the individual level. This Gram-negative bacterium has a great genetic variation with a number of virulence factors. In this review we focus in particular on the leukotoxin that, based on resent knowledge, might be one of the most important virulence factors of A. actinomycetemcomitans. PMID:25494963

  4. Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

    PubMed

    Stevens, Roy H; de Moura Martins Lobo Dos Santos, Caroline; Zuanazzi, David; de Accioly Mattos, Marcelo Barbosas; Ferreira, Davis Fernandes; Kachlany, Scott C; Tinoco, Eduardo M B

    2013-01-01

    Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens. PMID:23022667

  5. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

    PubMed

    Velusamy, Senthil Kumar; Sampathkumar, Vandana; Godboley, Dipti; Fine, Daniel H

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  6. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

    PubMed Central

    Godboley, Dipti; Fine, Daniel H.

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  7. Detection of antimicrobial activity of banana peel (Musa paradisiaca L.) on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Kapadia, Suraj Premal; Pudakalkatti, Pushpa S.; Shivanaikar, Sachin

    2015-01-01

    Introduction and Aim: Banana is used widely because of its nutritional values. In past, there are studies that show banana plant parts, and their fruits can be used to treat the human diseases. Banana peel is a part of banana fruit that also has the antibacterial activity against microorganisms but has not been studied extensively. Since, there are no studies that relate the antibacterial activity of banana peel against periodontal pathogens. Hence, the aim of this study is to determine the antimicrobial activity of banana peel extract on Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Material and Methods: Standard strains of P. gingivalis and A. actinomycetemcomitans were used in this study which was obtained from the in-house bacterial bank of Department of Molecular Biology and Immunology at Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre. The banana peel extract was prepared, and the antibacterial activity was assessed using well agar diffusion method and minimum inhibitory concentration was assessed using serial broth dilution method. Results: In the current study, both the tested microorganisms showed antibacterial activity. In well diffusion method, P. gingivalis and A. actinomycetemcomitans showed 15 mm and 12 mm inhibition zone against an alcoholic extract of banana peel, respectively. In serial broth dilution method P. gingivalis and A. actinomycetemcomitans were sensitive until 31.25 μg/ml dilutions. Conclusion: From results of the study, it is suggested that an alcoholic extract of banana peel has antimicrobial activity against P. gingivalis and A. actinomycetemcomitans. PMID:26681854

  8. TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

    PubMed Central

    Crosby, Juan A.; Kachlany, Scott C.

    2007-01-01

    Aggregatibacter actinomycetemcomitansW is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for β -hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxin-producing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer β -hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for “toxin and drug export”. Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system. PMID:17116373

  9. A Consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis Is Present in Sites Prior to Bone Loss in a Longitudinal Study of Localized Aggressive Periodontitis

    PubMed Central

    Markowitz, Kenneth; Fairlie, Karen; Tischio-Bereski, Debbie; Ferrendiz, Javier; Furgang, David; Paster, Bruce J.; Dewhirst, Floyd E.

    2013-01-01

    Aggregatibacter actinomycetemcomitans-induced localized aggressive periodontitis (LAP) in African-American adolescents has been documented but is poorly understood. Two thousand fifty-eight adolescents aged 11 to 17 years were screened for their periodontal status and the presence of A. actinomycetemcomitans in their oral cavity. Seventy-one A. actinomycetemcomitans-negative and 63 A. actinomycetemcomitans-positive periodontally healthy subjects were enrolled, sampled, examined, and radiographed yearly for 3 years. Gingival and periodontal pocket depth and attachment levels were recorded. Disease presentation was characterized by bone loss (BL). Subgingival sites were sampled every 6 months to assess (i) the role of A. actinomycetemcomitans in BL and (ii) the association of A. actinomycetemcomitans and other microbes in their relationships to BL. Sixteen of 63 subjects with A. actinomycetemcomitans developed BL (the other 47 subjects with A. actinomycetemcomitans had no BL). No A. actinomycetemcomitans-negative subjects developed BL. Human oral microbe identification microarray (HOMIM) was used for subgingival microbial assessment. On a subject level, pooled data from A. actinomycetemcomitans-positive subjects who remained healthy had higher prevalences of Streptococcus and Actinomyces species, while A. actinomycetemcomitans-positive subjects with BL had higher prevalences of Parvimonas micra, Filifactor alocis, A. actinomycetemcomitans, and Peptostreptococcus sp. human oral taxon 113 (HOT-113). At vulnerable sites, A. actinomycetemcomitans, Streptococcus parasanguinis, and F. alocis levels were elevated prior to BL. In cases where the three-organism consortium (versus A. actinomycetemcomitans alone) was detected, the specificity for detecting sites of future BL increased from 62% to 99%, with a sensitivity of 89%. We conclude that detecting the presence of A. actinomycetemcomitans, S. parasanguinis, and F. alocis together indicates sites of future BL in LAP. A

  10. Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Shanmugam, Mayilvahanan; El Abbar, Faiha; Ramasubbu, Narayanan

    2015-01-01

    Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans. PMID:26221956

  11. Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT

    PubMed Central

    Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Bueno-Silva, Bruno; DiRienzo, Joseph M.; Mayer, Marcia P. A.

    2016-01-01

    The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis. PMID:27064424

  12. Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

    PubMed

    Ragunath, C; DiFranco, K; Shanmugam, M; Gopal, P; Vyas, V; Fine, D H; Cugini, C; Ramasubbu, N

    2016-08-01

    Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae β-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms. PMID:26280561

  13. Occurrence of Aggregatibacter actinomycetemcomitans in Indian chronic periodontitis patients and periodontally healthy adults

    PubMed Central

    Joshi, Vinayak Mahableshwar; Bhat, Kishore Gajanan; Kugaji, Manohar Suresh; Ingalgi, Preeti Shivaji

    2016-01-01

    Background: Aggregatibacter actinomycetemcomitans (Aa), an important primary periodontal pathogen, is known for its strong virulence characteristics that cause periodontal disease. We investigated Aa occurrence in Indian individuals using culture and 16 s rDNA polymerase chain reaction (PCR). Materials and Methods: A cross-sectional study with 100 participants each in the healthy and chronic periodontitis (CP) groups was conducted. The subgingival plaque was collected and immediately plated on selective media for Aa. The remaining plaque samples were used for DNA extraction. PCR was performed using specific primers for Aa. Statistical Analysis Used: The detection of bacteria and the clinical parameters between the groups were compared using the Mann–Whitney U-test. For assessing the agreement between the results of anaerobic culture and PCR, Kappa analyses were performed. Results: Aa levels using culture and PCR was 51% and 69% in the CP group and 12% and 30% in the healthy group, respectively. The two groups showed significant differences (P < 0.00001). The detection accuracy of culture and PCR was assessed, and the coefficient of accuracy (k) was highly significant in the healthy (0.3103; P < 0.0001) and CP groups (0.1536; P < 0.0497). Conclusions: Aa was predominantly found in the CP group compared with the healthy group, which is consistent with previous findings. Our results showed that both techniques can be used for detecting Aa. An ideal technique for detecting subgingival microorganisms should be carefully selected depending on the scope of the intended future work. PMID:27143824

  14. Bioactive glass combined with bisphosphonates provides protection against biofilms formed by the periodontal pathogen Aggregatibacter actinomycetemcomitans.

    PubMed

    Hiltunen, Anna K; Skogman, Malena E; Rosenqvist, Kirsi; Juvonen, Helka; Ihalainen, Petri; Peltonen, Jouko; Juppo, Anne; Fallarero, Adyary

    2016-03-30

    Biofilms play a pivotal role in the progression of periodontitis and they can be treated with antiseptics (i.e. chlorhexidine) or antibiotics, but these therapeutic alternatives are unable of ameliorating periodontal alveolar bone loss, which has been, on the other hand, successfully treated with bone-preserving agents. The improved bone formation achieved in animal models by the combination of two such agents: bioactive glass (BAG) and bisphosphonates has attracted the interest for further exploring dental applications. However, the antimicrobial effects that may result from combining them have not been yet investigated. Here, our aim was to explore the anti-biofilm effects that could result from combining BAG with bisphosphonates, particularly in a dental biofilm model. The experiments were performed with an oral cavity single-specie (Aggregatibacter actinomycetemcomitans) biofilm assay, which was optimized in this contribution. Risedronate displayed an intrinsic anti-biofilm effect, and all bisphosphonates, except clodronate, reduced biofilm formation when combined with BAG. In particular, the anti-biofilm activity of risedronate was significantly increased by the combination with BAG. Since it has been proposed that some of the antimicrobial effects of BAG are caused by local pH changes, studies of pH variations were performed to gain a mechanistic understanding. However, the observed anti-biofilm effects could not be explained with lowered pHs. Overall, these results do provide further support for the promising use of bisphosphonate-BAG combinations in dental applications. These findings are particularly relevant for patients undergoing cancer chemotherapy, or osteoporotic patients, which are known to be more vulnerable to periodontitis. In such cases, bisphosphonate treatment could play a double positive effect: local treatment of periodontitis (in combination with BAG) and systemic treatment of osteoporosis, prevention of hypercalcemia and metastases. PMID

  15. Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.

    PubMed

    Balashova, N; Dhingra, A; Boesze-Battaglia, K; Lally, E T

    2016-02-01

    Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells. PMID:26361372

  16. Antibacterial Effect of an Herbal Product Persica on Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans: An In-Vitro Study

    PubMed Central

    Jelvehgaran Esfahani, Zahra; Kadkhoda, Zeinab; Eshraghi, Seyed Saeed; Salehi Surmaghi, Mohammad Hossein

    2014-01-01

    Objective: The plant Salvadora persica is used for oral hygiene in many parts of the world. It has been suggested that it has antibacterial properties, in addition to its ability to mechanically remove plaques. The aim of this study was to assess the antimicrobial activity of the herbal product Persica containing Salvadora persica against periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in vitro. Materials and Methods: Fifty patients with moderate and severe periodontitis were recruited. Using paper points, subgingival plaque samples were taken from pockets with attachment loss ≥ 3mm. The samples were subjected to microbial culture to yield P. gingivalis and A. actinomycetemcomitans. The ditch plate method was used for antimicrobial susceptibility testing of the bacteria to Persica compared to chlorhexidine and distilled water. The growth inhibition zones of microorganisms around the ditches were measured in millimeters. The data were analyzed using SPSS 16. Freidman test and Wilcoxon signed ranks test with Bonferroni adjustment were used for analysis of variance with 5% significance level. P<0.05 for main comparisons and P< 0.017 for multiple comparisons were considered statistically significant. Results: P. gingivalis was sensitive to chlorhexidine and persica. There was a significant difference (P=0.001) between antimicrobial activity of chlorhexidine (mean 28.733mm, SD 5.216) and Persica (mean 16.333mm, SD 5.259) compared to water against P. gingivalis. There was a significant difference (P< 0.001) between the antimicrobial activity of chlorhexidine (24.045mm, SD 3.897) and Persica (0.545mm, SD 2.558) with respect to A. actinomycetemcomitans. There was no significant difference (P=0.317) between the antimicrobial activity of Persica and water against A. actinomycetemcomitans. Conclusion: The herbal product Persica had significant antimicrobial activity against P. gingivalis and negligible antimicrobial activity against A

  17. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  18. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    ABSTRACT In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased

  19. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes.

    PubMed

    Alvarez, Carla; Benítez, Alvaro; Rojas, Leticia; Pujol, Myriam; Carvajal, Paola; Díaz-Zúñiga, Jaime; Vernal, Rolando

    2015-01-01

    In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a

  20. Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

    PubMed

    Sosroseno, Wihaskoro; Herminajeng, Endang; Susilowati, Heni; Budiarti, Sri

    2002-12-01

    The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases. PMID:16887678

  1. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans.

    PubMed

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness R a and R z were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2-5 µm) and protruding hills (10-50 µm) on Ti substrates were produced after electrochemical anodization with higher R a and R z surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  2. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans

    PubMed Central

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness Ra and Rz were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by  Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2–5 µm) and protruding hills (10–50 µm) on Ti substrates were produced after electrochemical anodization with higher Ra and Rz surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  3. The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans: evolutionary aspects, epidemiology and etiological role in aggressive periodontitis.

    PubMed

    Haubek, Dorte

    2010-09-01

    For many years, attention has been given to the oral bacterium Aggregatibacter actinomycetemcomitans, as a species possibly implicated in the etiology of aggressive periodontitis in adolescents. One of the major virulence factors of A. actinomycetemcomitans is the leukotoxin which is able to kill important cells of the immune system. As demonstrated in population genetic analyses, the population structure of A. actinomycetemcomitans is mainly clonal with evolutionary lineages corresponding to the serotypes. A particular highly leukotoxic clone (JP2) of serotype b has been discovered. The JP2 clone, with an estimated origin some 2400 years ago, is found to be highly conserved, based on analyses of a collection of JP2 clone strains collected through more than 20 years from individuals of diverse origin and living geographically widespread. Despite demonstration of minor evolutionary changes within the genome of JP2 clone strains of A. actinomycetemcomitans, the JP2 clone strains constitute a unique clonal type, the characteristics of which include a 530 basepair deletion in the leukotoxin operon implicated in the enhanced leukotoxic activity of the clone. Mapping of the geographic occurrence of the JP2 clone of A. actinomycetemcomitans has revealed that its colonization is largely restricted to individuals of African descent. Characteristic mutations, which allow JP2 clone isolates from the Mediterranean region to be distinguished from isolates from West Africa, including the Cape Verde islands, suggest that the JP2 clone initially emerged as a distinct genotype in the Mediterranean region of Africa and subsequently spread to West Africa, from where it might have been transferred to the American continent during the transatlantic slave trade. The finding of a sustained selective colonization of individuals of African descent, despite geographical separation from the African continent for centuries, suggests that the JP2 clone might have a distinct host tropism

  4. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160.

    PubMed

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  5. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160

    PubMed Central

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116–188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116–188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  6. Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

    PubMed Central

    Thay, Bernard; Damm, Anna; Kufer, Thomas A.; Wai, Sun Nyunt

    2014-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated by A. actinomycetemcomitans could deliver multiple proteins, including biologically active cytolethal distending toxin (CDT), into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work, we have used immunoelectron and confocal microscopy analysis and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). PMID:25024364

  7. Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material.

    PubMed

    Sosroseno, W; Bird, P S; Seymour, G J

    2011-10-01

    Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with L-N(6)-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A(2) (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. L-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA(2) but not PI-3K-dependent fashion. PMID:21736946

  8. Evaluation of chemical composition and efficacy of Chinese propolis extract on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Agarwal, Garima; Vemanaradhya, Gayathri G.; Mehta, Dhoom S.

    2012-01-01

    Background: Propolis as a natural remedy has maintained its popularity over long periods of time. The aim of this study was to determine the chemical composition in terms of total phenolic compounds and flavonoids present in Chinese propolis and to carry out an in vitro evaluation of its antimicrobial activity and the minimal inhibitory concentrations for Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: From the ethanolic extract of propolis (EEP), total phenol content was determined by the Folin–Ciocalteau method, flavones and flavonols by the modified aluminum chloride colorimetric method, and flavanones by the 2.4-dinitrophenylhydrazine (2,4-DNP) method. Agar well diffusion assay was used to evaluate the antimicrobial potential of propolis against Pg and Aa. The minimum inhibitory concentration of propolis against the two bacteria was determined using serial tube dilution technique. Results: The total concentration of phenol in the EEP was 19.44%, flavones and flavonols 2.616%, and flavanones 16.176%. The inhibitory zone depicting antimicrobial activity ranged from 18 to 25 mm for Pg and from 12 to 14 mm for Aa. The concentration range of Chinese propolis that is sensitive to inhibit the growth of Pg was 0.1–0.0125 μg/ml and for Aa it was 0.1–0.025 μg/ml. Conclusion: These data suggest that Chinese propolis has potent antimicrobial activity against the two periodontopathogens, suggesting its possible use as a natural alternative to the widely used synthetic antibiotics for periodontal therapy. PMID:23293477

  9. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis.

    PubMed

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35-76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants' health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0-0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1-57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0-88.2) and body mass index (OR = 3.0; 95%CI 1.7-5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is suggested. PMID

  10. Al(III), Pd(II), and Zn(II) phthalocyanines for inactivation of dental pathogen Aggregatibacter actinomycetemcomitans as planktonic and biofilm-cultures

    NASA Astrophysics Data System (ADS)

    Kussovski, V.; Mantareva, V.; Angelov, I.; Avramov, L.; Popova, E.; Dimitrov, S.

    2012-06-01

    The Gram-negative, oral bacterium Aggregatibacter actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. The new periodontal disease treatments are emergence in order to prevent infection progression. Antimicrobial photodynamic therapy (a-PDT) can be a useful tool for this purpose. It involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent in the presence of oxygen for eradication of target cells, and appears effective in photoinactivation of microorganisms. The phthalocyanine metal complexes of Pd(II)- (PdPcC) and Al(III)- (AlPc1) were evaluated as photodynamic sensitizers towards a dental pathogen A. actinomycetemcomitans in comparison to the known methylpyridyloxy-substituted Zn(II) phthalocyanine (ZnPcMe). The planktonic and biofilm-cultivated species of A. actinomycetemcomitans were treated. The photophysical results showed intensive and far-red absorbance with high tendency of aggregation for Pd(II)-phthalocyanine. The dark toxicities of both photosensitizers were negligible at concentrations used (< 0.5 log decrease of viable cells). The photodynamic response for planktonic cultured bacteria was full photoinactivation after a-PDT with ZnPcMe. In case of the newly studied complexes, the effect was lower for PdPcC (4 log) as well as for AlPc1 (1.5-2 log). As it is known the bacterial biofilms were more resistant to a-PDT, which was confirmed for A. actinomycetemcomitans biofilms with 3 log reductions of viable cells after treatment with ZnPcMe and approximately 1 log reduction of biofilms after PdPcC and AlPc1. The initial results suggest that a-PDT can be useful for effective inactivation of dental pathogen A. actinomycetemcomitans.

  11. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism

    PubMed Central

    Weigel, WA; Demuth, DR; Torres-Escobar, A; Juárez-Rodríguez, MD

    2015-01-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment. PMID:25923132

  12. Colonization and Persistence of Labeled and “Foreign” Strains of Aggregatibacter actinomycetemcomitans Inoculated into the Mouths of Rhesus Monkeys

    PubMed Central

    Fine, Daniel H.; Karched, Maribasappa; Furgang, David; Sampathkumar, Vandana; Velusamy, Senthil; Godboley, Dipti

    2015-01-01

    Aggregatibacter actinomycetemcomitans (Aa) is a pathobiont and part of a consortium of bacteria that can lead to periodontitis in humans. Our aim was to develop a model for oral inoculation of labeled Aa into a suitable host in order to study Aa traits and ecological factors that either enhance or repress its persistence. Primate species were screened for Aa to select a host for colonization studies. Macaca mulatta (Rhesus/Rh) was selected. Rh Aa strains were isolated, subjected to sequencing and functional analysis for comparison to human strains. “Best” methods for microbial decontamination prior to inoculation were assessed. Three groups were studied; Group 1 (N=5) was inoculated with Aa Spectinomycin resistant (SpecR) Rh strain 4.35, Group 2 (N=5) inoculated with Aa SpecR human strain IDH 781, and Group 3 (N=5) the un-inoculated control. Repeated feeding with pancakes spiked with SpecRAa followed high dose oral inoculation. Cheek, tongue, and plaque samples collected at baseline 1, 2, 3, and 4 weeks after inoculation were plated on agar; 1) selective for Aa, 2) enriched for total counts, and 3) containing 50 µg/ml of Spec. Aa was identified by colonial morphology and DNA analysis. Rh and human Aa had > 93–98 % genome identity. Rh Aa attached to tissues better than IDH 781 in vitro (p < 0.05). SpecR IDH 781 was not recovered from any tissue at any time; whereas, RhSpecR 4.35 was detected in plaque, but never tongue or cheek, in all monkeys at all times (> 1 × 105 colonies/ml; p < 0.001). In conclusion, the primate model provides a useful platform for studying integration of Aa strains into a reduced but established oral habitat. Primate derived SpecRAa was consistently detected in plaque at all collection periods; however, human derived Aa was never detected. The model demonstrated both microbial as well as tissue specificity. PMID:26213715

  13. Determination of antibacterial activity of green coffee bean extract on periodontogenic bacteria like Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Bharath, Nagaraj; Sowmya, Nagur Karibasappa; Mehta, Dhoom Singh

    2015-01-01

    Background: The aim of this study was to evaluate the antibacterial activity of pure green coffee bean extract on periodonto pathogenic bacteria Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were used to assess the antibacterial effect of pure green coffee bean extract against periodonto pathogenic bacteria by micro dilution method and culture method, respectively. Results: MIC values of Pg, Pi and Aa were 0.2 μg/ml whereas Fn showed sensitive at concentration of 3.125 μg/ml. MBC values mirrors the values same as that of MIC. Conclusion: Antimicrobial activity of pure green coffee bean extract against Pg, Pi, Fn and Aa suggests that it could be recommended as an adjunct to mechanical therapy in the management of periodontal disease. PMID:26097349

  14. Evaluation of antimicrobial action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans a major periodontal pathogen using polymerase chain reaction

    PubMed Central

    Kush, Anil; Thakur, Rachna; Patil, Sandya Devi S.; Paul, Santhosh T.; Kakanur, Madhu

    2015-01-01

    Background: In the present scenario, we are made available with chemomechanical caries removal system containing a natural proteolytic enzyme for the ease in the excavation of infected dentine. The additive action for these agents is providing antimicrobial and anti-inflammatory properties. Aim: This study was undertaken for assessing the action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans. Materials and Methods: The samples were collected for cultivation of the periodontal pathogen from the clinical periodontal pockets using sterile paper points. The samples cultured under suitable conditions were analyzed with quantitative polymerase chain reaction targeting 16s r-DNA. The samples were divided into three groups namely, Group A: Control, Group B: With Papacarie Duo, Group C: With Carie Care. The pathogen inoculums plugs were inserted in the petri dishes containing chemically defined medium and the experimental gels at different concentrations and were incubated under optimal conditions. The inhibition of growth of the pathogen was studied visually. Results: There was visual inhibition of growth for Group B and C and also exhibited a dose-dependent effect also. Conclusion: Based on the results of the present study, Carie Care™ gel demonstrated better antimicrobial action against A. actinomycetemcomitans which is a major periodontal disease causing pathogen. PMID:26681861

  15. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis

    PubMed Central

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35–76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants’ health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0–0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1–57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0–88.2) and body mass index (OR = 3.0; 95%CI 1.7–5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is

  16. Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results is pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with mass spectrometry was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild-type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild-type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the morC mutant strain is discussed. PMID:25684173

  17. Characterization of a natural mouse monoclonal antibody recognizing epitopes shared by oxidized low-density lipoprotein and chaperonin 60 of Aggregatibacter actinomycetemcomitans.

    PubMed

    Wang, Chunguang; Kankaanpää, Jari; Kummu, Outi; Turunen, S Pauliina; Akhi, Ramin; Bergmann, Ulrich; Pussinen, Pirkko; Remes, Anne M; Hörkkö, Sohvi

    2016-06-01

    Natural antibodies are predominantly antibodies of the IgM isotype present in the circulation of all vertebrates that have not been previously exposed to exogenous antigens. They are often directed against highly conserved epitopes and bind to ligands of varying chemical composition with low affinity. In this study we cloned and characterized a natural mouse monoclonal IgM antibody selected by binding to malondialdehyde acetaldehyde epitopes on low-density lipoprotein (LDL). Interestingly, the IgM antibody cross-reacted with Aggregatibacter actinomycetemcomitans (Aa) bacteria, a key pathogenic microbe in periodontitis reported to be associated with risk factor for atherosclerosis, thus being named as Aa_Mab. It is more intriguing that the binding molecule of Aa to Aa_Mab IgM was found to be Aa chaperonin 60 or HSP60, a member of heat-shock protein family, behaving not only as a chaperone for correct protein folding but also as a powerful virulence factor of the bacteria for inducing bone resorption and as a putative pathogenic factor in atherosclerosis. The findings will highlight the question of whether molecular mimicry between pathogen components and oxidized LDL could lead to atheroprotective immune activity, and also would be of great importance in potential application of immune response-based preventive and therapeutic strategies against atherosclerosis and periodontal disease. PMID:26786003

  18. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo. PMID:27594851

  19. Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription.

