Publications by authors named "Gary A Jarvis"

40 Publications

Predominant phosphorylation patterns in lipid A determined by top-down MS/MS.

J Lipid Res 2020 11 24;61(11):1437-1449. Epub 2020 Aug 24.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, CA, USA

Among the virulence factors in infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-κB via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.
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http://dx.doi.org/10.1194/jlr.RA120001014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604717PMC
November 2020

Cationic cell-penetrating peptide is bactericidal against Neisseria gonorrhoeae.

J Antimicrob Chemother 2019 11;74(11):3245-3251

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, USA.

Objectives: Cell-penetrating peptides (CPPs) have been evaluated for intracellular delivery of molecules and several CPPs have bactericidal activity. Our objectives were to determine the effect of a 12 amino acid CPPs on survival and on the invasive and inflammatory potential of Neisseria gonorrhoeae.

Methods: Survival of MDR and human challenge strains of N. gonorrhoeae grown in cell culture medium with 10% FBS was determined after treatment with the CPP and human antimicrobial peptide LL-37 for 4 h. Confocal microscopy was used to examine penetration of FITC-labelled CPP into bacterial cells. The ability of the CPP to prevent invasion of human ME-180 cervical epithelial cells and to reduce the induction of TNF-α in human THP-1 monocytic cells in response to gonococcal infection was assessed. Cytotoxicity of the CPP towards the THP-1 cells was determined.

Results: The CPP was bactericidal, with 95%-100% killing of all gonococcal strains at 100 μM. Confocal microscopy of gonococci incubated with FITC-labelled CPP revealed the penetration of the peptide. CPP treatment of N. gonorrhoeae inhibited gonococcal invasion of ME-180 cells and reduced the expression of TNF-α induced in THP-1 cells by gonococci. The CPP showed no cytotoxicity towards human THP-1 cells.

Conclusions: Based on these promising results, future studies will focus on testing of CPP in the presence of other types of host cells and exploration of structural modifications of the CPP that could decrease its susceptibility to proteolysis and increase its potency.
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http://dx.doi.org/10.1093/jac/dkz339DOI Listing
November 2019

Novel lipooligosaccharide is a determinant of inflammatory potential and virulence.

J Lipid Res 2018 10 26;59(10):1893-1905. Epub 2018 Jul 26.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, CA

The pathogenicity of , increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen , the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the LOS. Consistent with our structural findings, LOS and live bacteria induced less TNF-α secretion in human monocytes than did Furthermore, the bacteria were less virulent than in a infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of .
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http://dx.doi.org/10.1194/jlr.M085860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168313PMC
October 2018

Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors.

J Antimicrob Chemother 2018 08;73(8):2064-2071

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, USA.

Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae.

Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-α by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined.

Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at ≥5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-α probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor.

Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that warrants further investigation particularly owing to the rise in MDR gonorrhoea.
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http://dx.doi.org/10.1093/jac/dky151DOI Listing
August 2018

Innate immune response to lipooligosaccharide: pivotal regulator of the pathobiology of invasive Neisseria meningitidis infections.

Pathog Dis 2017 Apr;75(3)

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA.

Infections due to Neisseria meningitidis afflict more than one million people worldwide annually and cause death or disability in many survivors. The clinical course of invasive infections has been well studied, but our understanding of the cause of differences in patient outcomes has been limited because these are dependent on multiple factors including the response of the host, characteristics of the bacteria and interactions between the host and the bacteria. The meningococcus is a highly inflammatory organism, and the lipooligosaccharide (LOS) on the outer membrane is the most potent inflammatory molecule it expresses due to the interactions of the lipid A moiety of LOS with receptors of the innate immune system. We previously reported that increased phosphorylation of hexaacylated neisserial lipid A is correlated with greater inflammatory potential. Here we postulate that variability in lipid A phosphorylation can tip the balance of innate immune responses towards homeostatic tolerance or proinflammatory signaling that affects adaptive immune responses, causing disease with meningitis only, or septicemia with or without meningitis, respectively. Furthermore, we propose that studies of the relationship between bacterial virulence and gene expression should consider whether genetic variation could affect properties of biosynthetic enzymes resulting in LOS structural differences that alter disease pathobiology.
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http://dx.doi.org/10.1093/femspd/ftx030DOI Listing
April 2017

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding.

Proc Natl Acad Sci U S A 2017 02 13;114(9):2218-2223. Epub 2017 Feb 13.

School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia;

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from , determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.
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http://dx.doi.org/10.1073/pnas.1612927114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338521PMC
February 2017

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility.

J Am Soc Mass Spectrom 2016 07 7;27(7):1263-76. Epub 2