    PubMed

    Reddi, K; Nair, S P; White, P A; Hodges, S; Tabona, P; Meghji, S; Poole, S; Wilson, M; Henderson, B

    1996-03-15

    The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material. This material contained potent bone-resorbing activity. We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis. In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50%. This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS. The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion

  20. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

    PubMed Central

    Kalmar, J R; Arnold, R R

    1988-01-01

    Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen. PMID:3417349

  1. Classification, Identification, and Clinical Significance of Haemophilus and Aggregatibacter Species with Host Specificity for Humans

    PubMed Central

    2014-01-01

    SUMMARY The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology

  2. Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans

    PubMed Central

    Kaplan, Jeffrey B.; Meyenhofer, Markus F.; Fine, Daniel H.

    2003-01-01

    The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony. PMID:12562811

  3. Functional Mapping of an Oligomeric Autotransporter Adhesin of Aggregatibacter actinomycetemcomitans▿

    PubMed Central

    Yu, Chunxiao; Ruiz, Teresa; Lenox, Christopher; Mintz, Keith P.

    2008-01-01

    Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity. PMID:18310342

  4. A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease

    PubMed Central

    Schreiner, Helen; Li, Yu; Cline, Joshua; Tsiagbe, Vincent K.; Fine, Daniel H.

    2013-01-01

    Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4+ T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease. PMID:23936002

  5. Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

    PubMed Central

    Damek-Poprawa, Monika; Jang, Jae Yeon; Volgina, Alla; Korostoff, Jonathan

    2012-01-01

    The cytolethal distending toxin (Cdt), produced by some clinically important Gram-negative bacterial species, is related to the family of AB-type toxins. Three heterologous proteins (CdtA, CdtB, and CdtC) and a genotoxin mode of action distinguish the Cdt from others in this toxin class. Crystal structures of several species-specific Cdts have provided a basis for predicting subunit interactions and functions. In addition, empirical studies have yielded significant insights into the in vivo interactions of the Cdt subunits. However, there are still critical gaps in information about the intoxication process. In this study, a novel protein tagging technology was used to localize the subunits in Chinese hamster ovary cells (CHO-K1). A tetracysteine motif was engineered in each subunit, and in subunits with mutations in predicted functional domains, to permit detection with the fluorescein arsenical hairpin binding (FlAsH) dye Lumio green. Live-cell imaging, in conjunction with confocal microscopy, was used to capture the locations of the individual subunits in cells intoxicated, under various conditions, with hybrid heterotrimers. Using this approach, we observed the following. (i) The CdtA subunit remains on the cell surface of CHO cells in association with cholesterol-containing and cholesterol-depleted membrane. (ii) The CdtB subunit is exclusively in the cytosol and, after longer exposure times, localizes to the nucleus. (iii) The CdtC subunit is present on the cell surface and, to a greater extent, in the cytosol. These observations suggest that CdtC, but not CdtA, functions as a chaperone for CdtB entry into cells. PMID:22645284

  6. Immunosuppressive properties of Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Rabie, G; Lally, E T; Shenker, B J

    1988-01-01

    Actinobacillus actinomycetemcomitans produces a leukotoxin that kills human polymorphonuclear cells (PMNs) and monocytes but not lymphocytes. In this study, we examined A. actinomycetemcomitans leukotoxin for its ability to alter human peripheral blood lymphocyte (HPBL) responsiveness. After a 90-min exposure to the leukotoxin, all monocytes were killed and HPBL responsiveness to mitogens and antigens was significantly inhibited. The ability of the leukotoxin to inhibit HPBL responses was not surprising, since monocytes and macrophages are required for many lymphocyte functions. However, we were unable to totally restore HPBL responsiveness when adherent autologous monocytes were added back to cultures of leukotoxin-treated lymphocytes. These studies demonstrate that A. actinomycetemcomitans leukotoxin may also exert nonlethal effects directly on lymphocytes. Furthermore, impaired lymphocyte function did not appear to be the result of indirect effects of products released by dying monocytes. Although it is not clear how A. actinomycetemcomitans acts to cause disease, several investigators have proposed that impaired host defenses may play a pivotal role. Several studies have demonstrated defects in PMN, monocyte, and lymphocyte function in patients with periodontal disease. These findings, along with the data presented in this paper, support the hypothesis that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. The effects of this suppression may be to enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism. PMID:3335399

  7. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  8. Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Nowotny, A; Behling, U H; Hammond, B; Lai, C H; Listgarten, M; Pham, P H; Sanavi, F

    1982-01-01

    Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- and proteolysis-sensitive leukotoxin in similar preparations. Accordingly, the microvesicles released by strain Y4 may contain, in addition to endotoxin, several potent substances which are highly toxic and active in bone resorption, and these may be significant factors in the pathogenesis of periodontal diseases. PMID:7049947

  9. Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

    PubMed

    Nowotny, A; Behling, U H; Hammond, B; Lai, C H; Listgarten, M; Pham, P H; Sanavi, F

    1982-07-01

    Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- and proteolysis-sensitive leukotoxin in similar preparations. Accordingly, the microvesicles released by strain Y4 may contain, in addition to endotoxin, several potent substances which are highly toxic and active in bone resorption, and these may be significant factors in the pathogenesis of periodontal diseases. PMID:7049947

  10. Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells.

    PubMed Central

    Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells. Images PMID:8406899

  11. Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

    PubMed

    Kato, Satsuki; Sugimura, Norihiko; Nakashima, Keisuke; Nishihara, Tatsuji; Kowashi, Yusuke

    2005-03-01

    It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans

  12. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

    PubMed

    Lally, E T; Kieba, I R; Demuth, D R; Rosenbloom, J; Golub, E E; Taichman, N S; Gibson, C W

    1989-02-28

    The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity. PMID:2647082

  13. Lipopolysaccharide Endotoxins

    PubMed Central

    Raetz, Christian R. H.; Whitfield, Chris

    2008-01-01

    Summary Since lipopolysaccharide endotoxins of Gram-negative bacteria were last reviewed in this series in 1990, much has been learned about the assembly and signaling functions of these remarkable glycoconjugates. Lipopolysaccharides typically consist of a hydrophobic domain known as lipid A (or endotoxin), a non-repeating “core” oligosaccharide, and a distal polysaccharide (or O-antigen). The flood of recent genomic data has made it possible to study lipopolysaccharide assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and to create mutants or hybrid constructs with novel properties. Unexpectedly, key genes for lipid A biosynthesis have also been found in higher plants, indicating that eucaryotic lipid A-like molecules may exist. The carbohydrate diversity of lipopolysaccharides is better appreciated now than ten years ago, but much remains to be learned about function. Sequence comparisons suggest that extensive lateral transfer of genes for the assembly of O-antigens has occurred among bacteria. The most significant finding in the field of endotoxin biology since 1990 has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. The latter belongs to a family of innate immunity receptors, all of which possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking the inflammatory side effects of sepsis. Future progress will require insights into lipopolysaccharide-protein recognition at the atomic level, greater understanding of intra- and inter-cellular lipopolysaccharide trafficking, and incisive biological approaches that combine the tools of bacterial and animal genetics. PMID:12045108

  14. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  15. Recurrent infective endocarditis due to Aggregatibacter aphrophilus and Staphylococcus lugdunensis.

    PubMed

    Hidalgo-García, L; Hurtado-Mingo, A; Olbrich, P; Moruno-Tirado, A; Neth, O; Obando, I

    2015-03-01

    Uncommon microorganisms are increasingly being recognized as causative agents of paediatric infectious endocarditis (IE). We report a 4-year old girl with congenital heart disease, who suffered from 2 IE episodes secondary to Aggregatibacter aphrophilus (formerly Haemophilus aphrophilus) and Staphylococcus lugdunensis, both rarely reported pathogens in this age group. The patient was initially successfully treated with prolonged intravenous antibiotic courses, however removal of the Contegra valved conduit during the second episode was required due to recurrence of fever and development of pulmonary embolism despite completion of antibiotic therapy. A. aphrohilus is a member of the fastidious gram negative microorganisms of the HACEK group (Haemophilus spp., Aggregatibacter spp, Cardiobaterium hominis, Eikenella corrodens and Kingella kingae), that colonize the oropharynx and are a recognised cause of IE. Prognosis of children with IE due to HACEK group members varies, half of them suffering from complications and mortality rates of 10-12.5%. Although S. lugdunensis belongs to coagulase negative staphylococci (CONS), it behaves more like S. aureus species rather than CONS. This microorganism is a well-described cause of endocarditis in adult patients, associated with high requirements of surgical procedures and mortality (42-78%). In conclusion, paediatric IE can be caused by uncommon microorganisms associated with severe complications and potential fatality. The isolation of S. lugdunensis or A. aphrophilus in febrile patients should be considered clinically relevant and cardiac involvement must be ruled out. Those patients with proved IE will require prolonged intravenous antibiotic courses and in complicated cases surgical intervention. PMID:25751682

  16. Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism.

    PubMed

    Müller, H P; Lange, D E; Müller, R F

    1989-07-01

    The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a. PMID:2760252

  17. Biogenesis of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Holotoxin

    PubMed Central

    Ueno, Yoko; Ohara, Masaru; Kawamoto, Toru; Fujiwara, Tamaki; Komatsuzawa, Hitoshi; Oswald, Eric; Sugai, Motoyuki

    2006-01-01

    The cell cycle G2/M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA, cdtB, and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called “lipobox”, at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [3H]palmitate or [3H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA′), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA′ in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant. PMID:16714579

  18. Actinobacillus actinomycetemcomitans strains Y4 and N27 adhere to hydroxyapatite by distinctive mechanisms.

    PubMed Central

    Kagermeier, A S; London, J

    1985-01-01

    Actinobacillus actinomycetemcomitans strains Y4 and N27 absorb to spheroidal hydroxyapatite in roughly the same numbers per milligram of substrate and with the same tenacity as two previously tested Cytophaga species. Although the two strains of A. actinomycetemcomitans exhibited similar affinities and number of binding sites for SHA, their response to enzyme treatment and heating were very different. The capacity of strain Y4 to attach to spheroidal hydroxyapatite was diminished by treatment with proteases and phospholipases and was unaffected by neuraminidase, while strain N27 was unaffected by proteases and phospholipases and lost its binding capabilities when treated with neuraminidase. Images PMID:3972445

  19. Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors.

    PubMed

    Ingalls, R R; Heine, H; Lien, E; Yoshimura, A; Golenbock, D

    1999-06-01

    The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide. PMID:10340170

  20. The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed

    Hall, E R; Falkler, W A; Martin, S A; Suzuki, J B

    1991-12-01

    The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients. PMID:1765941

  1. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss. PMID:26817919

  2. Iron-Chelating Activity of Tetracyclines and Its Impact on the Susceptibility of Actinobacillus actinomycetemcomitans to These Antibiotics

    PubMed Central

    Grenier, Daniel; Huot, Marie-Pierre; Mayrand, Denis

    2000-01-01

    Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions. PMID:10681353

  3. Microbial ecology of Actinobacillus actinomycetemcomitans, Eikenella corrodens and Capnocytophaga spp. in adult periodontitis.

    PubMed

    Müller, H P; Heinecke, A; Borneff, M; Knopf, A; Kiencke, C; Pohl, S

    1997-08-01

    Information on intraoral distribution of putative periodontal pathogens might be essential for controlling different forms of periodontal disease. Colonization may be either promoted or impeded by other bacteria competing in the subgingival ecosystem. In recent investigations microbial associations between dental organisms have been determined in a multitude of subgingival plaque samples within multiple patients and described by odds ratios, in most circumstances without taking into account the correlated structure of the observations within a single individual. The present investigation had 3 major objectives: (i) to describe the intraoral distribution of some facultatively anaerobic, Gram-negative rods, i.e. Actinobacillus actinomycetemcomitans, Eikenella corrodens-like organisms and Capnocytophaga spp., in a multitude of subgingival and extracrevicular samples of 10 adult subjects with A. actinomycetemcomitans-associated periodontitis; (ii) to analyse possible inconsistencies of microbial associations between these periodontal organisms; and (iii) to determine factors increasing the likelihood of isolating these bacteria in a given subgingival site by employing Generalized Estimation Equation (GEE) methods. Clinical examinations were carried out at 6 sites of every tooth present. In each subject, 13 extracrevicular (2 cheek mucosa, 3 tongue, 4 gingival, 2 tonsillar samples, 1 palatinal, 1 saliva sample) and between 22 and 44 subgingival samples from deepest sites of every tooth present (n = 296) were selectively cultivated for A. actinomycetemcomitans, E. corrodens and Capnocytophaga spp. In extracrevicular material, A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens were isolated in 9, 10 and 6 patients, and from 65, 82 and 15% samples, respectively. The organisms were recovered from 51, 62 and 27% subgingival plaque samples, respectively. Heterogeneity tests did not reveal significant inconsistencies of microbial associations between bacteria in

  4. Oral and systemic immunoglobulin G-subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed

    Engström, P E; George, M; Larsson, P; Lally, E T; Taichman, N S; Norhagen, G

    1999-04-01

    Salivary, gingival crevicular fluid and serum-specific immunoglobulin G (IgG)-subclass antibodies to Actinobacillus actinomycetemcomitans leuktoxin were quantified by enzyme-linked immunosorbent assay. Samples were taken from six patients with periodontal pockets > or = 5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex- and age-matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1- and IgG2-subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P < 0.05) and in serum (P < 0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P < 0.05) and in serum (P < 0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin. PMID:10219169

  5. Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis.

    PubMed Central

    Haubek, D; Dirienzo, J M; Tinoco, E M; Westergaard, J; López, N J; Chung, C P; Poulsen, K; Kilian, M

    1997-01-01

    Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans. PMID:9399490

  6. Lipopolysaccharide rolls out the barrel

    PubMed Central

    Bishop, Russell E.

    2016-01-01

    Two crystal structures of the LptD–LptE protein complex reveal how the cell-wall component lipopolysaccharide is delivered and inserted into the external leaflet of the bacterial outer membrane. PMID:24990738

  7. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    SciTech Connect

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  8. Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus

    PubMed Central

    Rempe, Katherine A.; Spruce, Lynn A.; Porsch, Eric A.; Seeholzer, Steven H.; Nørskov-Lauritsen, Niels

    2015-01-01

    ABSTRACT Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes. The nontypeable Haemophilus influenzae glycosyltransferase HMW1C mediates unconventional N-linked glycosylation of the adhesive protein HMW1, which is encoded in a two-partner secretion system gene cluster that also encodes HMW1C. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. In the present study, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C that are not encoded near a gene encoding an obvious acceptor protein. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild-type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function. PMID:26307167

  9. Genetic and Functional Analyses of the Actinobacillus actinomycetemcomitans AfeABCD Siderophore-Independent Iron Acquisition System

    PubMed Central

    Rhodes, Eric R.; Tomaras, Andrew P.; McGillivary, Glen; Connerly, Pamela L.; Actis, Luis A.

    2005-01-01

    The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions. PMID:15908408

  10. Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitans isolates from geographically diverse populations

    PubMed Central

    Fabris, A. S.; DiRienzo, J. M.; Wïkstrom, M.; Mayer, M. P. A.

    2008-01-01

    A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium. The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A. actinomycetemcomitans from Brazil, Kenya, Japan and Sweden. Thirty-nine of 40 cell lysates caused distension of Chinese hamster ovary cells. At least one of the cdt genes was detected in all strains examined. The three cdt genes were detected, by PCR, in 34 DNA samples. DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity. Restriction analysis was performed on every amplicon obtained. PCR-RFLP patterns revealed that the three cdt genes were conserved. These data provided evidence that the cdt genes are found and expressed in the majority of the A. actinomycetemcomitans isolates. Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found. PMID:12121473

  11. Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

    PubMed Central

    Tran, Simon Dangtuan; Rudney, Joel D.

    1999-01-01

    Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. PMID

  12. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    PubMed

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens. PMID:9524322

  13. [Characterization of Budvicia aquatica lipopolysaccharides].

    PubMed

    Varbanets', L D; Brovars'ka, O S; Pokhil, S Y

    2011-01-01

    For the first time lipopolysaccharides (LPS) of 6 Budvicia aquatica strains--representatives of new Enterobacteriaceae species have been isolated. It was shown that the yield of LPS ranged from 0.9 to 7.0 % of cells dry weight. On the basis of monosaccharide composition LPS of tested strains may be referred to 3 groups. The serological studies indicated the immunochemical heterogeneity of B. aquatica species: LPS interacted only in homological system and showed no cross-reactivity with heterological antisera. PMID:21809684

  14. Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

    PubMed Central

    Cadieux, J E; Kuzio, J; Milazzo, F H; Kropinski, A M

    1983-01-01

    Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C. Images PMID:6409883

  15. The survival rate of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus following 4 randomized treatment modalities.

    PubMed

    Shiloah, J; Patters, M R; Dean, J W; Bland, P; Toledo, G

    1997-08-01

    The overall goal of this clinical study was to determine the short-term anti-infective effects of four randomized treatment modalities on Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Bacteroides forsythus (Bf) and determine the effects of bacterial survival on treatment outcomes in patients with adult periodontitis. Twelve adult patients requiring therapy for moderate periodontitis were selected for this study. All patients had at least one tooth in each quadrant that had an inflamed pocket of probing depth > or =5 mm with probing attachment loss that harbored at least one of the following three periodontal pathogens: Aa, Pg, or Bf. The number of target organisms per site was determined pre-operatively, at 1 week, and 1 month and 3 months postoperatively utilizing DNA probes. One quadrant in each patient was randomly assigned to each one of the following four treatment groups: 1) scaling and root planing (SRP group); 2) pocket reduction through osseous surgery and apically-positioned flap (OS group); 3) modified Widman flap (MWF group); and 4) modified Widman flap and topical application of saturated citric acid at pH 1 for 3 minutes (CA group). The 4 treatment modalities were performed in one appointment. No postoperative antibiotics were used. Patients were instructed to supplement their daily oral hygiene with chlorohexidine oral rinse during the study. The results of this investigation indicated that: 1) none of the treatment modalities was effective in eliminating the target species; 2) the incidence of infected sites for all groups was 100% preoperatively; 62.5%, 33.3%, and 31.3% at 1 week, and 1 and 3 months postoperatively, respectively; 3) these infected sites lost 1.1 +/- 0.4 mm of probing attachment compared to gain of 0.0 +/- 0.3 mm for uninfected sites; 4) the infected sites had higher plaque and bleeding on probing 0.9 +/- 0.3, 73 +/- 12%, respectively, compared to 0.3 +/- 0.1 and 30 +/- 8% for the uninfected sites

  16. Physical properties of defined lipopolysaccharide salts

    SciTech Connect

    Coughlin, R.T.; Haug, A.; McGroarty, E.J.

    1983-01-01

    The electron spin resonance probes 5-doxylstearate and 4-(dodecyldimethylammonia)-1-oxy-2,2,6,6-tetramethylpiperidine bromide were used to characterize the fluidity of the acyl chain and head-group regions, respectively, of defined salts of lipopolysaccharide (LPS) from Escherichia coli K12. The removal of the weakly bound divalent cations from native LPS by electrodialysis and their replacement by sodium had little effect on the midpoint of the lipid-phase transition or on head-group mobility. In contrast, lipopolysaccharide acyl chain mobility increased following electrodialysis. The replacement of most of the remaining cations with sodium resulted in a further dramatic increase in mobility in both the polar and nonpolar regions of lipopolysaccharide. Head-group mobility of the sodium salt of LPS was shown to be reduced with the addition of divalent cations. Furthermore, evidence is presented which suggests that low magnesium concentrations may induce phase separations in the sodium salt. The magnesium salt of lipopolysaccharide closely resembled the native form in both head-group and acyl chain mobility although the cation charge to phosphorus ratio in the magnesium salt was greater than that detected in the native isolate. Analyses of other lipopolysaccharide salts support our hypothesis that many of the observed differences in the physical and pathological properties of lipopolysaccharide salts may simply be explained by the degree of charge neutralization.

  17. Physical properties of defined lipopolysaccharide salts.

    PubMed

    Coughlin, R T; Haug, A; McGroarty, E J

    1983-04-12

    The electron spin resonance probes 5-doxylstearate and 4-(dodecyldimethylammonio)-1-oxy-2,2,6,6-tetramethylpiperidine bromide were used to characterize the fluidity of the acyl chain and head-group regions, respectively, of defined salts of lipopolysaccharide (LPS) from Escherichia coli K12. The removal of the weakly bound divalent cations from native LPS by electrodialysis and their replacement by sodium had little effect on the midpoint of the lipid-phase transition or on head-group mobility. In contrast, lipopolysaccharide acyl chain mobility increased following electrodialysis. The replacement of most of the remaining cations with sodium resulted in a further dramatic increase in mobility in both the polar and nonpolar regions of lipopolysaccharide. Head-group mobility of the sodium salt of LPS was shown to be reduced with the addition of divalent cations. Furthermore, evidence is presented which suggests that low magnesium concentrations may induce phase separations in the sodium salt. The magnesium salt of lipopolysaccharide closely resembled the native form in both head-group and acyl chain mobility although the cation charge to phosphorus ratio in the magnesium salt was greater than that detected in the native isolate. Analyses of other lipopolysaccharide salts support our hypothesis that many of the observed differences in the physical and pathological properties of lipopolysaccharide salts may simply be explained by the degree of charge neutralization. PMID:6303400

  18. [Characterization of Pantoea agglomerans lipopolysaccharides].

    PubMed

    Varbanets, L D; Brovarskaya, O S; Bulygina, T N; Garkavaya, E G; Zhitkevich, N V

    2014-01-01

    Lipopolysaccharides (LPS) from seven Pantoea agglomerans strains isolated from various plants were purified and chemically identified. LPS of the studied P. agglomerans strains were heterogeneous in monosaccharide composition. Thus, the LPS of P. agglomerans 8606 differed considerably from the LPSs of other strains, containing mannose as the predominant monosaccharide (69.8%), as well as ribose (15.1%) and xylose (12.6%), while the content of rhamnose, one of the predominant monosaccharides in other LPS samples, was 2.5%. Analysis of the fatty acid composition revealed the presence of C12-C16 acids. In lipids A of all the studied strains, 3-OH-C14:0 was the predominant acid (31.7 to 39.1%, depending on the strain). C12:0 (8.2 to 31.5%), C14:0 (12.9 to 30.8%), and C16:0 acids (3.4 to 16.9%) were also revealed. The studied P. agglomerans strains fell into three groups according to their fatty acid composition. The differences stemmed from the presence or absence of two fatty acids, 2-OH-C14:0 and C16:1. Ouchterlony double immunodiffusion in agar revealed that all the LPS under study exhibited antigenic activity in homologous systems. The results of serological cross reactions indicated immunochemical heterogeneity of the species P. agglomerans. Comparative investigation of the complex of parameters of peripheral blood cells from a healthy donor before and after treatment with LPS solutions showed that the values of no parameters exceeded the normal range. PMID:25941715

  19. Lipopolysaccharide Engineering in Neisseria meningitidis

    PubMed Central

    Pupo, Elder; Hamstra, Hendrik-Jan; Meiring, Hugo; van der Ley, Peter

    2014-01-01

    Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1− mutant, which lacks the 2′ secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB−/pagL+ mutant. Removal of remaining hexaacyl species exclusively present in lgtB−/pagL+ LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1− and pagL+ LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL+ LPS has significant potential as immune modulator in humans. PMID:24492609

  20. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

    PubMed Central

    Gustafsson, B; Rosén, A; Holme, T

    1982-01-01

    A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios. Images PMID:6183214

  1. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

    PubMed Central

    Yamaguchi, N; Yamashita, Y; Ikeda, D; Koga, T

    1996-01-01

    Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes. PMID:8698480

  2. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  3. [Phytotoxic properties of Ralstonia solanacearum lipopolysaccharides].

    PubMed

    Hrytsaĭ, R V; Iakovleva, L M; Varbanets', L D

    2014-01-01

    The study is dedicated to research of phytotoxic properties of Ralstonia solanacearum lipopolysaccharides. This causative agent is one of the most dangerous among potato bacterial diseases. It is revealed that the inhibitory effect of LPS solution on seedlings germination is more noticeable on crops susceptible to brown rot. Maximal total phytotoxic properties have been shown by LPS from strains 35, 52b, TX1 and TS3, which were characterized by relatively low rhamnose content. Relative to the control plants LPS may diminish and some ones--increase the root length, height and weight of seedlings, subject to particular strain. But the stimulation revealed is minor. PMID:25000727

  4. Lipopolysaccharide potentiates hyperthermia-induced seizures

    PubMed Central

    Eun, Baik-Lin; Abraham, Jayne; Mlsna, Lauren; Kim, Min Jung; Koh, Sookyong

    2015-01-01

    Background Prolonged febrile seizures (FS) have both acute and long-lasting effects on the developing brain. Because FS are often associated with peripheral infection, we aimed to develop a preclinical model of FS that simulates fever and immune activation in order to facilitate the implementation of targeted therapy after prolonged FS in young children. Methods The innate immune activator lipopolysaccharide (LPS) was administered to postnatal day 14 rat (200 μg/kg) and mouse (100 μg/kg) pups 2–2.5 h prior to hyperthermic seizures (HT) induced by hair dryer or heat lamp. To determine whether simulation of infection enhances neuronal excitability, latency to seizure onset, threshold temperature and total number of seizures were quantified. Behavioral seizures were correlated with electroencephalographic changes in rat pups. Seizure-induced proinflammatory cytokine production was assessed in blood samples at various time points after HT. Seizure-induced microglia activation in the hippocampus was quantified using Cx3cr1GFP/+ mice. Results Lipopolysaccharide priming increased susceptibility of rats and mice to hyperthemic seizures and enhanced seizure-induced proinflammatory cytokine production and microglial activation. Conclusions Peripheral inflammation appears to work synergistically with hyperthermia to potentiate seizures and to exacerbate seizure-induced immune responses. By simulating fever, a regulated increase in body temperature from an immune challenge, we developed a more clinically relevant animal model of prolonged FS. PMID:26357586

  5. Interaction of uranium(VI) with lipopolysaccharide.

    PubMed

    Barkleit, Astrid; Moll, Henry; Bernhard, Gert

    2008-06-01

    Bacteria have a great influence on the migration behaviour of heavy metals in the environment. Lipopolysaccharides form the main part of the outer membrane of Gram-negative bacteria. We investigated the interaction of the uranyl cation (UO2(2+)) with lipopolysaccharide (LPS) from Pseudomonas aeruginosa by using potentiometric titration and time-resolved laser-induced fluorescence spectroscopy (TRLFS) over a wide pH and concentration range. Generally, LPS consists of a high density of different functionalities for metal binding such as carboxyl, phosphoryl, amino and hydroxyl groups. The dissociation constants and corresponding site densities of these functional groups were determined using potentiometric titration. The combination of both methods, potentiometry and TRLFS, show that at an excess of LPS uranyl phosphoryl coordination dominates, whereas at a slight deficit on LPS compared to uranyl, carboxyl groups also become important for uranyl coordination. The stability constants of one uranyl carboxyl complex and three different uranyl phosphoryl complexes and the luminescence properties of the phosphoryl complexes are reported. PMID:18478152

  6. Epigenetic Alterations Induced by Bacterial Lipopolysaccharides.

    PubMed

    Chiariotti, Lorenzo; Coretti, Lorena; Pero, Raffaela; Lembo, Francesca

    2016-01-01

    Lipopolysaccharide (LPS) is one of the principal bacterial products known to elicit inflammation. Cells of myeloid lineage such as monocytes and macrophages, but also epithelial cells give rise to an inflammatory response upon LPS stimulation. This phenomenon implies reprogramming of cell specific gene expression that can occur through different mechanisms including epigenetic modifications. Given their intrinsic nature, epigenetic modifications may be involved both in the acute response to LPS and in the establishment of a preconditioned genomic state (epigenomic memory) that may potentially influence the host response to further contacts with microorganisms. Information has accumulated during the last years aimed at elucidating the epigenetic mechanisms which underlie the cellular LPS response. These findings, summarized in this chapter, will hopefully be a good basis for a definition of the complete cascade of LPS-induced epigenetic events and their biological significance in different cell types. PMID:26659265

  7. [Comparative characterisation of lipopolysaccharides of Rahnella aquatills].

    PubMed

    Skokliuk, L B; Varbanets', L D; Pokhyl, S I

    2009-01-01

    Lipopolysaccharides (LPS) of eight strains of R. aquatilis isolated from different sources have been studied. The studies of neutral monosaccharide composition evidence that all of LPS contain galactose (13.4-68.5%), glucose (5.7-29.8%) and heptose (2.6-8.3%) (depending on strains). Some monosaccharides, such as ribose (95U007), rhamnose (95U011, 95U012, 96U036), fucose (95U003, 95U004, 95U007) and mannose (95U012, 96U035, 96U036, 96U037) were absent in LPS. Arabinose was present in two strains--95U003 and 95U007. On the basis of monosaccharide composition all investigated LPS can be divided into six groups. It was shown by double immunodiffusion in agar that all R. aquatilis LPS displayed antigenic activity in homological systems. The results of serological cross reactions indicate the immunochemical heterogeneity of R. aquatilis species. PMID:20455427

  8. Anti-inflammatory, Antioxidant and Antimicrobial Effects of Artemisinin Extracts from Artemisia annua L.

    PubMed Central

    Kim, Wan-Su; Choi, Woo Jin; Lee, Sunwoo; Kim, Woo Joong; Lee, Dong Chae; Sohn, Uy Dong; Shin, Hyoung-Shik

    2015-01-01

    The anti-inflammatory, antioxidant, and antimicrobial properties of artemisinin derived from water, methanol, ethanol, or acetone extracts of Artemisia annua L. were evaluated. All 4 artemisinin-containing extracts had anti-inflammatory effects. Of these, the acetone extract had the greatest inhibitory effect on lipopolysaccharide-induced nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokine (IL-1β , IL-6, and IL-10) production. Antioxidant activity evaluations revealed that the ethanol extract had the highest free radical scavenging activity, (91.0±3.2%), similar to α-tocopherol (99.9%). The extracts had antimicrobial activity against the periodontopathic microorganisms Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum subsp. animalis, Fusobacterium nucleatum subsp. polymorphum, and Prevotella intermedia. This study shows that Artemisia annua L. extracts contain anti-inflammatory, antioxidant, and antimicrobial substances and should be considered for use in pharmaceutical products for the treatment of dental diseases. PMID:25605993

  9. [Regulation of thyroid and pituitary functions by lipopolysaccharide].

    PubMed

    Iaglova, N V; Berezov, T T

    2010-01-01

    Activation of toll-like receptors-4 by bacterial lipopolysaccharide downregulates pituitary and thyroid function. Besides decrease of thyroid-stimulating hormone secretion lipopolysaccharide affects secretion in follicular thyroid cells directly. The endotoxin partially activates and inhibits different phases of follicular thyrocytes' secretion. Lipopolysaccharide enhances thyroglobulin synthesis and exocytosis into follicular lumen and suppresses its resorbtion. It results in sharp drop of blood thyroxine concentration without decrease of deiodinases-mediated thiroxine to triiodothyronine conversion. Stimulation of the lipopolysaccharide-pretreated thyroid gland with thyroid-stimulating hormone increases resorbtion of thyroglobulin and thyroid hormone production. Combined stimulation of the thyroid gland increases protein bound thyroxine and triiodothyronine serum concentration unlike only TSH stimulation resulting in increase of free thyroid hormone levels. It also proves that binding capacity of thyroid hormone serum transport proteins during nonthyroidal illness syndrome remains normal. PMID:21341506

  10. [Modulation of immune response by bacterial lipopolysaccharides].

    PubMed

    Aldapa-Vega, Gustavo; Pastelín-Palacios, Rodolfo; Isibasi, Armando; Moreno-Eutimio, Mario A; López-Macías, Constantino

    2016-01-01

    Lipopolysaccharide (LPS) is a molecule that is profusely found on the outer membrane of Gram-negative bacteria and is also a potent stimulator of the immune response. As the main molecule on the bacterial surface, is also the most biologically active. The immune response of the host is activated by the recognition of LPS through Toll-like receptor 4 (TLR4) and this receptor-ligand interaction is closely linked to LPS structure. Microorganisms have evolved systems to control the expression and structure of LPS, producing structural variants that are used for modulating the host immune responses during infection. Examples of this include Helicobacter pylori, Francisella tularensis, Chlamydia trachomatis and Salmonella spp. High concentrations of LPS can cause fever, increased heart rate and lead to septic shock and death. However, at relatively low concentrations some LPS are highly active immunomodulators, which can induce non-specific resistance to invading microorganisms. The elucidation of the molecular and cellular mechanisms involved in the recognition of LPS and its structural variants has been fundamental to understand inflammation and is currently a pivotal field of research to understand the innate immune response, inflammation, the complex host-pathogen relationship and has important implications for the rational development of new immunomodulators and adjuvants. PMID:27560917

  11. Structure and Effects of Cyanobacterial Lipopolysaccharides

    PubMed Central

    Durai, Prasannavenkatesh; Batool, Maria; Choi, Sangdun

    2015-01-01

    Lipopolysaccharide (LPS) is a component of the outer membrane of mainly Gram-negative bacteria and cyanobacteria. The LPS molecules from marine and terrestrial bacteria show structural variations, even among strains within the same species living in the same environment. Cyanobacterial LPS has a unique structure, since it lacks heptose and 3-deoxy-d-manno-octulosonic acid (also known as keto-deoxyoctulosonate (KDO)), which are present in the core region of common Gram-negative LPS. In addition, the cyanobacterial lipid A region lacks phosphates and contains odd-chain hydroxylated fatty acids. While the role of Gram-negative lipid A in the regulation of the innate immune response through Toll-like Receptor (TLR) 4 signaling is well characterized, the role of the structurally different cyanobacterial lipid A in TLR4 signaling is not well understood. The uncontrolled inflammatory response of TLR4 leads to autoimmune diseases such as sepsis, and thus the less virulent marine cyanobacterial LPS molecules can be effective to inhibit TLR4 signaling. This review highlights the structural comparison of LPS molecules from marine cyanobacteria and Gram-negative bacteria. We discuss the potential use of marine cyanobacterial LPS as a TLR4 antagonist, and the effects of cyanobacterial LPS on humans and marine organisms. PMID:26198237

  12. Lipopolysaccharide induced conversion of recombinant prion protein

    PubMed Central

    Saleem, Fozia; Bjorndahl, Trent C; Ladner, Carol L; Perez-Pineiro, Rolando; Ametaj, Burim N; Wishart, David S

    2014-01-01

    The conformational conversion of the cellular prion protein (PrPC) to the β-rich infectious isoform PrPSc is considered a critical and central feature in prion pathology. Although PrPSc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrPC to proteinase K resistant PrPSc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrPC conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrPC to PrPSc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232). PMID:24819168

  13. Lipopolysaccharide (LPS) stimulation of fungal secondary metabolism

    PubMed Central

    Khalil, Zeinab G.; Kalansuriya, Pabasara; Capon, Robert J.

    2014-01-01

    We report on a preliminary investigation of the use the Gram-negative bacterial cell wall constituent lipopolysaccharide (LPS) as a natural chemical cue to stimulate and alter the expression of fungal secondary metabolism. Integrated high-throughput micro-cultivation and micro-analysis methods determined that 6 of 40 (15%) of fungi tested responded to an optimal exposure to LPS (0.6 ng/mL) by activating, enhancing or accelerating secondary metabolite production. To explore the possible mechanisms behind this effect, we employed light and fluorescent microscopy in conjunction with a nitric oxide (NO)-sensitive fluorescent dye and an NO scavenger to provide evidence that LPS stimulation of fungal secondary metabolism coincided with LPS activation of NO. Several case studies demonstrated that LPS stimulation can be scaled from single microplate well (1.5 mL) to preparative (>400 mL) scale cultures. For example, LPS treatment of Penicillium sp. (ACM-4616) enhanced pseurotin A and activated pseurotin A1 and pseurotin A2 biosynthesis, whereas LPS treatment of Aspergillus sp. (CMB-M81F) substantially accelerated and enhanced the biosynthesis of shornephine A and a series of biosynthetically related ardeemins and activated production of neoasterriquinone. As an indication of broader potential, we provide evidence that cultures of Penicillium sp. (CMB-TF0411), Aspergillus niger (ACM-4993F), Rhizopus oryzae (ACM-165F) and Thanatephorus cucumeris (ACM-194F) were responsive to LPS stimulation, the latter two examples being particular noteworthy as neither are known to produce secondary metabolites. Our results encourage the view that LPS stimulation can be used as a valuable tool to expand the molecular discovery potential of fungal strains that either have been exhaustively studied by or are unresponsive to traditional culture methodology. PMID:25379339

  14. Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin.

    PubMed Central

    Bélanger, M; Bégin, C; Jacques, M

    1995-01-01

    A previous study indicated that lipopolysaccharides (LPS) extracted from Actinobacillus pleuropneumoniae bind two low-molecular-mass proteins, of approximately 10 and 11 kDa, present in porcine respiratory tract secretions (M. Bélanger, D. Dubreuil, and M. Jacques, Infect. Immun. 62:868-873, 1994). In the present study, we determined the N-terminal amino acid sequences of these two proteins, which revealed high homology with the alpha and beta chains of pig hemoglobin. Some isolates of A. pleuropneumoniae were able to use hemoglobin from various animal species as well as other heme compounds as sole sources of iron for growth, while other isolates were unable to use them. Immunoelectron microscopy showed binding of pig hemoglobin at the surface of all A. pleuropneumoniae isolates as well as labeling of outer membrane blebs. We observed, using Western blotting (immunoblotting), that the lipid A-core region of LPS of all isolates was binding pig hemoglobin. Furthermore, lipid A obtained after acid hydrolysis of LPS extracted from A. pleuropneumoniae was able to bind pig hemoglobin and this binding was completely abolished by preincubation of lipid A with polymyxin B but was not inhibited by preincubation with glucosamines. Fatty acids constituting the lipid A of A. pleuropneumoniae, namely, dodecanoic acid, tetradecanoic acid, 3-hydroxytetradecanoic acid, hexadecanoic acid, and octadecanoic acid, were also binding pig hemoglobin. Our results indicate that LPS of all A. pleuropneumoniae isolates tested bind pig hemoglobin and that lipid A is involved in this binding. Our results also indicate that some A. pleuropneumoniae isolates are, in addition, able to use hemoglobin for growth. Binding of hemoglobin to LPS might represent an important means by which A. pleuropneumoniae acquires iron in vivo from hemoglobin released from erythrocytes lysed by the action of its hemolysins. PMID:7822035

  15. Further characterization of a lipopolysaccharide from Coxiella burneti.

    PubMed Central

    Chan, M L; McChesney, J; Paretsky, D

    1976-01-01

    The lipopolysaccharide previously isolated from the rickettsial agent of Q fever, Coxiella burneti, phase I, has been further characterized. The sugar residues ribose, mannose, gluclose, D-glycero-D-mannoheptose, and L-glycerto-D-mannoheptose are present. Two sugars remain unidentified, one of which is a minor and the other a major constituent. Isomyristic, palmitic, and beta-hydroxymyristic acids are the major fatty acid residues of the 15 identified. The nature and content of other lipopolysaccharide constituents are presented. Images PMID:971947

  16. Proteomic changes in chicken plasma induced by Salmonella typhimurium lipopolysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipopolysaccharides (LPS) are cell wall components of gram-negative bacteria that cause inflammation and sickness through genetic and proteomic activation. The objective of our study was to identify the proteomic changes in plasma associated with inflammation induced by LPS treatment. Five-week-old ...

  17. Proteomic analysis of macrophage activated with salmonella lipopolysaccharide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macrophages play pivotal role in immunity. They are activated by many pathogen derived molecules such as lipopolysaccharides (LPS) which trigger the production of various proteins and peptides that drive and resolve inflammation. There are numerous studies on the effect of LPS at the genome level bu...

  18. Bacterial lipopolysaccharides in plant and mammalian innate immunity.

    PubMed

    De Castro, Cristina; Holst, Otto; Lanzetta, Rosa; Parrilli, Michelangelo; Molinaro, Antonio

    2012-10-01

    This mini-review gives a structural view on the lipopolysaccharides (LPSs), the endotoxin from Gram negative bacteria, paying attention on the features that are relevant for their activity as elicitors of the innate immune system of humans, animals and plants. PMID:22533617

  19. Buccal microbiology analyzed by infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    de Abreu, Geraldo Magno Alves; da Silva, Gislene Rodrigues; Khouri, Sônia; Favero, Priscila Pereira; Raniero, Leandro; Martin, Airton Abrahão

    2012-01-01

    Rapid microbiological identification and characterization are very important in dentistry and medicine. In addition to dental diseases, pathogens are directly linked to cases of endocarditis, premature delivery, low birth weight, and loss of organ transplants. Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze oral pathogens Aggregatibacter actinomycetemcomitans ATCC 29523, Aggregatibacter actinomycetemcomitans-JP2, and Aggregatibacter actinomycetemcomitans which was clinically isolated from the human blood-CI. Significant spectra differences were found among each organism allowing the identification and characterization of each bacterial species. Vibrational modes in the regions of 3500-2800 cm-1, the 1484-1420 cm-1, and 1000-750 cm-1 were used in this differentiation. The identification and classification of each strain were performed by cluster analysis achieving 100% separation of strains. This study demonstrated that FTIR can be used to decrease the identification time, compared to the traditional methods, of fastidious buccal microorganisms associated with the etiology of the manifestation of periodontitis.

  20. Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

    PubMed

    Lin, Miao-Fang; Williams, Christie; Murray, Michael V; Ropp, Philip A

    2005-02-25

    During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes. PMID:15664347

  1. Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

    PubMed Central

    Tomás, J M; Jofre, J T

    1985-01-01

    Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage. Images PMID:3888963

  2. [Characterization of the lipopolysaccharides of serogroup II Azospirillum].

    PubMed

    Sigida, E N; Fedonenko, Iu P; Zdorovenko, É L; Butygin, G L; Konnova, S A; Ignatov, V V

    2014-01-01

    Lipopolysaccharides of six Azospirillum strains (A. brasilense SR50, SR80, SR88, SR109, SR111, SR115, and A. lipoferum SR 42) isolated from the rhizosphere of cereal plants of Saratov oblast, Russia and assigned to serogroup II by serological analysis were studied. In the lipid A fatty acid composition, the lipopolysaccharides under study were similar to those of other Azospirillum strains and were characterized by predominance of 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, and octadecenoic acids. Monosaccharide analysis of the O-specific polysaccharides (including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy) revealed the presence of two types of repeating units in varying ratios. High degree of serological similarity between the strains under study was shown to result from the presence of repeating units with identical structure in their O antigens. PMID:25844452

  3. Non-typhoidal Salmonella encephalopathy involving lipopolysaccharide in cattle.

    PubMed

    Xiong, N; Brewer, M T; Anderson, K L; Carlson, S A

    2013-02-22

    This study assessed the involvement of lipopolysaccharide (LPS) in the non-typhoidal Salmonella encephalopathy (NTSE) caused by a unique isolate of Salmonella enterica serovar Saint-paul (SstpNPG). NTSE was prevented by genetic (deletion of murE) or pharmacologic (polymyxin) disruption of LPS on SstpNPG although the disruption of LPS did not deter brain penetration of the strain. This is the first study to demonstrate that LPS is involved in the manifestations of NTSE. PMID:22939987

  4. Innate immunity probed by lipopolysaccharides affinity strategy and proteomics.

    PubMed

    Giangrande, Chiara; Colarusso, Lucia; Lanzetta, Rosa; Molinaro, Antonio; Pucci, Piero; Amoresano, Angela

    2013-01-01

    Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses. PMID:22752448

  5. SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

    SciTech Connect

    Lee, Ae Sin; Jung, Yu Jin; Kim, Dal; Nguyen-Thanh, Tung; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

    2014-08-08

    Highlights: • Knockout of SIRT2 attenuates lipopolysaccharide-induced iNOS expression. • Lipopolysaccharide-induced NO production is decreased in SIRT2 KO macrophage. • SIRT2 deficiency suppresses lipopolysaccharide-induced ROS production in macrophage. • M1-macrophage related factors are decreased in SIRT2 deficient cells. • SIRT2 deficiency decreases lipopolysaccharide-induced activation of NFκB. - Abstract: Introduction: SIRT2 is a NAD(+)-dependent deacetylases and associated with numerous processes such as infection, carcinogenesis, DNA damage and cell cycle regulation. However, the role of SIRT2 in inflammatory process in macrophage remains unclear. Materials and methods: In the present study, we have evaluated the regulatory effects of SIRT2 in lipopolysaccharide (LPS)-stimulated macrophages isolated from SIRT2 knockout (KO) and wild type (WT) mice or Raw264.7 macrophage cells. As inflammatory parameters, expression of inducible nitric oxide synthase (iNOS), the productions of nitric oxide, reactive oxygen species (ROS) and M1-macrophage-related factors were evaluated. We also examined the effects of SIRT2 on activation of nuclear factor-kappaB (NFκB) signaling. Results: SIRT2 deficiency inhibits LPS-induced iNOS mRNA and protein expression in bone marrow derived macrophages. SIRT2-siRNA transfection also suppressed LPS-induced iNOS expression in Raw264.7 macrophage cells. Bone marrow derived macrophages isolated from SIRT2 KO mice produced lower nitric oxide and expressed lower levels of M1-macrophage related markers including iNOS and CD86 in response to LPS than WT mice. Decrease of SIRT2 reduced the LPS-induced reactive oxygen species production. Deficiency of SIRT2 resulted in inhibition of NFκB activation through reducing the phosphorylation and degradation of IκBα. The phosphorylation and nuclear translocation of p65 was significantly decreased in SIRT2-deficient macrophages after LPS stimulation. Discussion: Our data suggested that

  6. Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

    PubMed Central

    Rivera, M; Bryan, L E; Hancock, R E; McGroarty, E J

    1988-01-01

    Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different. Images PMID:3123455

  7. N-Acetylneuraminic acid: a constituent of the lipopolysaccharide of Salmonella toucra.

    PubMed

    Kedzierska, B

    1978-11-15

    The composition of lipopolysaccharide and polysaccharides isolated after acidic or alkaline degradation of lipopolysaccharide of Salmonella toucra has been investigated. The following analytical methods were used in this study: gel-filtration and ion-exchange techniques, paper and gas-liquid chromatography as well as spectrophotometric analysis. The products of the lipopolysaccharide degradation were fractionated on the Sephadex G-25 and Sephadex G-50 columns. Lipopolysaccharide and products of its degradation besides glucosamine, galactose, glucose, heptose(s) and 3-deoxy-D-manno-octulosonate described as 'basal' sugars, also contained N-acetylneuraminic acid and an unidentified amino sugar. PMID:729582

  8. Lipopolysaccharide as a target for brucellosis vaccine design.

    PubMed

    Conde-Álvarez, Raquel; Arce-Gorvel, Vilma; Gil-Ramírez, Yolanda; Iriarte, Maite; Grilló, María-Jesús; Gorvel, Jean Pierre; Moriyón, Ignacio

    2013-05-01

    The gram-negative bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a world wide-distributed zoonotic disease that represents a serious problem for animal and human health. There is no human-to-human contagion and, since there is no human vaccine, animal vaccination is essential to control brucellosis. However, current vaccines (all developed empirically) do not provide 100% protection and are infectious in humans. Attempts to generate new vaccines by obtaining mutants lacking the lipopolysaccharide O-polysaccharide, in purine metabolism or in Brucella type IV secretion system have not been successful. Here we propose a new approach to develop brucellosis vaccines based on the concept that Brucella surface molecules evade efficient detection by innate immunity, thus delaying protective Th1 responses and opening a time window to reach sheltered intracellular compartments. We showed recently that a branch of the core oligosaccharide section of Brucella lipopolysaccharide hampers recognition by TLR4-MD2. Mutation of glycosyltransferase WadC, involved in the synthesis of this branch, results in a lipopolysaccharide that, while keeping the O-polysaccharide essential for optimal protection, shows a truncated core, is more efficiently recognized by MD2 and triggers an increased cytokine response. In keeping with this, the wadC mutant is attenuated in dendritic cells and mice. In the mouse model of brucellosis vaccines, the Brucella abortus wadC mutant conferred protection similar to that provided by S19, the best cattle vaccine available. The properties of the wadC mutant provide the proof of concept for this new approach and open the way for more effective brucellosis vaccines. PMID:23219811

  9. Lipopolysaccharide-Induced Toxic Shock Syndrome in Rabbits.

    PubMed

    Stach, Christopher S; Schlievert, Patrick M

    2016-01-01

    Enhancement of susceptibility to lipopolysaccharide (LPS; endotoxin) is a defining characteristic of Staphylococcus aureus superantigens. At the time of this publication, there are 24 identified staphylococcal superantigens (SAgs), some of which have yet to be fully characterized. Testing the capacity of superantigens to potentiate LPS sensitivity is essential to characterize the role of these proteins in disease development. Here we describe how to perform studies of the enhancement of LPS-induced toxic shock syndrome in rabbits. This protocol also provides information on a second important activity of superantigens: the production of fever. PMID:26676037

  10. Computer Simulation of the Rough Lipopolysaccharide Membrane of Pseudomonas Aeruginosa

    SciTech Connect

    Lins, Roberto D.; Straatsma, TP

    2001-08-01

    Lipopolysaccharides (LPS) form the major constituent of the outer membrane of Gram-negative bacteria, and are believed to play a key role in processes that govern microbial metal binding, microbial adsorption to mineral surfaces, and microbe mediated oxidation/reduction reactions at the bacterial exterior surface. A computational modeling capability is being developed for the study of geochemical reactions at the outer bacterial envelope of Gram-negative bacteria. The understanding of these mechanisms is crucial for the development of successful environmental bioremediation strategies. A molecular model for the rough LPS of Pseudomonas aeruginosa has been designed based on available experimentally determined structural information.

  11. Novel change in the carbohydrate portion of Myxococcus xanthus lipopolysaccharide during development.

    PubMed Central

    Panasenko, S M; Jann, B; Jann, K

    1989-01-01

    We have examined the alterations in lipopolysaccharide during aggregation and early development in Myxococcus xanthus. The lipopolysaccharide was isolated and characterized from cells developing on agar during glycerol induction and vegetative growth. A methylated amino sugar was identified as 6-O-methylgalactosamine by gas-liquid chromatography-mass spectrometry. This novel sugar was enriched in cells developing on agar. Images PMID:2495265

  12. Lipopolysaccharide deacylation by an endogenous lipase controls innate antibody responses to Gram-negative bacteria.

    PubMed

    Lu, Mingfang; Zhang, Mei; Takashima, Akira; Weiss, Jerrold; Apicella, Michael A; Li, Xiang-Hong; Yuan, Dorothy; Munford, Robert S

    2005-10-01

    T cell-independent type 1 agonists such as Gram-negative bacterial lipopolysaccharides can stimulate B lymphocytes to proliferate and produce antibodies by signaling through Toll-like receptors. This phenomenon is well established in vitro, yet polyclonal B cell responses after bacterial infection in vivo are often weak and short-lived. We show here that B cell proliferation and polyclonal antibody production in response to Gram-negative bacterial infection are modulated by acyloxyacyl hydrolase, a host enzyme that deacylates bacterial lipopolysaccharides. Deacylation of lipopolysaccharide occurred over several days, allowing lipopolysaccharide to act as an innate immune stimulant yet limiting the eventual amount of B cell proliferation and polyclonal antibody production. Control of lipopolysaccharide activation by acyloxyacyl hydrolase indicates that mammals can regulate immune responses to bacterial infection by chemical modification of a Toll-like receptor agonist. PMID:16155573

  13. Chemical structure and inhalation toxicity of lipopolysaccharides from bacteria on cotton.

    PubMed Central

    Helander, I; Salkinoja-Salonen, M; Rylander, R

    1980-01-01

    Lipopolysaccharides from different bacteria isolated from cotton were purified and chemically analyzed. Their pulmonary toxicity to animals was tested in inhalation tests. Lipopolysaccharides from Agrobacterium and Xanthomonas were shown to differ from the others in that they contained no heptose and no non-hydroxylated fatty acids with a chain length of 12, 14, or 16 carbon atoms. Lipopolysaccharides from Pseudomonas putida, Enterobacter agglomerans, and Klebsiella oxytoca were found to cause an influx of polymorphonuclear leukocytes into the airways. Lipopolysaccharides from Agrobacterium sp. and Xanthomonas sinensis caused no significant invasion. The data point to substances in both the lipid A part and the core part of the lipopolysaccharides being responsible for the capacity to induce leukocyte invasion into the airways. PMID:7000706

  14. Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium

    PubMed Central

    Mohamed, Kettani-Halabi; Daniel, Tran; Aurélien, Dauphin; El-Maarouf-Bouteau, Hayat; Rafik, Errakhi; Arbelet-Bonnin, Delphine; Biligui, Bernadette; Florence, Val; Mustapha, Ennaji Moulay; François, Bouteau

    2015-01-01

    Lipopolysaccharides (LPS) are a component of the outer cell surface of almost all Gram-negative bacteria and play an essential role for bacterial growth and survival. Lipopolysaccharides represent typical microbe-associated molecular pattern (MAMP) molecules and have been reported to induce defense-related responses, including the expression of defense genes and the suppression of the hypersensitive response in plants. However, depending on their origin and the challenged plant, LPS were shown to have complex and different roles. In this study we showed that LPS from plant pathogens Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum induce common and different responses in A. thaliana cells when compared to those induced by LPS from non-phytopathogens Escherichia coli and Pseudomonas aeruginosa. Among common responses to both types of LPS are the transcription of defense genes and their ability to limit of cell death induced by Pectobacterium carotovorum subsp carotovorum. However, the differential kinetics and amplitude in reactive oxygen species (ROS) generation seemed to regulate defense gene transcription and be determinant to induce programmed cell death in response to LPS from the plant pathogenic Pectobacterium. These data suggest that different signaling pathways could be activated by LPS in A. thaliana cells. PMID:25760034

  15. Prolonged sleep fragmentation of mice exacerbates febrile responses to lipopolysaccharide

    PubMed Central

    Ringgold, Kristyn M.; Barf, R. Paulien; George, Amrita; Sutton, Blair C.; Opp, Mark R.

    2013-01-01

    Background Sleep disruption is a frequent occurrence in modern society. Whereas many studies have focused on the consequences of total sleep deprivation, few have investigated the condition of sleep disruption. New Method We disrupted sleep of mice during the light period for 9 consecutive days using an intermittently-rotating disc. Results Electroencephalogram (EEG) data demonstrated that non-rapid eye movement (NREM) sleep was severely fragmented and REM sleep was essentially abolished during the 12 h light period. During the dark period, when sleep was not disrupted, neither NREM sleep nor REM sleep times differed from control values. Analysis of the EEG revealed a trend for increased power in the peak frequency of the NREM EEG spectra during the dark period. The fragmentation protocol was not overly stressful as body weights and water consumption remained unchanged, and plasma corticosterone did not differ between mice subjected to 3 or 9 days of sleep disruption and home cage controls. However, mice subjected to 9 days of sleep disruption by this method responded to lipopolysaccharide with an exacerbated febrile response. Comparison with existing methods Existing methods to disrupt sleep of laboratory rodents often subject the animal to excessive locomotion, vibration, or sudden movements. This method does not suffer from any of these confounds. Conclusions This study demonstrates that prolonged sleep disruption of mice exacerbates febrile responses to lipopolysaccharide. This device provides a method to determine mechanisms by which chronic insufficient sleep contributes to the etiology of many pathologies, particularly those with an inflammatory component. PMID:23872243

  16. Neuroprotective Activity of (-)-Epigallocatechin Gallate against Lipopolysaccharide-Mediated Cytotoxicity.

    PubMed

    Liu, Jin-Biao; Zhou, Li; Wang, Yi-Zhong; Wang, Xu; Zhou, Yu; Ho, Wen-Zhe; Li, Jie-Liang

    2016-01-01

    Lipopolysaccharide- (LPS-) mediated systemic inflammation plays a critical role in neurodegenerative diseases. The present study was conducted to evaluate the protective effects of epigallocatechin gallate (EGCG), the major component in green tea, on LPS-mediated inflammation and neurotoxicity. LPS treatment of macrophages induced expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). However, EGCG pretreatment of macrophages significantly inhibited LPS-mediated induction of these cytokines. In addition, EGCG significantly diminished LPS-induced inflammatory cytokines in the peripheral mononuclear blood cells (PBMCs). Supernatant from EGCG-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-EGCG-pretreated and LPS-activated macrophage cultures. Furthermore, EGCG treatment of neurons could inhibit LPS-induced production of reactive oxygen species (ROS). Thus EGCG represents a potent and useful neuroprotective agent for inflammation-mediated neurological disorders. PMID:27191001

  17. [Immunostimulating activity of the lipopolysaccharides of blue-green algae].

    PubMed

    Besednova, N N; Smolina, T P; Mikheĭskaia, L V; Ovodova, R G

    1979-12-01

    The whole cells of blue-gree algae and lipopolysaccharides isolated from these cells were shown to stimulate the production of macro-(mainly) and microglobulin antibodies in rabbits. The macro- and microphage indices in rabbits increased significantly after the injection of LPS isolated from blue-green algae 24--48 hours before infecting the animals with a virulent Y. pseudotuberculosis strain. Besides, the inhibiting action of this strain on the migration of phagocytes to the site of infection was abolished immediately after the injection. The use of the indirect hemagglutination test allowed to prove the absence of close antigenic interrelations between blue-green algae and the following organisms: Spirulina platensis, Microcystis aeruginosa, Phormidium africanum and P. uncinatum. PMID:117655

  18. Sirtuin 4 Regulates Lipopolysaccharide Mediated Leydig Cell Dysfunction.

    PubMed

    Ramatchandirin, Balamurugan; Sadasivam, Mohanraj; Kannan, Arun; Prahalathan, Chidambaram

    2016-04-01

    Bacterial lipopolysaccharide (LPS) is the most important contributing factor in pathogenesis of bacterial infection in male accessory glands; and it has shown to inhibit testicular steroidogenesis and induce apoptosis. The present study demonstrates that LPS causes mitochondrial dysfunction via suppression of sirtuin 4 (SIRT4); which in turn affects Leydig cell function by modulating steroidogenesis and apoptosis. LC-540 Leydig cells treated with LPS (10 µg/ml) showed impaired steroidogenesis and increased cellular apoptosis. The mRNA and protein expression of SIRT4 were decreased in LPS treated cells when compared to controls. The obtained data suggest that the c-Jun N-terminal kinase (JNK) activation suppresses SIRT4 expression in LPS treated Leydig cells. Furthermore, the overexpression of SIRT4 prevented LPS induced impaired steroidogenesis and cellular apoptosis by improving mitochondrial function. These findings provide valuable information that SIRT4 regulates LPS mediated Leydig cell dysfunction. PMID:26365714

  19. Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis

    SciTech Connect

    Clover, R.H.; Kieber, J.; Signer, E.R. )

    1989-07-01

    Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix{sup +} on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix{sup {minus}} in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix{sup {minus}} on alfalfa.

  20. Lipopolysaccharide modification in Gram-negative bacteria during chronic infection.

    PubMed

    Maldonado, Rita F; Sá-Correia, Isabel; Valvano, Miguel A

    2016-07-01

    The Gram-negative bacterial lipopolysaccharide (LPS) is a major component of the outer membrane that plays a key role in host-pathogen interactions with the innate immune system. During infection, bacteria are exposed to a host environment that is typically dominated by inflammatory cells and soluble factors, including antibiotics, which provide cues about regulation of gene expression. Bacterial adaptive changes including modulation of LPS synthesis and structure are a conserved theme in infections, irrespective of the type or bacteria or the site of infection. In general, these changes result in immune system evasion, persisting inflammation and increased antimicrobial resistance. Here, we review the modifications of LPS structure and biosynthetic pathways that occur upon adaptation of model opportunistic pathogens (Pseudomonas aeruginosa, Burkholderia cepacia complex bacteria, Helicobacter pylori and Salmonella enterica) to chronic infection in respiratory and gastrointestinal sites. We also discuss the molecular mechanisms of these variations and their role in the host-pathogen interaction. PMID:27075488

  1. Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

    PubMed Central

    Burmann, Björn M.; Holdbrook, Daniel A.; Callon, Morgane; Bond, Peter J.; Hiller, Sebastian

    2015-01-01

    The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function. PMID:25809264

  2. [Biological activity of native and modified lipopolysaccharides of Pragia fontium].

    PubMed

    Varbanets', L D; Shubchyns'kyĭ, V V; Pokhyl, S I; Seĭfullina, I I; Shmatkova, N V; Samburs'kyĭ, S E

    2009-01-01

    Modified lipopolysaccharides (LPS) of Pragia fontium were obtained with germanium complexes (IV) of 2-aminobenzoylhydrazon of salicylic aldehyde (2-NH2-H2Bs), 2-hydroxybenzoylhydrazon salicylic aldehyde (2-OH-H2Bs) and nicotinoylhydrazon of salicylic aldehyde (H2Ns). The modification of LPS was confirmed by IR spectroscopy. Comparative investigations of pyrogenic activity of native and modified LPS showed, that only P. fontium 20125 LPS, modified by germanium complexes with 2-hydroxybenzoylhydrazon of salicylic aldehyde (2-OH-H2Bs) has lost the pyrogenic activity. In the homological reactions of double immunodiffusion in agar it was shown that all modified LPS unlike the native ones lose completely antigenic activity. PMID:19877414

  3. Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides

    PubMed Central

    Maranha, Ana; Moynihan, Patrick J.; Miranda, Vanessa; Correia Lourenço, Eva; Nunes-Costa, Daniela; Fraga, Joana S.; José Barbosa Pereira, Pedro; Macedo-Ribeiro, Sandra; Ventura, M. Rita; Clarke, Anthony J.; Empadinhas, Nuno

    2015-01-01

    Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. Enzymatic studies, synthetic chemistry, NMR spectroscopy and mass spectrometry approaches suggest that, in contrast to the prevailing consensus, octanoate is not esterified to the primary hydroxyl group of glycerate but instead to the C6 OH of the second glucose in DGG. These observations raise important new questions about the MGLP reducing end architecture and about subsequent biosynthetic steps. Functional characterization of this unique octanoyltransferase, whose gene has been proposed to be essential for M. tuberculosis growth, adds new insights into a vital mycobacterial pathway, which may inspire new drug discovery strategies. PMID:26324178

  4. Pleurotus eryngii Ameliorates Lipopolysaccharide-Induced Lung Inflammation in Mice

    PubMed Central

    Andoh, Tsugunobu; Ouchi, Kenji; Inatomi, Satoshi

    2014-01-01

    Pleurotus eryngii (P. eryngii) is consumed as a fresh cultivated mushroom worldwide and demonstrated to have multiple beneficial effects. We investigated the anti-inflammatory effect of P. eryngii in mice with acute lung injury (ALI). Intranasal instillation of lipopolysaccharide (LPS) (10 μg/site/mouse) induced marked lung inflammation (increase in the number of inflammatory cells, protein leakage, and production of nitric oxide in bronchoalveolar lavage fluid) as well as histopathological damage in the lung, 6 h after treatment. Mice administered heat-treated P. eryngii (0.3–1 g/kg, p.o. (HTPE)) 1 h before LPS challenge showed decreased pulmonary inflammation and ameliorated histopathological damage. These results suggest that HTPE has anti-inflammatory effects against ALI. Thus, P. eryngii itself may also have anti-inflammatory effects and could be a beneficial food for the prevention of ALI induced by bacterial infection. PMID:24799939

  5. Cyanobacterial lipopolysaccharides and human health – a review

    PubMed Central

    Stewart, Ian; Schluter, Philip J; Shaw, Glen R

    2006-01-01

    Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae. We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans. There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation. PMID:16563160

  6. Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide.

    PubMed

    Casabuono, Adriana; Petrocelli, Silvana; Ottado, Jorgelina; Orellano, Elena G; Couto, Alicia S

    2011-07-22

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo₂Hex₆GalA₃Fuc3NAcRha₄ and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response. PMID:21596742

  7. Examination of Lipopolysaccharide (O-Antigen) Populations of Thiobacillus ferrooxidans from Two Mine Tailings

    PubMed Central

    Southam, G.; Beveridge, T. J.

    1993-01-01

    Net acid-generating capacities of 39.74 kg of H2SO4 per ton (ca. 0.05 kg/kg) (pH 2.68) for the Lemoine copper mine tailings (closed ca. 8 years ago; located 40 km west of Chibougamau, Quebec, Canada) and 16.07 kg of H2SO4 per ton (ca. 0.02 kg/kg) (pH 3.01) for the Copper Rand tailings (in current use and 50 km distant [east] from those of Lemoine) demonstrate that these sulfide tailings can support populations of acidophilic thiobacilli. Oxidized regions in both tailings environments were readily visible, were extremely acidic (Lemoine, pH 2.36; Copper Rand, pH 3.07), and provided natural isolates for our study. A 10% (wt/vol) oxalic acid treatment, which solubilizes both ferric sulfate and ferric hydroxide precipitates (B. Ramsay, J. Ramsay, M. deTremblay, and C. Chavarie, Geomicrobiol. J. 6:171-177, 1988), enabled the recovery of intact bacterial cells from the tailings material and from liquid synthetic medium for lipopolysaccharide analysis. No viable cells could be cultured after this oxalic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electro-phoretic profiles of lipopolysaccharides extracted from the Lemoine tailings were complex, indicating a heterogeneous population of Thiobacillus ferrooxidans. Six T. ferrooxidans subspecies as identified by lipopolysaccharide analysis (i.e., lipopolysaccharide chemotypes) were eventually isolated from a total of 112 cultures from the Lemoine tailings. Using the same isolate and lipopolysaccharide typing techniques, we identified only a single lipopolysaccharide chemotype from 20 cultures of T. ferrooxidans isolated from the Copper Rand tailings. This homogeneity of lipopolysaccharide chemotype was much different from what was found for the older Lemoine tailings and may reflect a progressive lipopolysaccharide heterogeneity of Thiobacillus isolates as tailings leach and age. Images PMID:16348925

  8. Blockade of the N-Methyl-D-Aspartate Glutamate Receptor Ameliorates Lipopolysaccharide-Induced Renal Insufficiency

    PubMed Central

    Huang, Ho-Shiang; Ma, Ming-Chieh

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor activation in rat kidney reduces renal perfusion and ultrafiltration. Hypoperfusion-induced ischemia is the most frequent cause of functional insufficiency in the endotoxemic kidney. Here, we used non-hypotensive rat model of lipopolysaccharide-induced endotoxemia to examine whether NMDA receptor hyperfunction contributes to acute kidney injury. Lipopolysaccharide-induced renal damage via increased enzymuria and hemodynamic impairments were ameliorated by co-treatment with the NMDA receptor blocker, MK-801. The NMDA receptor NR1 subunit in the rat kidney mainly co-localized with serine racemase, an enzyme responsible for synthesizing the NMDA receptor co-agonist, D-serine. The NMDA receptor hyperfunction in lipopolysaccharide-treated kidneys was demonstrated by NR1 and serine racemase upregulation, particularly in renal tubules, and by increased D-serine levels. Lipopolysaccharide also induced cell damage in cultured tubular cell lines and primary rat proximal tubular cells. This damage was mitigated by MK-801 and by small interfering RNA targeting NR1. Lipopolysaccharide increased cytokine release in tubular cell lines via toll-like receptor 4. The release of interleukin-1β from these cells are the most abundant. An interleukin-1 receptor antagonist not only attenuated cell death but also abolished lipopolysaccharide-induced NR1 and serine racemase upregulation and increases in D-serine secretion, suggesting that interleukin-1β-mediated NMDA receptor hyperfunction participates in lipopolysaccharide-induced tubular damage. The results of this study indicate NMDA receptor hyperfunction via cytokine effect participates in lipopolysaccharide-induced renal insufficiency. Blockade of NMDA receptors may represent a promising therapeutic strategy for the treatment of sepsis-associated renal failure. PMID:26133372

  9. Structural Analysis and Involvement in Plant Innate Immunity of Xanthomonas axonopodis pv. citri Lipopolysaccharide*

    PubMed Central

    Casabuono, Adriana; Petrocelli, Silvana; Ottado, Jorgelina; Orellano, Elena G.; Couto, Alicia S.

    2011-01-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response. PMID:21596742

  10. A method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide.

    PubMed

    Roos, Abraham B; Berg, Tove; Ahlgren, Kerstin M; Grunewald, Johan; Nord, Magnus

    2014-01-01

    Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology. PMID:25548888

  11. Genomic and Proteomic Studies on Plesiomonas shigelloides Lipopolysaccharide Core Biosynthesis

    PubMed Central

    Aquilini, Eleonora; Merino, Susana; Regué, Miguel

    2014-01-01

    We report here the identification of waa clusters with the genes required for the biosynthesis of the core lipopolysaccharides (LPS) of two Plesiomonas shigelloides strains. Both P. shigelloides waa clusters shared all of the genes besides the ones flanking waaL. In both strains, all of the genes were found in the waa gene cluster, although one common core biosynthetic gene (wapG) was found in a different chromosome location outside the cluster. Since P. shigelloides and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up at least to the second outer-core residue, the functions of the common P. shigelloides genes were elucidated by genetic complementation studies using well-defined K. pneumoniae mutants. The function of strain-specific inner- or outer-core genes was identified by using as a surrogate acceptor LPS from three well-defined K. pneumoniae core LPS mutants. Using this strategy, we were able to assign a proteomic function to all of the P. shigelloides waa genes identified in the two strains encoding six new glycosyltransferases (WapA, -B, -C, -D, -F, and -G). P. shigelloides demonstrated an important variety of core LPS structures, despite being a single species of the genus, as well as high homologous recombination in housekeeping genes. PMID:24244003

  12. Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein.

    PubMed Central

    Larrick, J W; Hirata, M; Balint, R F; Lee, J; Zhong, J; Wright, S C

    1995-01-01

    CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-amino-acid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18(104-140). A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18(104-140) and a more active truncated fragment, CAP18(104-135), were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18(104-140) may have therapeutic utility for conditions associated with elevated concentrations of LPS. PMID:7890387

  13. Lipopolysaccharide does not affect acoustic startle reflex in mice.

    PubMed

    Juszczak, Grzegorz R; Blaszczyk, Janusz; Sadowski, Bogdan; Sliwa, Adam T; Wolak, Patrycja; Tymosiak-Zielinska, Agnieszka; Lisowski, Pawel; Swiergiel, Artur H

    2008-01-01

    Bacterial endotoxin (lipopolysaccharide; LPS) evokes in rodents an adaptive sickness behavior. It also produces changes in stress hormones secretion and activity of brain serotonergic and noradrenergic systems that have been implicated in stress responses, fear, and anxiety. Acoustic startle reflex (ASR) is regarded as a protective behavioral response that is enhanced in threatening situations or following an aversive event, and it can be modulated by physiological and emotional state of an animal. Effects of intraperitoneal injections of LPS on ASR, prepulse inhibition (PPI), locomotor activity in open field, and blood plasma corticosterone concentration were studied in lines of mice that display high (HA line) or low (LA line) swim stress-induced analgesia and also differ in emotional behaviors, including the magnitude of ASR. In both lines LPS produced robust sickness behavior, as evidenced by a decrease in locomotion and body weight, and an increase in corticosterone concentration. However, in neither line LPS injections affected responses to acoustic stimuli as assessed by the ASR and PPI magnitudes. The findings suggest that in sickness behavior induced by LPS the protective responses to salient environmental stimuli are not impaired. The significance of this finding for the concept of sickness behavior is discussed. PMID:17651939

  14. Effects of lipopolysaccharide on the catabolic activity of macrophages

    SciTech Connect

    Cluff, C.; Ziegler, H.K.

    1986-03-05

    The ability of macrophages to degrade and catabolize antigens is of relevance both as a means to process complex antigens prior to presentation to T cells, as well as a way to down regulate immune responses by destroying the antigenicity of polypeptides. With these considerations, the authors have investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of /sup 125/-I labelled surface components of heat-killed Listeria monocytogenes (HKLM) subsequent to their uptake by macrophages. They have compared the catabolic activity of macrophages from peritoneal exudates of mice injected i.p. with saline or LPS and have found that LPS-elicited macrophages display a greatly enhanced (3 fold) rate of catabolism. This increase in catabolic activity peaks 3 days after LPS injection and steadily declines thereafter, approaching a baseline level after 3 weeks. The enhancement of catabolic activity is under LPS gene control. LPS-elicited macrophages rapidly destroy the antigenicity of bacterial antigens and function poorly as antigen presenting cells in vitro. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions with negative regulatory effects on the induction of specific immune responses.

  15. Proteomic Changes in Chicken Plasma Induced by Salmonella typhimurium Lipopolysaccharides

    PubMed Central

    Packialakshmi, Balamurugan; Liyanage, Rohana; Lay, Jackson O.; Makkar, Sarbjeet K.; Rath, Narayan C.

    2016-01-01

    Lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria that produce inflammation and sickness in higher animals. The objective was to identify plasma proteomic changes in an avian model of inflammation. Chickens were treated with either saline or LPS, and blood was collected at 24 hours postinjection. The pooled plasma samples were depleted of high-abundant proteins and analyzed by matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS). MALDI analyses showed an increase in fibrinogen beta-derived peptide and a decrease in apolipoprotein-AII-derived peptide in LPS samples. Label-free quantitation of LC–MS/MS spectra revealed an increase in the levels of α1-acid glycoprotein, a chemokine CCLI10, and cathelicidin-2, but a decrease in an interferon-stimulated gene-12-2 protein in the LPS group. These differentially expressed proteins are associated with immunomodulation, cytokine changes, and defense mechanisms, which may be useful as candidate biomarkers of infection and inflammation. PMID:27053921

  16. [SEROLOGICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF PANTOEA AGGLOMERANS LIPOPOLYSACCHARIDES].

    PubMed

    Bulygina, T V; Yakovleva, L M; Brovarska, O S; Varbanets, L D

    2015-01-01

    The serological and phytotoxic properties of lipopolysaccharide (LPS) of plant pathogens--Pantoea agglomerans were studied. It is known that the thin variations in the structure of the O-specific polysaccharides determining serological specificity of gram- negative bacteria and used as a molecular basis of serological classification schemes. For P. agglomerans still does not exist a classification scheme based on serology specificity of their LPS. The results of cross serological tests demonstrate immunochemical heterogeneity of species P agglomerans. Only three strains of the 8488, 8490 and 7969 according to the agglutination of O-antigens and direct hemagglutination and inhibition direct hemagglutination can be attributed to a single serogroup. Other strains--each separate group, although some have a relationship. Compared with control plants under the influence of seed treatment of LPS in plants may be reduced, and in some cases increased root length, height and weight sprout, depending on the strain from which the selected LPS. Dive seedlings of tomatoes in the solutions of the studied preparations FSC caused the loss, and after some time, restore turgor. PMID:26829835

  17. Lipopolysaccharide aggravates bisphosphonate-induced osteonecrosis in rats.

    PubMed

    Sakaguchi, O; Kokuryo, S; Tsurushima, H; Tanaka, J; Habu, M; Uehara, M; Nishihara, T; Tominaga, K

    2015-04-01

    The pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is highly controversial. We have previously reported the development of osteonecrosis by periodontal pathogenic stimulation in the jaw and femur of rats treated with bisphosphonate. Since the major toxicity factor of Gram-negative bacteria is lipopolysaccharide (LPS), the present study aimed to evaluate the relationship between osteonecrosis and LPS in a rat model of BRON-like lesions. Seventeen male rats were injected subcutaneously with zoledronic acid weekly for 4 weeks and divided into three groups: LPS (LPS administered into the bone marrow of the mandible and femur) and LPS plus polymyxin B (PMB) and saline groups (given neutralized LPS with PMB or saline, respectively, using the same protocol). At 4 weeks after the procedure, harvested specimens were analyzed using histomorphology (n=5 from each group) and histochemistry (n=1 each from LPS and LPS plus PMB groups). There was a significantly wider area of osteonecrosis in the LPS group as compared to the saline and LPS plus PMB groups in both the mandible (P=0.030 and P=0.009, respectively) and femur (P=0.002 and P=0.020, respectively). Our results indicate that LPS stimulation is deeply involved in the development and promotion of BRON. PMID:25442743

  18. Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages

    SciTech Connect

    Handman, E.; Schnur, L.F.; Spithill, T.W.; Mitchell, G.F.

    1986-12-01

    Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. The authors have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study they have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here they show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, they show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.

  19. Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline.

    PubMed

    Sharma, Arpita; Patro, Nisha; Patro, Ishan K

    2016-05-01

    Astrocytes are most abundant glial cell type in the brain and play a main defensive role in central nervous system against glutamate-induced toxicity by virtue of numerous transporters residing in their membranes and an astrocyte-specific enzyme glutamine synthetase (GS). In view of that, a dysregulation in the astrocytic activity following an insult may result in glutamate-mediated toxicity accompanied with astrocyte and microglial activation. The present study suggests that the lipopolysaccharide (LPS)-induced inflammation results in significant astrocytic apoptosis compared to other cell types in hippocampus and minocycline could not efficiently restrict the glutamate-mediated toxicity and apoptosis of astrocytes. Upon LPS exposure 76 % astrocytes undergo degeneration followed by 44 % oligodendrocytes, 26 % neurons and 10 % microglia. The pronounced astrocytic apoptosis resulted from the LPS-induced glutamate excitotoxicity leading to their hyperactivation as evident from their hypertrophied morphology, glutamate transporter 1 upregulation and downregulation of GS. Therapeutic minocycline treatment to LPS-infused rats efficiently restricted the inflammatory response and degeneration of other cell types but could not significantly combat with the apoptosis of astrocytes. Our study demonstrates a novel finding on cellular degeneration in the hippocampus revealing more of astrocytic death and suggests a more careful consideration on the protective efficacy of minocycline. PMID:26188416

  20. Wogonoside ameliorates lipopolysaccharide-induced acute lung injury in mice.

    PubMed

    Zhang, Liang; Ren, Yi; Yang, Chengliang; Guo, Yue; Zhang, Xiaojing; Hou, Gang; Guo, Xinjin; Sun, Nan; Liu, Yongyu

    2014-12-01

    Wogonoside has been reported to have anti-inflammatory properties. In this study, we evaluated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside 1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α, IL-6, and IL-1β levels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the TNF-α, IL-6, and IL-1β levels were significantly decreased. Meanwhile, wogonoside significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways. PMID:24854163

  1. Immobilization and molecular interactions between bacteriophage and lipopolysaccharide bilayers.

    PubMed

    Handa, Hitesh; Gurczynski, Stephen; Jackson, Matthew P; Mao, Guangzhao

    2010-07-20

    The paper describes immobilization methods of bacteriophage P22 and tailspike gp9 proteins isolated from P22 on atomic force microscope (AFM) probes. The paper also reports single molecule force spectroscopy (SMFS) using AFM of the immobilized P22 (or gp9) interactions with substrate-supported O-antigenic lipopolysaccharides (LPS) bilayers. LPS covers the outer membrane of gram-negative bacteria, such as Salmonella typhimurium. Evidence from AFM imaging and SMFS shows that immobilized P22 (or gp9) are capable of strong and multivalent binding to supported LPS. The most common rupture forces between P22 and LPS were identified to be 72, 130, 206, and 279 pN at force loading rate of 12,000 pN/s. The quantized unbinding force was found to decrease with decreasing force loading rate as predicted by the Bell model. By fitting the force data with the Bell model, an energy barrier of 55 kJ/mol was obtained. Evidence is also provided that demonstrates the resilience of phage to pH and temperature fluctuation as well as dehydration/rehydration cycles. The biospecific interactions between P22 and the LPS are relevant to cell infection, inflammation, cancer progression and metastasis, food safety, pharmaceuticals, and biosensor development. PMID:20481467

  2. Allergen immunotherapy with nanoparticles containing lipopolysaccharide from Brucella ovis.

    PubMed

    Gómez, Sara; Gamazo, Carlos; San Roman, Beatriz; Ferrer, Marta; Sanz, Maria Luisa; Espuelas, Socorro; Irache, Juan M

    2008-11-01

    The adjuvant and protective capacity against anaphylactic shock of the association between rough lipopolysaccharide of Brucella ovis (LPS) coencapsulated with ovalbumin (OVA), as a model allergen, in Gantrez AN nanoparticles was investigated. Several strategies were performed in order to study the adjuvant effect of the LPS either encapsulated or coating the nanoparticles. OVA, as well as LPS, was incorporated either during the manufacturing process (OVA-encapsulated or LPS-encapsulated nanoparticles, respectively) or after the preparation (OVA-coated or LPS-coated nanoparticles, respectively). After the administration of 10 microg of OVA incorporated in the different formulations, all the nanoparticles, with or without LPS, were capable of amplifying the immune response (IgG(1) and IgG(2a)). However, in a model of sensitized mice to OVA, the formulation with OVA and LPS-entrapped inside the nanoparticles administered intradermally in three doses of 3 microg of OVA each was the only treatment that totally protected the mice from death after a challenge with an intraperitoneal injection of OVA. In contrast, the control group administered with OVA adsorbed onto a commercial alhydrogel adjuvant showed 80% mortality. These results are highly suggestive for the valuable use of Gantrez nanoparticles combined with rough LPS of B. ovis in immunotherapy. PMID:18582571

  3. Early morphofunctional plasticity of microglia in response to acute lipopolysaccharide.

    PubMed

    Madore, C; Joffre, C; Delpech, J C; De Smedt-Peyrusse, V; Aubert, A; Coste, L; Layé, S; Nadjar, A

    2013-11-01

    Within the central nervous system (CNS) the traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. This led to the concept of "resting" versus "activated" microglia. However, this is misleading because multiple phenotypic and morphological stages of microglia can influence neuronal structure and function in any condition and recent evidence extends their role to healthy brain homeostasis. The present work was thus aimed at reappraising the concept of morphofunctional activity of microglia in a context of peripheral acute immune challenge, where microglial activity is known to be modified, using the new state-of-the-art techniques available. To do so, mice were injected peripherally with lipopolysaccharide, a potent inducer of cerebral inflammation, and we assessed early cytokines production, phenotype, motility and morphology of microglial cells. Our results showed that LPS induced a widespread inflammatory response both peripherally and centrally, as revealed by the quantification of cytokines levels. We also found an alteration of microglial motility that was confirmed by in vivo studies showing an overall reduction of microglial processes length in the hippocampus of LPS-treated animals. Finally, analysis of various surface receptors expression revealed that LPS did not significantly impact microglial phenotype 2h after the injection but rather induced an increase of CD11b(+)/CD45(high) cells. These latter may be at the vasculature, at the CNS vicinity, or may have invaded the CNS. PMID:23994463

  4. Macrophage cytokine response to particles and lipopolysaccharide in vitro.

    PubMed

    Daniels, A U; Barnes, F H; Charlebois, S J; Smith, R A

    2000-03-15

    Several investigators have suggested that biologic molecules adsorbed onto particles may play a key role in determining macrophage response. Adsorbed endotoxins (bacterial debris) may be of particular importance since they are widely present exogenously and endogenously and adhere strongly to many materials. Murine-transformed peritoneal macrophages (IC-21) were used in this in vitro study. Secretions of IL-1 beta, TNF alpha, and IL-6 were used as a measure of macrophage response to micron-range particles of high-density polyethylene and Co-Cr-Mo alloy, with and without adsorbed lipopolysaccharide (LPS) endotoxin. Little cytokine secretion was measured in response to particles (and to polypropylene experimental chambers) cleaned with ethanol and saline and not exposed to LPS. The lack of macrophage response to cleaned particles has been reported by others and may help reconcile conflicting reports in the literature. Cytokine secretion levels were high in all cases if the chambers (with or without particles) were exposed to LPS (and rinsed to minimize nonbound LPS). Secretion patterns were different with particles present and for polymer versus metal particles. Overall, these results suggest that (1) adsorbed molecules on material surfaces strongly affect macrophage response and (2) particle surface chemistry and microstructure affect the concentration and configuration of adsorbed molecules, further influencing particle interaction with macrophage surface receptors. PMID:10602080

  5. Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

    SciTech Connect

    Li Song; Zhang Junjie

    2009-01-09

    Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the {beta} isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment.

  6. Meningococcal lipopolysaccharides: virulence factor and potential vaccine component.

    PubMed Central

    Verheul, A F; Snippe, H; Poolman, J T

    1993-01-01

    Lipopolysaccharides (LPS) are surface components of the outer membrane of Neisseria meningitidis. Today, 12 different types of meningococcal LPS (immunotypes) are known, of which 3 are prevalent in the western world. The differences between these immunotypes are in the oligosaccharide part of the LPS molecule and consist of small differences in the oligosaccharide structure, the amount and location of phosphoethanolamine groups, and the degree of O acetylation of individual monosaccharides. Although the differences between the various immunotypes are small, they have a profound influence on the immunochemical and immunological properties of these molecules. Furthermore, each individual strain synthesizes a number of different LPS molecules. The expression of the various components (protective epitopes) is influenced by growth conditions and growth phase. Meningococci can endogenously sialyate their LPS, which constitutes one of the mechanisms by which N. meningitidis can evade the response of the human host. Meningococcal LPS play a key role in the induction of septic shock and can probably enhance the invasiveness of meningococcal strains and shield protective epitopes. Therefore, incorporation of (detoxified) LPS or oligosaccharide components derived therefrom might be very beneficial for the efficacy of a vaccine against group B meningococci. An overview of the development of vaccines against group B meningococci is given, and the status and potential of meningococcal LPS-derived (synthetic) oligosaccharide-protein conjugate vaccines are discussed. PMID:8464406

  7. Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria.

    PubMed

    Lee, Kyung-Jo; Lee, Mi-Ae; Hwang, Won; Park, Hana; Lee, Kyu-Ho

    2016-08-01

    The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria. PMID:27294580

  8. Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions.

    PubMed Central

    Jarrell, K F; Kropinski, A M

    1981-01-01

    We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations. PMID:6798225

  9. Lipopolysaccharide-induced inflammatory liver injury in mice.

    PubMed

    Hamesch, K; Borkham-Kamphorst, E; Strnad, P; Weiskirchen, R

    2015-04-01

    The intraperitoneal application of lipopolysaccharide (LPS) alone or in combination with other hepatotoxins is an experimental model for inducing systemic and hepatic inflammation in rodents applied worldwide. The endotoxin is recognized by the LPS-binding protein. This complex binds together with the lymphocyte antigen 96 (MD2) and the pattern-recognition receptor CD14 to members of the toll-like receptor family. The activated receptor complex in turn transduces signals to well characterized intracellular cascades that result in a multifaceted network of intracellular responses ending in inflammation. The most prominent among these is the activation of the NF-κB pathway and the production of a multitude of inflammatory cytokines. Although the application of LPS is in general easy to perform, unintended variations in preparation of the injection solution or in handling of the animals might affect the reproducibility or the outcome of a specific experiment. Here, we present a well-standardized protocol that allows for an induction of highly reproducible acute hepatic inflammation in mice. Furthermore, examples of appropriate readouts for the resulting inflammatory response are given. PMID:25835737

  10. Inhibition of lipopolysaccharide induced acute inflammation in lung by chlorination.

    PubMed

    Zhang, Jinshan; Xue, Jinling; Xu, Bi; Xie, Jiani; Qiao, Juan; Lu, Yun

    2016-02-13

    Lipopolysaccharide (LPS, also called endotoxin) is a pro-inflammatory constituent of gram negative bacteria and cyanobacteria, which causes a potential health risk in the process of routine urban application of reclaimed water, such as car wash, irrigation, scenic water refilling, etc. Previous studies indicated that the common disinfection treatment, chlorination, has little effect on endotoxin activity removal measured by Limulus amebocyte lysate (LAL) assay. However, in this study, significant decrease of acute inflammatory effects was observed in mouse lung, while LAL assay still presented a moderate increase of endotoxin activity. To explore the possible mechanisms, the nuclear magnetic resonance (NMR) results showed the chlorination happened in alkyl chain of LPS molecules, which could affect the interaction between LPS and LPS-binding protein. Also the size of LPS aggregates was found to drop significantly after treatment, which could be another results of chlorination caused polarity change. In conclusion, our observation demonstrated that chlorination is effective to reduce the LPS induced inflammation in lung, and it is recommended to use health effect-based methods to assess risk removal of water treatment technologies. PMID:26530889

  11. Visualization and analysis of lipopolysaccharide distribution in binary phospholipid bilayers

    SciTech Connect

    Henning, Maria Florencia; Sanchez, Susana; Bakas, Laura

    2009-05-22

    Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram-negative bacteria during infections. It have been reported that LPS may play a role in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes. In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4 {sup o}C. The LPS distribution was analyzed on GUVs of DPPC:DOPC using FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains. Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several components of the LPS-sensing machinery.

  12. Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

    PubMed Central

    Mintz, C S; Schultz, D R; Arnold, P I; Johnson, W

    1992-01-01

    Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. PMID:1612744

  13. Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

    PubMed Central

    Norman, R. Sean; Frontera-Suau, Roberto; Morris, Pamela J.

    2002-01-01

    Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation. PMID:12324360

  14. Arctigenin attenuates lipopolysaccharide-induced acute lung injury in rats.

    PubMed

    Shi, Xianbao; Sun, Hongzhi; Zhou, Dun; Xi, Huanjiu; Shan, Lina

    2015-04-01

    Arctigenin (ATG) has been reported to possess anti-inflammatory properties. However, the effects of ATG on lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains not well understood. In the present study, our investigation was designed to reveal the effect of ATG on LPS-induced ALI in rats. We found that ATG pretreatment attenuated the LPS-induced ALI, as evidenced by the reduced histological scores, myeloperoxidase activity, and wet-to-dry weight ratio in the lung tissues. This was accompanied by the decreased levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-1 (IL-6) in the bronchoalveolar lavage fluid. Furthermore, ATG downregulated the expression of nuclear factor kappa B (NF-κB) p65, promoted the phosphorylation of inhibitor of nuclear factor-κB-α (IκBα) and activated the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPKα) in the lung tissues. Our results suggested that ATG attenuates the LPS-induced ALI via activation of AMPK and suppression of NF-κB signaling pathway. PMID:25008149

  15. Retention of bacterial lipopolysaccharide at the site of subcutaneous injection.

    PubMed Central

    Yokochi, T.; Inoue, Y.; Yokoo, J.; Kimura, Y.; Kato, N.

    1989-01-01

    The tissue distribution of Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) was studied in mice injected subcutaneously (s.c.) or intraperitoneally (i.p.) with 125I-labeled KO3 LPS. Marked retention of KO3 LPS radioactivity could be found at the site of s.c. injection for several weeks. On the other hand, about 85% of the radioactivity rapidly disappeared from the peritoneal cavity within 6 h after i.p. injection. The long-term presence of KO3 LPS at the injection site was also supported by experiments with 51Cr-labeled KO3 LPS and immunoblotting and immunofluorescence staining methods. The R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS and the lipid A fraction of KO3 LPS seemed to remain at the site in larger amounts and for longer times than KO3 LPS. There were no marked differences in the retention pattern at the injection site among KO3 LPS, Escherichia coli LPS, Salmonella typhosa LPS, and Salmonella enteritidis LPS. However, much less radioactivity accumulated in the livers and spleens of mice injected with either KO3 LPS or S. typhosa LPS compared with the other LPS preparations. It was suggested that retention of LPS at the site of s.c. injection may play an important role in the development of various biological actions of s.c. injected LPS. Images PMID:2722239

  16. Lipopolysaccharide Attenuates the Cytotoxicity of Resveratrol in Transformed Mouse Macrophages.

    PubMed

    Achy-Brou, Christelle A Adiabouah; Billack, Blase

    2016-09-01

    Resveratrol and pterostilbene are natural products that are present in plants and have been incorporated into various dietary supplements. Numerous beneficial pharmacologic effects have been reported for these stilbenes; however, the mechanism by which these compounds exert a cytotoxic effect in RAW 264.7 macrophages has not been well characterized. We have previously described that resveratrol is toxic to these tumor-derived macrophages and that stimulation with lipopolysaccharide (LPS) reduces resveratrol toxicity via a mechanism that involves activation of toll like receptor 4. In the present work, we examined the cellular and molecular effects of resveratrol and the related compound pterostilbene by determining cell viability and caspase 3 activity in control and LPS-stimulated RAW 264.7 macrophages incubated with these stilbenes for 24 h. We found that LPS stimulation reduced the cytotoxicity of resveratrol but not of pterostilbene in these cells. When examined for effects on caspase 3 activation after a 24 h incubation, resveratrol and pterostilbene were each found to separately and significantly increase caspase 3 activity in these cells. LPS stimulation prevented caspase 3 activation by pterostilbene and reduced caspase 3 activation by resveratrol in RAW 264.7 macrophages. The data presented here indicate that LPS induces a phenotype switch in tumor-derived RAW 264.7 macrophages in which cells experiencing LPS in the presence of resveratrol or pterostilbene become less likely to activate the pro-apoptotic factor caspase 3. PMID:27277074

  17. Chikusetsusaponin V attenuates lipopolysaccharide-induced liver injury in mice.

    PubMed

    Dai, Yan Wen; Zhang, Chang Cheng; Zhao, Hai Xia; Wan, Jing Zhi; Deng, Li Li; Zhou, Zhi Yong; Dun, Yao Yan; Liu, Chao Qi; Yuan, Ding; Wang, Ting

    2016-06-01

    Chikusetsusaponin V (CsV), a saponin from Panax japonicus, has been reported to inhibit inflammatory responses in lipopolysaccharide (LPS)-induced macrophage cells. However, whether CsV could alleviate LPS-induced liver injury in vivo and the potential mechanisms involved remain unclear. In the present study, we investigated the anti-inflammatory effects of CsV on LPS-induced acute liver injury in mice and further explored the potential mechanisms involved. Our results showed that CsV significantly attenuated elevation of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and improved liver histopathological changes in LPS-induced mice. In addition, CsV decreased serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels and inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in LPS challenged mice. Furthermore, CsV inhibited nuclear factor kappa B (NF-κB) activation by downregulating phosphorylated NF-κB, IκB-α, ERK, c-Jun N-terminal kinase (JNK) and p38 levels in the liver tissue, which ultimately decreased nucleus NF-κB protein level. In conclusion, our data suggested that CsV could be a promising drug for preventing LPS challenged liver injury since it attenuated LPS-induced inflammatory responses, partly via inhibiting NF-κB and MAPK signaling pathways. PMID:26981791

  18. Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation

    PubMed Central

    Sun, Yue; Shang, Dejing

    2015-01-01

    Antimicrobial peptides (AMPs) are usually small molecule peptides, which display broad-spectrum antimicrobial activity, high efficiency, and stability. For the multiple-antibiotic-resistant strains, AMPs play a significant role in the development of novel antibiotics because of their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. Besides broad-spectrum antibacterial activity, AMPs also have anti-inflammatory activity. The neutralization of lipopolysaccharides (LPS) plays a key role in anti-inflammatory action of AMPs. On the one hand, AMPs can readily penetrate the cell wall barrier by neutralizing LPS to remove Gram-negative bacteria that can lead to infection. On the contrary, AMPs can also inhibit the production of biological inflammatory cytokines to reduce the inflammatory response through neutralizing circulating LPS. In addition, AMPs also modulate the host immune system by chemotaxis of leukocytes, to promote immune cell proliferation, epithelialization, and angiogenesis and thus play a protective role. This review summarizes some recent researches about anti-inflammatory AMPs, with a focus on the interaction of AMPs and LPS on the past decade. PMID:26612970

  19. Lipopolysaccharide induces expression of collagen VI in the rat lung.

    PubMed

    Okawa, Sayuri; Unuma, Kana; Yamada, Atsushi; Aki, Toshihiko; Uemura, Koichi

    2015-01-01

    The involvement of the lung during the septic systemic inflammatory response elicited by administration of lipopolysaccharide (LPS) was investigated. Eight-week-old male Sprague-Dawley rats were injected i.p. with 15 mg/kg LPS. After 24 h, the lungs were excised to evaluate the cellular responses to LPS. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis revealed that type VI collagen (ColVI) was extremely upregulated during sepsis in the rat lung within the first 24 h of LPS administration. Upregulation of ColVI protein and its mRNA was demonstrated by Western blot analysis, real time PCR, and immunohistochemistry. To the best of our knowledge, this is the first report demonstrating the activation of ColVI in the rat lung at the early stage of systemic inflammation. Activation of ColVI might be involved in sepsis-mediated lung fibrosis at an early stage. PMID:26023260

  20. Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production.

    PubMed

    Lin, Jisheng; Smith, Mark A; Benjamin, William H; Kaminski, Robert W; Wenzel, Heather; Nahm, Moon H

    2016-08-01

    There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency. PMID:27280622

  1. Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis *

    PubMed Central

    Xu, Hai-sheng; Lyu, Sun-jian; Xu, Jie-hao; Lu, Bin-jie; Zhao, Jing; Li, Song; Li, Yi-qun; Chen, Yu-yin

    2015-01-01

    In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes. PMID:26642180

  2. Interleukin-1 in Lipopolysaccharide Induced Chorioamnionitis in the Fetal Sheep

    PubMed Central

    Berry, Clare A.; Nitsos, Ilias; Hillman, Noah H.; Pillow, J. Jane; Polglase, Graeme R.; Kramer, Boris W.; Kemp, Matthew W.; Newnham, John P.; Jobe, Alan H.; Kallapur, Suhas G.

    2011-01-01

    We tested the hypothesis that interleukin 1 (IL-1) mediates intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis in preterm fetal sheep. Time-mated Merino ewes with singleton fetuses received IL-1α, LPS, or saline (control) by intra-amniotic injection 1 to 2 days before operative delivery at 124 ± 1 days gestational age (N = 5-9/group; term = 150 days). Recombinant human IL-1 receptor antagonist (rhIL-1ra) was given into the amniotic fluid 3 hours before intra-amniotic LPS or saline to block IL-1 signaling. Inflammation in the chorioamnion was determined by histology, cytokine messenger RNA (mRNA), protein expression, and by quantitation of activated inflammatory cells. Intra-amniotic IL-1 and LPS both induced chorioamnionitis. However, IL-1 blockade with IL-1ra did not decrease intra-amniotic LPS-induced increases in pro-inflammatory cytokine mRNAs, numbers of inflammatory cells, myeloperoxidase, or monocyte chemotactic protein-1-expressing cells in the chorioamnion. We conclude that IL-1 and LPS both can cause chorioamnionitis, but IL-1 is not an important mediator of LPS-induced chorioamnionitis in fetal sheep. PMID:21493953

  3. Lipopolysaccharide, central in vivo biogenic amine variations, and anhedonia.

    PubMed

    Borowski, T; Kokkinidis, L; Merali, Z; Anisman, H

    1998-12-01

    Systemic administration of lipopolysaccharide (LPS), a non-specific activator of proinflammatory cytokine release from macrophages, provokes sickness characterized by anorexia, soporific effects, and disturbances of locomotor activity and exploration. In addition, endotoxin treatment may provoke an anhedonic response. Assessment of anhedonia in appetitive paradigms, however, is compromised by the anorexia provoked by the treatment. The present investigation assessed the anhedonic effects of LPS on rewarding lateral hypothalamic brain stimulation. Using a simultaneous discrimination, current titration procedure in the assessment of intracranial self-stimulation (ICSS), it was found that acute systemic administration of LPS (50 microg, 100 microg or 200 microg) reduced ICSS during the ascending sequence of current presentations, but had little effect on responding to a series of descending currents. In a parallel experiment, peripheral administration of LPS (100 microg) increased in vivo dopamine (DA) efflux from the nucleus accumbens, a region thought to be involved in goal-directed responding to positively reinforcing stimuli. It is suggested that LPS alters ICSS in a manner different than that observed following stressor exposure or peripheral IL-2 treatment. Furthermore, LPS may engender an anhedonic effect (possibly secondary to sickness), and the decline of responding reflects the relation between the cost of responding given in the face of sickness and the reward received for responding. PMID:9875707

  4. Characterization of the lipopolysaccharide from Vibrio cholerae 395 (Ogawa).

    PubMed Central

    Kabir, S

    1982-01-01

    The chemical structure and biological properties of the lipopolysaccharide (LPS) from Vibrio cholerae 395 (Ogawa), isolated by the phenol-water procedure, were studied. Upon acid hydrolysis, the LPS was split into its polysaccharide and lipid A moieties. The polysaccharide contained both neutral (glucose, heptose, fructose) and amino (glucosamine, quinovosamine) sugars. The LPS contained the acid-labile amino sugar, 4-amino-arabinose, which was absent in the Inaba serotype of V. cholerae. The LPS differed from the LPSs of Enterobacteriaceae by the absence of 2-keto-3-deoxyoctonate and the presence of fructose. Analysis of the methylated polysaccharide by gas-liquid chromatography and mass spectrometry showed that it had a branched structure with glucose and heptose residues primarily appearing at the nonreducing-end groups. Interactions with lectins, concanavalin A. and wheat germ agglutinin suggested that terminal glucose residues were alpha linked, whereas terminal glucosamine residues were connected by alpha-1,3 linkages. The major fatty acids of the LPS were C14:0, C16:0, C12h:0, and C14h:0 compounds, of which only the C14h:0 were amide linked, the remainder being ester linked to the backbone. Biological studies showed that the LPS possessed endotoxic properties such as lethality, pyrogenicity, limulus lysate gelation, and ability to induce non-specific resistance to infection. Thus, the LPS from V. cholerae 395 (Ogawa) possessed both common and distinct features as compared with the LPSs from the Enterobacteriaceae. Images PMID:7152669

  5. Biological and physicochemical properties of the lipopolysaccharide of Chromatium vinosum.

    PubMed Central

    Hurlbert, R E; Hurlbert, I M

    1977-01-01

    The lipopolysaccharide (LPS) of Chromatium vinosum has anticomplementary activity. This anticomplementary activity is destroyed by alkaline digestion of the LPS and is suppressed by both Mg2+ and Ca2+ ions. Treatment of the LPS with ethylenediaminetetraacetic acid, sodium deoxycholate, or dimethyl sulfoxide did not affect its toxicity toward mice; however, alkaline-treated LPS was not toxic. Treatment of the LPS with sodium deoxycholate, dimethyl sulfoxide, or sodium dodecyl sulfate resulted in reversible dissociation into subunits. Aggregation of the subunits into the original form was achieved by removing the dispersing agent by dialysis against distilled water followed by freezing and thawing. Electron micrographs of phenol-extracted LPS showed long filaments. Electron micrographs of sodium deoxycholate- and sodium dodecyl sulfate-treated and dialyzed LPS showed a mixture of small subunits and short filaments, whereas dimethyl sulfoxide-treated and dialyzed LPS contained only small ovoid spheres. The LPS produced an ordered series of multiple bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar banding pattern was observed for Salmonella abortus-equi and Proteus mirabilis LPS. The C. vinosum LPS appears to be mitogenic for mouse spleen cells. Images PMID:892903

  6. Astrocytes Release Polyunsaturated Fatty Acids by Lipopolysaccharide Stimuli.

    PubMed

    Aizawa, Fuka; Nishinaka, Takashi; Yamashita, Takuya; Nakamoto, Kazuo; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2016-01-01

    We previously reported that levels of long-chain fatty acids (FAs) including docosahexaenoic acids (DHA) increase in the hypothalamus of inflammatory pain model mice. However, the precise mechanisms underlying the increment of free fatty acids (FFAs) in the brain during inflammation remains unknown. In this study, we characterized FFAs released by inflammatory stimulation in rat primary cultured astrocytes, and tested the involvement of phospholipase A2 (PLA2) on these mechanisms. Lipopolysaccharide (LPS) stimulation significantly increased the levels of several FAs in the astrocytes. Under these conditions, mRNA expression of cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) in LPS-treated group increased compared with the control group. Furthermore, in the culture media, the levels of DHA and arachidonic acid (ARA) significantly increased by LPS stimuli compared with those of a vehicle-treated control group whereas the levels of saturated FAs (SFAs), namely palmitic acid (PAM) and stearic acid (STA), did not change. In summary, our findings suggest that astrocytes specifically release DHA and ARA by inflammatory conditions. Therefore astrocytes might function as a regulatory factor of DHA and ARA in the brain. PMID:27374285

  7. Bacterial lipopolysaccharide promotes destabilization of lung surfactant-like films.

    PubMed

    Cañadas, Olga; Keough, Kevin M W; Casals, Cristina

    2011-01-01

    The airspaces are lined with a dipalmitoylphosphatidylcholine (DPPC)-rich film called pulmonary surfactant, which is named for its ability to maintain normal respiratory mechanics by reducing surface tension at the air-liquid interface. Inhaled airborne particles containing bacterial lipopolysaccharide (LPS) may incorporate into the surfactant monolayer. In this study, we evaluated the effect of smooth LPS (S-LPS), containing the entire core oligosaccharide region and the O-antigen, on the biophysical properties of lung surfactant-like films composed of either DPPC or DPPC/palmitoyloleoylphosphatidylglycerol (POPG)/palmitic acid (PA) (28:9:5.6, w/w/w). Our results show that low amounts of S-LPS fluidized DPPC monolayers, as demonstrated by fluorescence microscopy and changes in the compressibility modulus. This promoted early collapse and prevented the attainment of high surface pressures. These destabilizing effects could not be relieved by repeated compression-expansion cycles. Similar effects were observed with surfactant-like films composed of DPPC/POPG/PA. On the other hand, the interaction of SP-A, a surfactant membrane-associated alveolar protein that also binds to LPS, with surfactant-like films containing S-LPS increased monolayer destabilization due to the extraction of lipid molecules from the monolayer, leading to the dissolution of monolayer material in the aqueous subphase. This suggests that SP-A may act as an LPS scavenger. PMID:21190662

  8. Antioxidant Effects of Cranberry Powder in Lipopolysaccharide Treated Hypercholesterolemic Rats

    PubMed Central

    Kim, Mi Joung; Kim, Jung Hee; Kwak, Ho-Kyung

    2014-01-01

    This study was conducted to investigate the effects of cranberry power on antioxidant defense system in rats fed an atherogenic diet and injected with lipopolysaccharide (LPS). Sprague-Dawley rats were divided into the following 5 groups: normal diet+saline (NS), atherogenic diet+saline (AS), atherogenic diet+LPS (AL), atherogenic diet with 5% cranberry powder+LPS (AL-C5), and atherogenic diet with 10% cranberry powder+LPS (AL-C10). Total antioxidant status measured by ferric reducing ability of plasma (FRAP) was significantly reduced by LPS injection (24%) and was restored by the cranberry powder treatment (P<0.05). In addition, the mean level of plasma total phenolics was significantly decreased by LPS injection (P<0.05) and tended to be increased when cranberry powder was incorporated in to the diet. Activity of serum superoxide dismutase (SOD) tended to be lowered by LPS injection and declined further in cranberry powder fortified groups. Overall results indicate that dietary cranberry powder may provide appropriate antioxidants to counter the diminished antioxidant status induced by exposing hypercholesterolemic rats to LPS. PMID:25054105

  9. Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.

    PubMed

    Brandenburg, K; Jürgens, G; Müller, M; Fukuoka, S; Koch, M H

    2001-08-01

    The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed. PMID:11592403

  10. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    PubMed Central

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine J.; Orr, Galya; Geiger, Franz M.; Haynes, Christy L.; Pedersen, Joel A.

    2015-01-01

    Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides. PMID:26207769

  11. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes.

    PubMed

    Jacobson, Kurt H; Gunsolus, Ian L; Kuech, Thomas R; Troiano, Julianne M; Melby, Eric S; Lohse, Samuel E; Hu, Dehong; Chrisler, William B; Murphy, Catherine J; Orr, Galya; Geiger, Franz M; Haynes, Christy L; Pedersen, Joel A

    2015-09-01

    Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides. PMID:26207769

  12. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    SciTech Connect

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine J.; Orr, Galya; Geiger, Franz M.; Haynes, Christy L.; Pedersen, Joel A.

    2015-07-24

    We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

  13. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    DOE PAGESBeta

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine J.; Orr, Galya; et al

    2015-07-24

    We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) andmore » second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.« less

  14. Lipopolysaccharide density and structure govern the extent and distance of nanoparticle interaction with actual and model bacterial outer membranes

    SciTech Connect

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine; Orr, Galya; Geiger, Franz M.; Haynes, Christy L.; Pedersen, Joel A.

    2015-07-24

    Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

  15. Montelukast attenuates lipopolysaccharide-induced cardiac injury in rats.

    PubMed

    Khodir, A E; Ghoneim, H A; Rahim, M A; Suddek, G M

    2016-04-01

    This study investigates the possible protective effects of montelukast (MNT) against lipopolysaccharide (LPS)-induced cardiac injury, in comparison to dexamethasone (DEX), a standard anti-inflammatory. Male Sprague Dawley rats (160-180 g) were assigned to five groups (n = 8/group): (1) control; (2) LPS (10 mg/kg, intraperitoneal (i.p.)); (3) LPS + MNT (10 mg/kg, per os (p.o.)); (4) LPS + MNT (20 mg/kg, p.o.); and (5) LPS + DEX (1 mg/kg, i.p.). Twenty-four hours after LPS injection, heart/body weight (BW) ratio and percent survival of rats were determined. Serum total protein, creatine kinase muscle/brain (CK-MB), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) activities were measured. Heart samples were taken for histological assessment and for determination of malondialdehyde (MDA) and glutathione (GSH) contents. Cardiac tumor necrosis factor α (TNF-α) expression was evaluated immunohistochemically. LPS significantly increased heart/BW ratio, serum CK-MB, ALP, and LDH activities and decreased percent survival and serum total protein levels. MDA content increased in heart tissues with a concomitant reduction in GSH content. Immunohistochemical staining of heart specimens from LPS-treated rats revealed high expression of TNF-α. MNT significantly reduced percent mortality and suppressed the release of inflammatory and oxidative stress markers when compared with LPS group. Additionally, MNT effectively preserved tissue morphology as evidenced by histological evaluation. MNT (20 mg/kg) was more effective in alleviating LPS-induced heart injury when compared with both MNT (10 mg/kg) and DEX (1 mg/kg), as evidenced by decrease in positive staining by TNF-α immunohistochemically, decrease MDA, and increase GSH content in heart tissue. This study demonstrates that MNT might have cardioprotective effects against the inflammatory process during endotoxemia. This effect can be attributed to its antioxidant and/or anti-inflammatory properties. PMID:26089034

  16. Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo

    PubMed Central

    Poujol, Fanny; Monneret, Guillaume; Pachot, Alexandre; Textoris, Julien; Venet, Fabienne

    2015-01-01

    Context Sepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved. Study The aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli. Results We showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3. Conclusion We conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic. PMID:26642057

  17. Sickness behaviour after lipopolysaccharide treatment in ghrelin deficient mice.

    PubMed

    Szentirmai, Éva; Krueger, James M

    2014-02-01

    Ghrelin is an orexigenic hormone produced mainly by the gastrointestinal system and the brain. Much evidence also indicates a role for ghrelin in sleep and thermoregulation. Further, ghrelin was recently implicated in immune system modulation. Administration of bacterial lipopolysaccharide (LPS) induces fever, anorexia, and increased non-rapid-eye movement sleep (NREMS) and these actions are mediated primarily by proinflammatory cytokines. Ghrelin reduces LPS-induced fever, suppresses circulating levels of proinflammatory cytokines and reduces the severity and mortality of various models of experimental endotoxemia. In the present study, we determined the role of intact ghrelin signaling in LPS-induced sleep, feeding, and thermoregulatory responses in mice. Sleep-wake activity was determined after intraperitoneal, dark onset administration of 0.4, 2 and 10 μg LPS in preproghrelin knockout (KO) and wild-type (WT) mice. In addition, body temperature, motor activity and changes in 24-h food intake and body weight were measured. LPS induced dose-dependent increases in NREMS, and suppressed rapid-eye movement sleep, electroencephalographic slow-wave activity, motor activity, food intake and body weight in both Ppg KO and WT mice. Body temperature changes showed a biphasic pattern with a decrease during the dark period followed by an increase in the light phase. The effects of the low and middle doses of LPS were indistinguishable between the two genotypes. Administration of 10 μg LPS, however, induced significantly larger changes in NREMS and wakefulness amounts, body temperature, food intake and body weight in the Ppg KO mice. These findings support a role for ghrelin as an endogenous modulator of inflammatory responses and a central component of arousal and feeding circuits. PMID:24309634

  18. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed Central

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-01-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age. PMID:1500196

  19. Inflammatory effects of Edwardsiella ictaluri lipopolysaccharide modifications in catfish gut.

    PubMed

    Santander, Javier; Kilbourne, Jacquelyn; Park, Jie-Yeun; Martin, Taylor; Loh, Amanda; Diaz, Ignacia; Rojas, Robert; Segovia, Cristopher; DeNardo, Dale; Curtiss, Roy

    2014-08-01

    Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to

  20. Btk Regulates Macrophage Polarization in Response to Lipopolysaccharide

    PubMed Central

    Ní Gabhann, Joan; Hams, Emily; Smith, Siobhán; Wynne, Claire; Byrne, Jennifer C.; Brennan, Kiva; Spence, Shaun; Kissenpfennig, Adrien; Johnston, James A.; Fallon, Padraic G.; Jefferies, Caroline A.

    2014-01-01

    Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk−\\−) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk−/− macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk−/− macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk−/− macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk−/− mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation. PMID:24465735

  1. Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria.

    PubMed

    Hug, Isabelle; Feldman, Mario F

    2011-02-01

    Bacteria generate and attach countless glycan structures to diverse macromolecules. Despite this diversity, the mechanisms of glycoconjugate biosynthesis are often surprisingly similar. The focus of this review is on the commonalities between lipopolysaccharide (LPS) and glycoprotein assembly pathways and their evolutionary relationship. Three steps that are essential for both pathways are completed by membrane proteins. These include the initiation of glycan assembly through the attachment of a first sugar residue onto the lipid carrier undecaprenyl pyrophosphate, the translocation across the plasma membrane and the final transfer onto proteins or lipid A-core. Two families of initiating enzymes have been described: the polyprenyl-P N-acetylhexosamine-1-P transferases and the polyprenyl-P hexosamine-1-P transferases, represented by Escherichia coli WecA and Salmonella enterica WbaP, respectively. Translocases are either Wzx-like flippases or adenosine triphosphate (ATP)-binding cassette transporters (ABC transporters). The latter can consist either of two polypeptides, Wzt and Wzm, or of a single polypeptide homolog to the Campylobacter jejuni PglK. Finally, there are two families of conjugating enzymes, the N-oligosaccharyltransferases (N-OTase), best represented by C. jejuni PglB, and the O-OTases, including Neisseria meningitidis PglL and the O antigen ligases involved in LPS biosynthesis. With the exception of the N-OTases, probably restricted to glycoprotein synthesis, members of all these transmembrane protein families can be involved in the synthesis of both glycoproteins and LPS. Because many translocation and conjugation enzymes display relaxed substrate specificity, these bacterial enzymes could be exploited in engineered living bacteria for customized glycoconjugate production, generating potential vaccines and therapeutics. PMID:20871101

  2. Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

    USGS Publications Warehouse

    Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

    2004-01-01

    Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

  3. Impaired phagocyte responses to lipopolysaccharide in paroxysmal nocturnal hemoglobinuria.

    PubMed Central

    Duchow, J; Marchant, A; Crusiaux, A; Husson, C; Alonso-Vega, C; De Groote, D; Neve, P; Goldman, M

    1993-01-01

    Bone marrow-derived cells from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) show a defect in the expression of phosphatidylinositol-anchored membrane proteins, including the CD14 molecule. Blocking experiments with anti-CD14 monoclonal antibodies have shown that lipopolysaccharide (LPS)-induced tumor necrosis factor alpha production by monocytes depends on the interaction between CD14 and a complex formed by LPS and LPS-binding protein. We used a whole-blood model to examine the LPS-induced production of tumor necrosis factor alpha and interleukin-6 in PNH patients and healthy volunteers. At low endotoxin concentrations (1 ng/ml), PNH patients displayed a marked defect in the production of both cytokines, whereas at high LPS concentrations (100 ng/ml), cytokine production was similar to that in healthy volunteers. Using flow cytometry, we also studied the expression of the adhesion molecules Mac-1 (CD11b/CD18) and ICAM-1 (CD54) by monocytes and granulocytes after LPS stimulation. Compared with phagocytes from healthy volunteers, CD14-deficient cells showed poor Mac-1 and ICAM-1 upregulation when whole blood was stimulated with LPS (1 ng/ml), whereas their response to higher LPS doses (100 and 1,000 ng/ml) was essentially normal. The importance of the CD14 molecule in the activation of phagocytes by low LPS concentrations was confirmed by the inhibitory effect of an anti-CD14 antibody both in healthy volunteers and in PNH patients. Since these patients produce the soluble form of the CD14 molecule, these data suggest that soluble CD14 could play a role in phagocyte responses to LPS. We conclude that, in whole blood, phagocytes from PNH patients show impaired responsiveness to LPS and this phenomenon is most probably related to their defect in expression of membrane CD14. PMID:7691746

  4. Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment

    PubMed Central

    2014-01-01

    Background Systemic bacterial infections often result in enduring cognitive impairment and are a risk factor for dementia. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that intermittent fasting (IF) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of IF on the cognitive sequelae of systemic and brain inflammation is unknown. Methods Rats on IF for 30 days received 1 mg/kg of lipopolysaccharide (LPS) or saline intravenously. Half of the rats were subjected to behavioral tests and the other half were euthanized two hours after LPS administration and the hippocampus was dissected and frozen for analyses. Results Here, we report that IF ameliorates cognitive deficits in a rat model of sepsis by a mechanism involving NF-κB activation, suppression of the expression of pro-inflammatory cytokines, and enhancement of neurotrophic support. Treatment of rats with LPS resulted in deficits in cognitive performance in the Barnes maze and inhibitory avoidance tests, without changing locomotor activity, that were ameliorated in rats that had been maintained on the IF diet. IF also resulted in reduced levels of mRNAs encoding the LPS receptor TLR4 and inducible nitric oxide synthase (iNOS) in the hippocampus. Moreover, IF prevented LPS-induced elevation of IL-1α, IL-1β and TNF-α levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1β, IFN-γ, RANTES, TNF-α and IL-6 levels. Conclusions Taken together, our results suggest that IF induces adaptive responses in the brain and periphery that can suppress inflammation and preserve cognitive function in an animal model of systemic bacterial infection. PMID:24886300

  5. Lumican overexpression exacerbates lipopolysaccharide-induced renal injury in mice.

    PubMed

    Lu, Xiao-Mei; Ma, Ling; Jin, Yu-Nan; Yu, Yan-Qiu

    2015-09-01

    The present study aimed to investigate the role of lumican in mice with endotoxin-induced acute renal failure (ARF). Lumican transgenic mice and wild‑type mice were injected with lipopolysaccharide (LPS; 10 mg/kg) to establish a model of ARF. The mice were sacrificed at 24 h and the blood and renal tissue samples were collected. The value of serum creatinine (SCr) and blood urea nitrogen (BUN) were measured to determine renal function. An ELISA was used to determined the concentrations of renal cytokines, including tumor necrosis factor (TNF)α, interleukin (IL)‑6, IL‑4 and IL‑10. The protein expression levels of Toll-like receptor (TLR4) and nuclear factor (NF)κB in renal tissues were assessed using western blot analysis. Terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling was performed to monitor apoptosis of renal tissue. Light microscopy and electron microscopy were used to observe structural changes in the renal tissues. Following the administration of LPS, the SCr and BUN values of mice in the lumican transgenic group were higher compared with those in the control group. The expression levels of renal TLR4, NFκB, TNFα, IL‑6, IL‑4 and IL‑10 were upregulated in the lumican transgenic mice compared with those in the wild‑type control group. Apoptosis was detected predominantly on the renal tubule. There was a significant difference in the optical density of apoptotic bodies between the control mice and the lumican transgenic mice. Light and electron microscopy demonstrated more severe renal tissue injury in the lumican transgenic mice compared with that in the control mice. In conclusion, LPS may cause excessive apoptosis in the renal tubular cells via the TLR4 signal transduction pathway, a decrease in the number of renal tubular cells and ARF. Lumican may be important in mice with LPS-induced ARF. PMID:26081832

  6. Astragalus saponins Inhibits Lipopolysaccharide-Induced Inflammation in Mouse Macrophages.

    PubMed

    Wang, Yue; Ren, Tianjing; Zheng, Lucong; Chen, Hubiao; Ko, Joshua Kashun; Auyeung, Kathy Kawai

    2016-01-01

    Excessive nitric oxide (NO) and pro-inflammatory cytokines are produced during the pathogenesis of inflammatory diseases and cancer. It has been demonstrated that anti-inflammation contributes Astragalus membranaceus saponins (AST)'s beneficial effects in combination of conventional anticancer drugs. However, the immunomodulating property of AST has not been well characterized. In this study, we found that AST suppressed lipopolysaccharide (LPS)-induced generation of NO without causing cytotoxicity in the mouse macrophage RAW264.7. The gene and protein overexpression of inducible NO synthase (iNOS) as well as the production of tumor necrosis factor-[Formula: see text], evoked by LPS, was consistently down-regulated by AST. AST also inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and suppressed nuclear factor (NF)-[Formula: see text]B activation and the associated I[Formula: see text]B[Formula: see text] degradation during LPS insult. Furthermore, AST induced growth inhibition in promyelocytic leukemic HL-60 cells and T-lymphocyte leukemic Jurkat cells, but exerted no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMC). It is known that the chemotherapeutic drug 5-FU can suppress the immune system, which can be identified by a reduced white blood cell count and decreased hematocrit, while the combination of AST and 5-FU can reverse the above hematologic toxicities. To summarize, non-cytotoxic concentrations of AST suppress LPS-induced inflammatory responses via the modulation of p38 MAPK signaling and the inhibition of NO and cytokine release. Importantly, AST can alleviate the hematologic side effects of current chemotherapeutic agents. These findings can facilitate the establishment of AST in the treatment of inflammatory diseases and inflammation-mediated tumor development. PMID:27109155

  7. Chemical Structure of Lipid A Isolated from Flavobacterium meningosepticum Lipopolysaccharide

    PubMed Central

    Kato, Hitomi; Haishima, Yuji; Iida, Takatoshi; Tanaka, Akira; Tanamoto, Ken-ichi

    1998-01-01

    The chemical structure of the lipid A of the lipopolysaccharide component isolated from Flavobacterium meningosepticum IFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the free lipid A: a β (1→6)-linked 2-amino-2-deoxy-d-glucose, which is usually present in enterobacterial lipid A’s, and a 2-amino-6-O-(2,3-diamino-2,3-dideoxy-β-d-glucopyranosyl)-2-deoxy-d-glucose, in a molar ratio of 1.00:0.35. Both backbones were α-glycosidically phosphorylated in position 1, and the hydroxyl groups at positions 4, 4′, and 6′ were unsubstituted. Liquid secondary ion-mass spectrometry revealed a pseudomolecular ion at m/z 1673 [M-H]− as a major monophosphoryl lipid A component carrying five acyl groups. Fatty acid analysis showed that the lipid A contained 1 mol each of amide-linked (R)-3-OH iC17:0, ester-linked (R)-3-OH iC15:0, amide-linked (R)-3-O-(iC15:0)-iC17:0, and both amide- and ester-linked (R)-3-OH C16:0. Fatty acid distribution analyses using several mass spectrometry determinations demonstrated that the former two constituents were distributed on positions 2 and 3 of the reducing terminal unit of the backbones and that the latter two were attached to the 2′ and 3′ positions in the nonreducing terminal residue. PMID:9683486

  8. Ethanol versus lipopolysaccharide-induced hypothermia: involvement of urocortin.

    PubMed

    Turek, V F; Ryabinin, A E

    2005-01-01

    The urocortin1 (Ucn1) neurons of the mid-brain-localized Edinger-Westphal nucleus (EW) are robustly responsive to ethanol (EtOH) administration, and send projections to the dorsal raphe nucleus (DRN), which contains corticotropin-releasing factor type 2 receptors (CRF2) that are responsive to Ucn1. In addition, the DRN has been shown to be involved in regulation of body temperature, a function greatly affected by EtOH administration. The goal of the present study was to identify the role that the urocortinergic projections from the EW to the DRN have in mediating EtOH-induced and lipopolysaccharide (LPS)-induced hypothermia. Male C57BL6/J mice were used. Groups of mice underwent cannulation of the DRN, and then received i.p. injections of EtOH (2g/kg) or LPS (600 microg/kg or 400 microg/kg), followed by intra-DRN injections of artificial cerebrospinal fluid (aCSF) or anti-sauvagine (aSVG) (55 pmol), a CRF2 antagonist. Separate groups of mice received single intra-DRN injections of Ucn1 (20 pmol), CRF (20 pmol) or aCSF. For all experiments, core temperatures were monitored rectally every 30 min for several hours post-injection. Both EtOH and LPS induced hypothermia, and aSVG significantly attenuated this effect after EtOH; however, there was no significant attenuation of hypothermia after either dose of LPS. Ucn1 injection also caused hypothermia, while CRF injection did not. These data demonstrate that EtOH-induced hypothermia, but not LPS-induced hypothermia, may involve Ucn1 from EW acting at CRF2 receptors in the DRN. PMID:15964490

  9. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-09-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age. PMID:1500196

  10. Ligustrazine effect on lipopolysaccharide-induced pulmonary damage in rats.

    PubMed

    Wang, Huiqi; Chen, Yuanzhuo; Li, Wenjie; Li, Congye; Zhang, Xiangyu; Peng, Hu; Gao, Chengjin

    2015-09-01

    We investigated the effectiveness of ligustrazine (tetramethylpyrazine, TMP) in alleviating pulmonary damage induced by lipopolysaccharide (LPS). Twenty-four healthy male Sprague-Dawley rats were randomly divided into three groups: the blank group, LPS group, and TMP treatment group (TMP group). The LPS group was intraperitoneally injected with LPS (20mg/kg), and the TMP group was intraperitoneally injected with LPS (20mg/kg) and ligustrazine (80mg/kg). Blood gas analysis, hematoxylin-eosin staining dye extravasation and diffuse alveolar damage (DAD) score, the wet/dry lung tissue (W/D) ratios, the expression of CD31+ vascular endothelial microparticles (EMPs), tumor necrosis factor alpha (TNF-α) levels, and the protein expression of Rho-associated coiled-coil-forming protein kinase (ROCK) II and Toll-like receptor 4 (TLR4) were analyzed. Compared with the blank group, the arterial partial pressure of oxygen (PaO2) declined in both 1 and 4h (P<0.01), the W/D ratio and DAD score increased (P<0.01), and the TNF-α levels in serum, CD31+ EMPs, and protein expression of ROCK II and TLR4 were significantly increased (P<0.01) in the LPS group. Compared with the LPS group, PaO2 significantly increased in the TMP group at 4h (P<0.05), while the W/D ratio and DAD score were significantly decreased in the TMP group (P<0.01). TNF-α levels, CD31+ EMPs, and protein expression of ROCK II and TLR4 were significantly decreased in the TMP group compared with the LPS group (P<0.01). This study demonstrated that TMP can alleviate LPS-induced pulmonary damage by attenuating pulmonary vascular permeability and CD31+ EMP levels in the plasma, reducing the release of the inflammatory mediator TNF-α and inhibiting the protein expression of ROCK II and TLR4. PMID:26088147

  11. Lipopolysaccharides as Determinants of Serological Variability in Pseudomonas corrugata

    PubMed Central

    Siverio, F.; Cambra, M.; Gorris, M. T.; Corzo, J.; Lopez, M. M.

    1993-01-01

    The variation in biochemical and serological features of 128 isolates of Pseudomonas corrugata has been studied with 56 isolates from Spain and 72 isolates from other countries. Isolates were analyzed with common diagnostic tests and with the AP150CHE system. Variability among isolates for some standard tests usually listed as positive or negative for this species, such as arginine dihydrolase and gelatin hydrolysis, lipase and lecithinase activities, pigment production, and wrinkled colony morphology, was observed. Three antisera were raised against the type strain and two Spanish isolates from tomato and pepper plants. Serological reactions were studied by indirect immunofluorescence and indirect enzyme-linked immunosorbent assay. Eighty-three isolates reacted with a single antiserum, 6 reacted with two antisera, and none reacted with three antisera. Thirty-nine isolates did not react with any of the three antisera. These results suggest that serology will not be a useful method for routine diagnosis of P. corrugata unless common antigens can be identified. Electrophoresis and immunoelectrotransfer were used to study the antigens involved. Each antiserum reacted with whole-cell lysates, giving two common bands for P. corrugata isolates and other Pseudomonas species and a ladder-like pattern characteristic of lipopolysaccharides (LPS). Common bands were not observed after proteinase K treatment. More than 10 LPS patterns were distinguished in 98 isolates after silver staining of polyacrylamide gels. There was no correlation between the geographical origin or host of the isolates and the LPS patterns. A correlation between LPS groups and serological reaction was observed. Images PMID:16348957

  12. Immunomodulatory Effects of Yersinia pestis Lipopolysaccharides on Human Macrophages ▿

    PubMed Central

    Matsuura, Motohiro; Takahashi, Hideyuki; Watanabe, Haruo; Saito, Shinji; Kawahara, Kazuyoshi

    2010-01-01

    In the current study, we investigated the activity of lipopolysaccharide (LPS) purified from Yersinia pestis grown 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 from Y. pestis cells 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 that Y. pestis causes infection in humans without stimulating the TLR4-based defense system via bacterial 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 system via the bacterial binding forms. PMID:19889939

  13. Peripheral tumors alter neuroinflammatory responses to lipopolysaccharide in female rats

    PubMed Central

    Pyter, Leah M.; Bih, Sarah El Mouatassim; Sattar, Husain; Prendergast, Brian J.

    2014-01-01

    Cancer is associated with an increased prevalence of depression. Peripheral tumors induce inflammatory cytokine production in the brain and depressive-like behaviors. Mounting evidence indicates that cytokines are part of a pathway by which peripheral inflammation causes depression. Neuroinflammatory responses to immune challenges can be exacerbated (primed) by prior immunological activation associated with aging, early-life infection, and drug exposure. This experiment tested the hypothesis that peripheral tumors likewise induce neuroinflammatory sensitization, or priming. Female rats with chemically-induced mammary carcinomas were injected with either saline or lipopolysaccharide (LPS, 250 μg/kg; i.p.), and expression of mRNAs involved in the pathway linking inflammation and depression (interleukin-1beta [Il-1β], CD11b, IκBα, indolamine 2,3-deoxygenase [Ido]) was quantified by qPCR in the hippocampus, hypothalamus, and frontal cortex, 4 or 24 h post-treatment. In the absence of LPS, hippocampal Il-1β and CD11b mRNA expression were elevated in tumor-bearing rats, whereas Ido expression was reduced. Moreover, in saline-treated rats basal hypothalamic Il-1β and CD11b expression were positively correlated with tumor weight; heavier tumors, in turn, were characterized by more inflammatory, necrotic, and granulation tissue. Tumors exacerbated CNS proinflammatory gene expression in response to LPS: CD11b was greater in hippocampus and frontal cortex of tumor-bearing relative to tumor-free rats, IκBβ was greater in hippocampus, and Ido was greater in hypothalamus. Greater neuroinflammatory responses in tumor-bearing rats were accompanied by attenuated body weight gain post-LPS. The data indicate that neuroinflammatory pathways are potentiated, or primed, in tumor-bearing rats, which may exacerbate future negative behavioral consequences. PMID:24457042

  14. Lipopolysaccharide hyporesponsiveness: protective or damaging response to the brain?

    PubMed

    Pardon, Marie Christine

    2015-01-01

    Lipopolysaccharide (LPS) endotoxins are widely used as experimental models of systemic bacterial infection and trigger robust inflammation by potently activating toll-like receptors 4 (TLR4) expressed on innate immune cells. Their ability to trigger robust neuroinflammation despite poor brain penetration can prove useful for the understanding of how inflammation induced by viral infections contributes to the pathogenesis of neurodegenerative diseases. A single LPS challenge often result in a blunted inflammatory response to subsequent stimulation by LPS and other TLR ligands, but the extent to which endotoxin tolerance occur in the brain requires further clarification. LPS is also thought to render the brain transiently resistant to subsequent brain injuries by attenuating the concomitant pro-inflammatory response. While LPS hyporesponsiveness and preconditioning are classically seen as protective mechanisms limiting the toxic effects of sustained inflammation, recent research casts doubt as to whether they have beneficial or detrimental roles on the brain and in neurodegenerative disease. These observations suggest that spatio-temporal aspects of the immune responses to LPS and the disease status are determinant factors. Endotoxin tolerance may lead to a late pro-inflammatory response with potential harmful consequences. And while reduced TLR4 signaling reduces the risk of neurodegenerative diseases, up-regulation of anti-inflammatory cytokines associated with LPS hyporesponsiveness can have deleterious consequences to the brain by inhibiting the protective phenotype of microglia, aggravating the progression of some neurodegenerative conditions such as Alzheimer's disease. Beneficial effects of LPS preconditioning, however appear to require a stimulation of anti-inflammatory mediators rather than an attenuation of the pro-inflammatory response. PMID:26662122

  15. Beryllium Alters Lipopolysaccharide-Mediated Intracellular Phosphorylation and Cytokine Release in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M.; Gupta, Goutam; McCleskey, T. Mark; Chaudhary, Anu

    2013-01-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide - mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We find that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1β is enhanced. Additionally, not all lipopolysaccharide - mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1β secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling. PMID:19894180

  16. Lipopolysaccharide-Related Stimuli Induce Expression of the Secretory Leukocyte Protease Inhibitor, a Macrophage-Derived Lipopolysaccharide Inhibitor

    PubMed Central

    Jin, Fenyu; Nathan, Carl F.; Radzioch, Danuta; Ding, Aihao

    1998-01-01

    Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-κB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417–426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins—interleukin-10 (IL-10) and IL-6—also induced SLPI, while TNF and IL-1β did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules—taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls—also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI’s slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria. PMID:9596701

  17. Chemical composition of lipopolysaccharides isolated from various endophytic nitrogen-fixing bacteria of the genus Herbaspirillum.

    PubMed

    Serrato, R V; Sassaki, G L; Cruz, L M; Carlson, R W; Muszyński, A; Monteiro, R A; Pedrosa, F O; Souza, E M; Iacomini, M

    2010-04-01

    Bacteria from the genus Herbaspirillum are endophytes responsible for nitrogen fixation in gramineous plants of economic importance such as maize, sugarcane, sorghum, rice, and wheat. Some species are known to produce plant growth substances. In contrast, Herbaspirillum rubrisubalbicans strains are known to be mild plant pathogens. The molecular communication between the plant and the microbes might involve lipopolysaccharides present in the outer membrane of these gram-negative bacteria. Phenol-water extraction was used to obtain lipopolysaccharides from 7 strains of Herbaspirillum seropedicae (SmR1, Z67, Z78, ZA95, and M2) and H. rubrisubalbicans (M1 and M4). The electrophoretic profiles and chemical composition of the lipopolysaccharides obtained in the phenol and aqueous extracts were shown herein. PMID:20453901

  18. Unilamellar liposomes modulate secretion of tumor necrosis factor by lipopolysaccharide-stimulated macrophages.

    PubMed Central

    Brisseau, G F; Kresta, A; Schouten, D; Bohnen, J M; Shek, P N; Fok, E; Rotstein, O D

    1994-01-01

    Liposomal encapsulation of antimicrobial agents has been used to improve drug delivery, particularly against intracellular pathogens. The effect of unilamellar liposomes on macrophage activation in response to Escherichia coli lipopolysaccharide was examined. Liposomes caused a dose- and time-dependent inhibition of tumor necrosis factor release by lipopolysaccharide-treated cells. The accumulation of tumor necrosis factor mRNA transcripts was unaffected, suggesting a posttranscriptional mechanism for this effect. However, induction of macrophage procoagulant activity was unaffected by liposomes, indicating a selective rather than a global inhibition. These data suggest that liposomes used for drug delivery may modulate the host response to infection. Images PMID:7872768

  19. Interaction between complement subcomponent C1q and bacterial lipopolysaccharides.

    PubMed

    Zohair, A; Chesne, S; Wade, R H; Colomb, M G

    1989-02-01

    The heptose-less mutant of Escherichia coli, D31m4, bound complement subcomponent C1q and its collagen-like fragments (C1qCLF) with Ka values of 1.4 x 10(8) and 2.0 x 10(8) M-1 respectively. This binding was suppressed by chemical modification of C1q and C1qCLF using diethyl pyrocarbonate (DEPC). To investigate the role of lipopolysaccharides (LPS) in this binding, biosynthetically labelled [14C]LPS were purified from E. coli D31m4 and incorporated into liposomes prepared from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [PC/PE/LPS, 2:2:1, by wt.]. Binding of C1q or its collagen-like fragments to the liposomes was estimated via a flotation test. These liposomes bound C1q and C1qCLF with Ka values of 8.0 x 10(7) and 2.0 x 10(7) M-1; this binding was totally inhibited after chemical modification of C1q and C1qCLF by DEPC. Liposomes containing LPS purified from the wild-strain E. coli K-12 S also bound C1q and C1qCLF, whereas direct binding of C1q or C1qCLF to the bacteria was negligible. Diamines at concentrations which dissociate C1 into C1q and (C1r, C1s)2, strongly inhibited the interaction of C1q or C1qCLF with LPS. Removal of 3-deoxy-D-manno-octulosonic acid (2-keto-3-deoxyoctonic acid; KDO) from E. coli D31m4 LPS decreases the binding of C1qCLF to the bacteria by 65%. When this purified and modified LPS was incorporated into liposomes, the C1qCLF binding was completely abolished. These results show: (i) the essential role of the collagen-like moiety and probably its histidine residues in the interaction between C1q and the mutant D31m4; (ii) the contribution of LPS, particularly the anionic charges of KDO, to this interaction. PMID:2649081

  20. Nilotinib ameliorates lipopolysaccharide-induced acute lung injury in rats

    SciTech Connect

    El-Agamy, Dina S.

    2011-06-01

    The present study aimed to investigate the effect of the new tyrosine kinase inhibitor, nilotinib on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and explore its possible mechanisms. Male Sprague-Dawley rats were given nilotinib (10 mg/kg) by oral gavage twice daily for 1 week prior to exposure to aerosolized LPS. At 24 h after LPS exposure, bronchoalveolar lavage fluid (BALF) samples and lung tissue were collected. The lung wet/dry weight (W/D) ratio, protein level and the number of inflammatory cells in the BALF were determined. Optical microscopy was performed to examine the pathological changes in lungs. Malondialdehyde (MDA) content, superoxidase dismutase (SOD) and reduced glutathione (GSH) activities as well as nitrite/nitrate (NO{sub 2}{sup -}/NO{sub 3}{sup -}) levels were measured in lung tissues. The expression of inflammatory cytokines, tumor necrosis factor-{alpha} (TNF-{alpha}), transforming growth factor-{beta}{sub 1} (TGF-{beta}{sub 1}) and inducible nitric oxide synthase (iNOS) were determined in lung tissues. Treatment with nilotinib prior to LPS exposure significantly attenuated the LPS-induced pulmonary edema, as it significantly decreased lung W/D ratio, protein concentration and the accumulation of the inflammatory cells in the BALF. This was supported by the histopathological examination which revealed marked attenuation of LPS-induced ALI in nilotinib treated rats. In addition, nilotinib significantly increased SOD and GSH activities with significant decrease in MDA content in the lung. Nilotinib also reduced LPS mediated overproduction of pulmonary NO{sub 2}{sup -}/NO{sub 3}{sup -} levels. Importantly, nilotinib caused down-regulation of the inflammatory cytokines TNF-{alpha}, TGF-{beta}{sub 1} and iNOS levels in the lung. Taken together, these results demonstrate the protective effects of nilotinib against the LPS-induced ALI. This effect can be attributed to nilotinib ability to counteract the inflammatory cells

  1. Lipopolysaccharide Pretreatment Protects from Renal Ischemia/Reperfusion Injury

    PubMed Central

    Heemann, Uwe; Szabo, Attila; Hamar, Peter; Müller, Veronika; Witzke, Oliver; Lutz, Jens; Philipp, Thomas

    2000-01-01

    In vivo administration of low doses of lipopolysaccharide (LPS) to rodents can protect these animals from subsequently administrated, usually lethal doses of endotoxin or LPS. In this study we tested the effects of LPS pretreatment on ischemia/reperfusion injury in the kidney. Male C57/B1 mice were pretreated with different doses of LPS or phosphate-buffered saline on days −4 and −3. The right kidney was removed, and the vessels of the left kidney were clamped for 30 or 45 minutes on day 0. Creatinine levels and survival of animals were monitored. To test the involvement of cytokines, additional animals were harvested before (“time 0”) and 15 minutes, 1, 2, 8, and 16 hours after reperfusion for histology, immunohistochemistry, terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay, and reverse transcriptase-polymerase chain reaction analysis (including tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, inducible nitric oxide synthase (iNOS), and interferon (IFN)-γ messenger RNA (mRNA)). In controls, renal ischemia of 30 minutes was nonlethal, whereas 73% of the animals died within 48 ± 18 hours, after 45 minutes of ischemia. All different doses of LPS protected the animals from lethal renal ischemia/reperfusion injury. Starting at similar levels, serum creatinine increased significantly in controls but not in LPS-pretreated animals over time. As early as 2 hours after reperfusion, tubular cell damage was significantly more pronounced in controls than in LPS-treated mice. In controls, tubules deteriorated progressively until 8 hours of reperfusion. At this time, more than 50% of tubular cells were destroyed. This destruction was accompanied by a pronounced leukocytic infiltration, predominantly by macrophages. In contrast, LPS pretreatment prevented the destruction of kidney tissue and infiltration by leukocytes. The terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay revealed significantly more apoptotic cells in

  2. Moesin Functions as a Lipopolysaccharide Receptor on Human Monocytes

    PubMed Central

    Tohme, Ziad N.; Amar, Salomon; Van Dyke, Thomas E.

    1999-01-01

    Bacterial endotoxin (lipopolysaccharide [LPS]), a glycolipid found in the outer membranes of gram-negative bacteria, induces the secretion of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 by monocytes/macrophages. The secretion of these biologically active compounds leads to multiple pathological conditions, such as septic shock. There is substantial evidence that chronic exposure to LPS mediates, at least in part, the tissue destruction associated with gram-negative infection. CD14, a 55-kDa protein, has been identified as an LPS receptor. In conjunction with a serum protein, LPS binding protein (LBP), LPS-CD14 interactions mediate many LPS functions in the inflammatory response. However, CD14 lacks a cytoplasmic domain, or any known signal transduction sequence motif, suggesting the existence of another cell surface domain capable of transducing signals. In this paper, we report a second, CD14-independent LPS binding site, which, based on biological activity, appears to be a functional LPS receptor. Cross-linking experiments were performed to identify LPS binding sites. Two molecules were identified: a 55-kDa protein (CD14) and a second, 78-kDa band. Sequencing of the 78-kDa protein by mass spectroscopic analysis revealed 100% homology with moesin (membrane-organizing extension spike protein). Antibody to CD14 induced partial blocking of the LPS response. However, antimoesin monoclonal antibody completely blocked the LPS-induced TNF-α response in human monocytes, without blocking CD14 binding of LPS. Irrelevant isotype controls had no effect. Additional experiments were performed to evaluate the specificity of the antimoesin blocking. Separate experiments evaluated antimoesin effects on monocyte chemotaxis, IL-1 production in response to IL-1 stimulation, and TNF-α secretion in response to Staphylococcus aureus stimulation. Antimoesin blocked only LPS-mediated events. The data suggest that moesin

  3. Human monocyte CD14 is upregulated by lipopolysaccharide.

    PubMed Central

    Landmann, R; Knopf, H P; Link, S; Sansano, S; Schumann, R; Zimmerli, W

    1996-01-01

    Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low-dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first 3 h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5-fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with > or = 1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 micrograms/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor alpha, anti-interleukin-6, anti-gamma interferon, and anti-LPS-binding protein. LPS-induced tumor necrosis factor alpha production was abolished after a second 4-h challenge. In contrast, the LPS-induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents. PMID:8613389

  4. Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves.

    PubMed

    Benjamin, A L; Korkmaz, F T; Elsasser, T H; Kerr, D E

    2016-07-01

    The innate immune response following experimental mastitis is quite variable between individual dairy cattle. An inflammatory response that minimizes collateral damage to the mammary gland while still effectively resolving the infection following pathogen exposure is beneficial to dairy producers. The ability of a lipopolysaccharide (LPS) exposure in early life to generate a low-responding phenotype and thus reduce the inflammatory response to a later-life LPS challenge was investigated in neonatal bull calves. Ten Holstein bull calves were randomly assigned to either an early life LPS (ELL) group (n=5) or an early life saline (ELS) group (n=5). At 7d of age, calves received either LPS or saline, and at 32d of age, all calves were challenged with an intravenous dose of LPS to determine the effect of the early life treatment (LPS or saline) on the immune response generated toward a subsequent LPS challenge. Dermal fibroblast and monocyte-derived macrophage cultures from each calf were established at age 20 and 27d, respectively, to model sustained effects from the early life LPS exposure on gene expression and protein production of components within the LPS response pathway. The ELL calves had greater levels of plasma IL-6 and tumor necrosis factor-α than the ELS calves following the early life LPS or saline treatments. However, levels of these 2 immune markers were similar between ELL and ELS calves when both groups were subsequently challenged with LPS. A comparison of the in vitro LPS responses of the ELL and ELS calves revealed similar patterns of protein production and gene expression following an LPS challenge of both dermal fibroblast and monocyte-derived macrophage cultures established from the treatment groups. Whereas an early life exposure to LPS did not result in a dampened inflammatory response toward a later LPS challenge in these neonatal bull calves, the potential that exposure to inflammation or stress in early life or in utero can create an

  5. A natural therapeutic approach for the treatment of periodontitis by MK615.

    PubMed

    Morimoto-Yamashita, Yoko; Kawakami, Yoshiko; Tatsuyama, Syoko; Miyashita, Keiko; Emoto, Makiko; Kikuchi, Kiyoshi; Kawahara, Ko-ichi; Tokuda, Masayuki

    2015-11-01

    Periodontitis is a chronic inflammatory disease that affects the tooth-supporting tissues. Gingival fibroblasts are the most abundant cells in periodontal tissues and they participate actively in the host inflammatory response to periodontal pathogens that is known to mediate local tissue destruction in periodontitis. The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries and is a familiar and commonly consumed food. The health benefits of Ume are widely recognized and have been confirmed in recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in oral health is unknown. We hypothesized that the anti-inflammatory activities of MK615 could be exploited to inhibit the effects of lipopolysaccharide (LPS) produced by periodontal bacterial pathogens, such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Here, we show that LPS-induced interleukin (IL)-6 and IL-8 production by gingival fibroblasts was dose-dependently inhibited by MK615. As a potent inhibitor of the inflammatory responses induced by periodontal pathogens, MK615 merits further testing as a therapeutic agent in inflammatory diseases such as periodontitis. PMID:26305447

  6. Anti-inflammatory and wound healing potential of citrus auraptene.

    PubMed

    La, Vu Dang; Zhao, Lei; Epifano, Francesco; Genovese, Salvatore; Grenier, Daniel

    2013-10-01

    Auraptene is the most abundant naturally occurring geranyloxycoumarin. It is primarily isolated from plants in the Rutaceae family, many of which, like citrus fruits, are used as food in many countries. Auraptene is a biologically active secondary metabolite with valuable properties. The aim of our study was to identify novel properties of auraptene with potential for managing periodontal diseases, an inflammatory disease of bacterial origin affecting the tissues surrounding and supporting the teeth. In vitro assays showed that auraptene decreased, in a dose-dependent manner, the secretion of matrix metalloproteinase 2 as well as key inflammatory mediators, including interleukin-6 (IL-6), IL-8, and chemokine (C-C motif) ligand-5 secreted by Aggregatibacter actinomycetemcomitans lipopolysaccharide-stimulated oral epithelial cells. Using gingival fibroblasts, auraptene showed a significant (P<.05) wound healing effect by its capacity to increase cell migration. In conclusion, auraptene shows promise for promoting wound healing and controlling periodontal diseases through its capacity to interfere with inflammatory mediator secretion. PMID:24070132

  7. Dietary L-arginine supplementation modulates lipopolysaccharide-induced systemic inflammatory response in broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to evaluate whether dietary supplementation with L-arginine (Arg) could attenuate lipopolysaccharide (LPS)-induced systemic inflammatory response through LPS/TLR-4 signaling pathway in broilers. The experiment was designed as a 2 × 3 factorial arrangement (n = 8 cages/treatm...

  8. EFFECTS OF DIESEL EXHAUST PARTICLES ON HUMAN ALVEOLAR MACROPHAGE RESPONSIVENESS TO LIPOPOLYSACCHARIDE

    EPA Science Inventory

    Effects of diesel exhaust particles on human alveolar macrophage responsiveness to lipopolysaccharide
    S. Mundandhara1 , S. Becker2 and M. Madden2, 1UNC Center for Environmental Medicine, Asthma, and Lung Biology, 2US EPA, NHEERL, HSD, Chapel Hill, NC, US

    Epidemiological...

  9. GENE EXPRESSION PROFILING OF BOVINE MACROPHAGES IN RESPONSE TO ESCHERICHIA COLI O157:H7 LIPOPOLYSACCHARIDE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5,644 (79.8%)...

  10. Lipopolysaccharide Sequestrants: Structural Correlates of Activity and Toxicity in Novel Acylhomospermines

    PubMed Central

    Miller, Kelly A.; Kumar, E.V.K. Suresh; Wood, Stewart J.; Cromer, Jens R.; Datta, Apurba; David*, Sunil A.

    2005-01-01

    Lipopolysaccharides (LPS), otherwise termed ‘endotoxins’, are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of ‘Septic Shock’, a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a series of acylated homologated spermine compounds with lipopolysaccharide (LPS) have been characterized. The optimal acyl chain length for effective sequestration of LPS was identified to be C16 for the mono-acyl compounds. The most promising of these compounds, 4e, binds LPS with an ED50 of 1.37 μM. Nitric oxide production in murine J774A.1 cells, as well as TNF-α in human blood, are inhibited in a dose-dependent manner by 4e at concentrations orders of magnitude lower than toxic doses. Administration of 4e to d-galactosamine-sensitized mice challenged with supralethal doses of LPS provided significant protection against lethality. Potent anti-endotoxic activity, low toxicity, and ease of synthesis render this class of compounds candidate endotoxin-sequestering agents of potential significant therapeutic value. PMID:15801849

  11. Arginine supplementation does not alter nitrogen metabolism of beef steers during a lipopolysaccharide challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Demand for Arg is reported to increase during immune challenge. This study evaluated the effects of lipopolysaccharide (LPS) and abomasal Arg infusion on N metabolism and immune response of 20 ruminally cannulated steers (369 ± 46 kg BW) in a randomized block design. Each block was 20 d and consiste...

  12. EFFECTS OF CHRONIC EXERCISE CONDITIONING ON THERMAL RESPONSES TO LIPOPOLYSACCHARIDE AND TURPENTINE ABSCESS IN FEMALE RATS.

    EPA Science Inventory

    Chronic exercise conditioning has been shown to alter basal thermoregulatory processes as well as the response to inflammatory agents. Two such agents, lipopolysaccharide (LPS) and turpentine (TPT) are inducers of fever in rats. LPS, given intraperitoneally (i.p.), involves a sys...

  13. Prenatal transportation alters the metabolic response of Brahman bull calves exposed to a lipopolysaccharide (LPS) challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was designed to determine if prenatal transportation influences the metabolic response to a postnatal lipopolysaccharide (LPS) challenge. Pregnant Brahman cows (n=96) matched by age and parity were separated into transported (TRANS; n=48; transported for 2 hours on gestational day 60, 80,...

  14. EFFECTS OF DIESEL EXHAUST PARTICLES ON HUMAN MACROPHAGE RESPONSIVENESS TO LIPOPOLYSACCHARIDE

    EPA Science Inventory

    EFFECTS OF DIESEL EXHAUST PARTICLES ON HUMAN MACROPHAGE RESPONSIVENESS TO LIPOPOLYSACCHARIDE
    S. Mundandhara1 and M.C. Madden2, 1UNC Center for Environmental Medicine, Asthma, and Lung Biology, 2US EPA, NHEERL, Human Studies Division, Chapel Hill, NC, USA

    Epidemiologica...

  15. Effect of prenatal stress on subsequent response to mixing stress and a lipopolysaccharide challenge in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sows subjected to prenatal stress have been found to produce offspring that alter the manner in which they respond to stress. Our objective was to determine if exposing a sow to stress altered the response of the offspring to lipopolysaccharide (LPS) at 2 mo of age or their response to mixing stres...

  16. Garlic (Allium sativum) Extracts Inhibits Lipopolysaccharide-Induced Toll-Like Receptor 4 Dimerization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Garlic has been used as a folk medicine for a long history. Numerous studies demonstrated that garlic extracts and its sulfur-containing compounds inhibit nuclear factor-kappa B (NF-kB) activation induced by various receptor agonist including lipopolysaccharide (LPS). These effects suggest that garl...

  17. Effect of Sodium Butyrate on Growth Performance and Response to Lipopolysaccharide in Weanling Pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two experiments were conducted to determine the effects of dietary sodium butyrate on growth performance and response to E. coli. lipopolysaccharide (LPS) in weanling pigs. In the first 28 d experiment, 180 pigs (initial BW 6.3 kg) were fed 0, 0.05, 0.1, 0.2, and 0.4% sodium butyrate, or 110 mg/kg d...

  18. Arginine supplementation does not alter nitrogen metabolism of beef steers during a lipopolysaccharide challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Demand for arginine (Arg) is reported to increase during immune challenges. This study evaluated effects of lipopolysaccharide (LPS) and abomasal Arg infusion on nitrogen (N) metabolism and immune response of 20 ruminally cannulated steers (369 ± 46 kg BW) in a randomized block design. Each block co...

  19. Profile of the bovine acute-phase response following an intravenous bolus-dose lipopolysaccharide challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two experiments were conducted to further define the acute-phase response to a lipopolysaccharide (LPS) challenge in beef steers. In Exp. 1, 9 crossbred beef steers (449 ± 12 kg BW) were used in a completely random design to determine the effects of 0.5, 1.0, or 2.0 micrograms of LPS/kilogram of bod...

  20. Alpha-lipoic acid protects mitochondrial enzymes and attenuates lipopolysaccharide-induced hypothermia in mice

    EPA Science Inventory

    Abstract: Hypothermia is a key symptom of sepsis and the mechanism(s) leading to hypothermia during sepsis is largely unknown. To investigate a potential mechanism and find an effective treatment for hypothermia in sepsis, we induced hypothermia in mice by lipopolysaccharide (LP...

  1. Interaction of peptide-bound beads with lipopolysaccharide and lipoproteins.

    PubMed

    Suzuki, Masatsugu M; Matsumoto, Megumi; Omi, Hiroyuki; Kobayashi, Tomomi; Nakamura, Akio; Kishi, Hiroko; Kobayashi, Sei; Takagi, Takashi

    2014-05-01

    We previously reported the generation of lipopolysaccharide (LPS)-binding peptides by phage display and chemical modification. Among them, a dodecapeptide designated Li5-025 (K'YSSSISSIRAC'; K' and C' denote d-lysine and d-cysteine, respectively) showed a high binding affinity for LPS and was resistant to protease digestion (Suzuki et al., 2010). In the current study, Li5-025-bound silica beads, hereafter referred to as P-beads, were generated and found to be devoid of LPS-neutralizing activity. Thus, LPS bound to the P-beads could be directly used in the Limulus amebocyte lysate (LAL) assay. P-beads bound LPS dissolved in solutions of ethanol, pH4, pH10, and 0.5M NaCl and LPS bound to the P-beads was quantitatively assayed. The sensitivity of this assay was observed to be approximately 0.1pg/mL LPS. P-beads bound LPS dissolved in antithrombin III (AT III) solution which is a strong inhibitor of activated factors C and B as well as the clotting enzyme in the LAL assay; the inhibitory effect of AT III was completely reversed upon washing the P-beads with 25% acetonitrile. This was employed as the first step for the detection of free LPS in plasma using the LAL assay. LPS added to human plasma at 0°C followed by application to the P-beads and subsequent washing with 25% acetonitrile resulted in low LPS activity as detected by the LAL assay. However, further washing of the P-beads with 0.1% Triton X100 in 25% acetonitrile resulted in high LPS activity. This is the first instance of quantitative detection of free LPS in plasma using the LAL assay, and the sensitivity of this method was observed to be 1pg/mL of LPS. The proteins eluted in the 0.1% Triton X-100 wash were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two protein bands of 28kDa and 18kDa were predominantly observed. Mass spectrometry analysis revealed that the 28kDa and 18kDa bands corresponded to apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), respectively. Apo

  2. Cerium dioxide nanoparticles do not modulate the lipopolysaccharide-induced inflammatory response in human monocytes

    PubMed Central

    Hussain, Salik; Al-Nsour, Faris; Rice, Annette B; Marshburn, Jamie; Ji, Zhaoxia; Zink, Jeffery I; Yingling, Brenda; Walker, Nigel J; Garantziotis, Stavros

    2012-01-01

    Background Cerium dioxide (CeO2) nanoparticles have potential therapeutic applications and are widely used for industrial purposes. However, the effects of these nanoparticles on primary human cells are largely unknown. The ability of nanoparticles to exacerbate pre-existing inflammatory disorders is not well documented for engineered nanoparticles, and is certainly lacking for CeO2 nanoparticles. We investigated the inflammation-modulating effects of CeO2 nanoparticles at noncytotoxic concentrations in human peripheral blood monocytes. Methods CD14+ cells were isolated from peripheral blood samples of human volunteers. Cells were exposed to either 0.5 or 1 μg/mL of CeO2 nanoparticles over a period of 24 or 48 hours with or without lipopolysaccharide (10 ng/mL) prestimulation. Modulation of the inflammatory response was studied by measuring secreted tumor necrosis factor-alpha, interleukin-1beta, macrophage chemotactic protein-1, interferon-gamma, and interferon gamma-induced protein 10. Results CeO2 nanoparticle suspensions were thoroughly characterized using dynamic light scattering analysis (194 nm hydrodynamic diameter), zeta potential analysis (−14 mV), and transmission electron microscopy (irregular-shaped particles). Transmission electron microscopy of CD14+ cells exposed to CeO2 nanoparticles revealed that these nanoparticles were efficiently internalized by monocytes and were found either in vesicles or free in the cytoplasm. However, no significant differences in secreted cytokine profiles were observed between CeO2 nanoparticle-treated cells and control cells at noncytotoxic doses. No significant effects of CeO2 nanoparticle exposure subsequent to lipopolysaccharide priming was observed on cytokine secretion. Moreover, no significant difference in lipopolysaccharide-induced cytokine production was observed after exposure to CeO2 nanoparticles followed by lipopolysaccharide exposure. Conclusion CeO2 nanoparticles at noncytotoxic concentrations neither

  3. Influence of Core Oligosaccharide of Lipopolysaccharide to Outer Membrane Behavior of Escherichia coli

    PubMed Central

    Wang, Zhou; Wang, Jianli; Ren, Ge; Li, Ye; Wang, Xiaoyuan

    2015-01-01

    Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior. PMID:26023839

  4. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants.

    PubMed Central

    Smit, J; Kamio, Y; Nikaido, H

    1975-01-01

    The outer membrane layer of the cell wall was isolated from wild-type Salmonella typhimurium LT2 as well as from its mutants producing lipopolysaccharides with shorter saccharide chains. Chemical analysis of these preparations indicated the following. (i) The number of lipopolysaccharide molecules per unit area was constant, regardless of the length of the saccharide side chain in lipopolysaccharide. (ii) In contrast, in "deep rough" (Rd or Re) mutants producing the lipopolysaccharides with very short saccharide chains, the amount of outer membrane protein per unit surface area decreased to about 60% of the value in the wild type. (iii) In the wild type, the amount of phospholipids is slightly less than what is needed to cover one side of the membrane as a monolayer. In comparison with the wild type, the outer membrane of Rd and Re mutants contains about 70% more phospholipids, which therefore must be distributed in both the outer and inner leaflets of the membrane. Freeze-fracture studies showed that the outer membrane of Re mutants were easily fractured, but fracture became increasingly difficult in strains producing lipopolysaccharides with longer side chains. The convex fracture face was always nearly smooth, but the concave fracture face or the outer half of the membrane was densely covered with particles 8 to 10 nm in diameter. The density of particles was decreased in Re mutants to the same extent as the reduction in proteins, suggesting the largely proteinaceous nature of particles. A model for the supramolecular structure of the outer membrane is presented on the basis of these and other results. Images PMID:1102538

  5. Methylprednisolone Protects Cardiac Pumping Mechanics from Deteriorating in Lipopolysaccharide-Treated Rats

    PubMed Central

    Ko, Ya-Hui; Tsai, Ming-Shian; Chang, Ru-Wen; Chang, Chun-Yi; Wang, Chih-Hsien; Wu, Ming-Shiou; Liang, Jin-Tung; Chang, Kuo-Chu

    2015-01-01

    It has been shown that a prolonged low-dose corticosteroid treatment attenuates the severity of inflammation and the intensity and duration of organ system failure. In the present study, we determined whether low-dose methylprednisolone (a synthetic glucocorticoid) can protect male Wistar rats against cardiac pumping defects caused by lipopolysaccharide-induced chronic inflammation. For the induction of chronic inflammation, a slow-release ALZET osmotic pump was subcutaneously implanted to infuse lipopolysaccharide (1 mg kg−1 d−1) for 2 weeks. The lipopolysaccharide-challenged rats were treated on a daily basis with intraperitoneal injection of methylprednisolone (5 mg kg−1 d−1) for 2 weeks. Under conditions of anesthesia and open chest, we recorded left ventricular (LV) pressure and ascending aortic flow signals to calculate the maximal systolic elastance (Emax) and the theoretical maximum flow (Qmax), using the elastance-resistance model. Physically, Emax reflects the contractility of the myocardium as an intact heart, whereas Qmax has an inverse relationship with the LV internal resistance. Compared with the sham rats, the cardiodynamic condition was characterized by a decline in Emax associated with the increased Qmax in the lipopolysaccharide-treated rats. Methylprednisolone therapy increased Emax, which suggests that the drug may have protected the contractile status from deteriorating in the inflamed heart. By contrast, methylprednisolone therapy considerably reduced Qmax, indicating that the drug may have normalized the LV internal resistance. In parallel, the benefits of methylprednisolone on the LV systolic pumping mechanics were associated with the reduced cardiac levels of negative inotropic molecules such as peroxynitrite, malondialdehyde, and high-mobility group box 1 protein. Based on these data, we suggested that low-dose methylprednisolone might prevent lipopolysaccharide-induced decline in cardiac intrinsic contractility and LV internal

  6. Quercetin inhibits inflammatory bone resorption in a mouse periodontitis model.

    PubMed

    Napimoga, Marcelo H; Clemente-Napimoga, Juliana T; Macedo, Cristina G; Freitas, Fabiana F; Stipp, Rafael N; Pinho-Ribeiro, Felipe A; Casagrande, Rubia; Verri, Waldiceu A

    2013-12-27

    Periodontitis is a disease that leads to bone destruction and represents the main cause of tooth loss in adults. The development of aggressive periodontitis has been associated with increased inflammatory response that is induced by the presence of a subgingival biofilm containing Aggregatibacter actinomycetemcomitans. The flavonoid quercetin (1) is widespread in vegetables and fruits and exhibits many biological properties for possible medical and clinical applications such as its anti-inflamatory and antioxidant effects. Thus, in the present study, the properties of 1 have been evaluated in bone loss and inflammation using a mouse periodontitis model induced by A. actinomycetemcomitans infection. Subcutaneous treatment with 1 reduced A. actinomycetemcomitans-induced bone loss and IL-1β, TNF-α, IL-17, RANKL, and ICAM-1 production in the gingival tissue without affecting bacterial counts. These results demonstrated that quercetin exhibits protective effects in A. actinomycetemcomitans-induced periodontitis in mice by modulating cytokine and ICAM-1 production. PMID:24246038

  7. Inhibitory activity of plant stilbenoids against nitric oxide production by lipopolysaccharide-activated microglia.

    PubMed

    Nassra, Merian; Krisa, Stéphanie; Papastamoulis, Yorgos; Kapche, Gilbert Deccaux; Bisson, Jonathan; André, Caroline; Konsman, Jan-Pieter; Schmitter, Jean-Marie; Mérillon, Jean-Michel; Waffo-Téguo, Pierre

    2013-07-01

    Microglia-driven inflammatory processes are thought to play an important role in ageing and several neurological disorders. Since consumption of a diet rich in polyphenols has been associated with anti-inflammatory and neuroprotective effects, we studied the effects of twenty-five stilbenoids isolated from Milicia excelsa, Morus alba, Gnetum africanum, and Vitis vinifera. These compounds were tested at 5 and 10 µM on BV-2 microglial cells stimulated with bacterial lipopolysaccharide. Ten stilbenoids reduced lipopolysaccharide-induced nitric oxide production at 5 and/or 10 µM. Two tetramers, E-vitisin A and E-vitisin B, were the most effective molecules. Moreover, they attenuated the expression of the inducible NO synthase protein and gene. PMID:23807809

  8. Transcriptional Activation of Mucin by Pseudomonas aeruginosa Lipopolysaccharide in the Pathogenesis of Cystic Fibrosis Lung Disease

    NASA Astrophysics Data System (ADS)

    Li, Jian-Dong; Dohrman, Austin F.; Gallup, Marianne; Miyata, Susumu; Gum, James R.; Kim, Young S.; Nadel, Jay A.; Prince, Alice; Basbaum, Carol B.

    1997-02-01

    An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a CI ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

  9. Morphological damage induced by Escherichia coli lipopolysaccharide in cultured hepatocytes: localization and binding properties.

    PubMed Central

    Pagani, R.; Portolés, M. T.; Díaz-Laviada, I.; Municio, A. M.

    1988-01-01

    Lipopolysaccharides (LPS) from Gram-negative bacteria are considered to be the responsible agents for the induction of endotoxic shock, affecting the liver as a target organ. In this study, the cell morphology and some biochemical properties of 24 h-culture-hepatocyte monolayers treated with Escherichia coli 0111:B4 lipopolysaccharide, were observed. Cell morphology was observed by scanning electron microscopy and immunofluorescence methods. LPS interaction induced an increase in rounded cells with diminished adhesion capacity. As biochemical parameters, albumin synthesis and 2-deoxyglucose uptake were measured. LPS decreased the hexose uptake in a dose-dependent manner. Binding of (14C)LPS to cultured hepatocytes showed that LPS binds to non-specific constituents of the membrane bilayer. Images Fig. 1 Fig. 2 Fig. 3 Fig. 7 PMID:3052562

  10. [Immune-regulating effect of phenibut under lipopolysaccharide-induced immune stress conditions].

    PubMed

    Samotrueva, M A; Tiurenkov, I N; Teplyĭ, D L; Kuleshevskaia, N R; Khlebtsova, E V

    2010-05-01

    The immunoregulating effect of phenibut has been demonstrated on the model of immune stress caused by the injection of lipopolysaccharide from Pseudomonas aeruginosa. The degree of expression of the specific (in a delayed-type hypersensitivity reaction and passive hemagglutination) and nonspecific (phagocytic activity of neutrophils) links of immunomodulation was studied. The formation of lipopolysaccharide (LPS) induced immune stress is characterized by the increase of the indicated parameters of immunity. It is found that phenibut (under intraabdominal injection of 25 mg/kg within 5 days) removes the manifestations of hyperreactivity of the cellular link of immunity, and also restores the amount of phagocytic cells, which is evidence of the immunomodulating properties of the drug under conditions of hyperimmunization. PMID:20597368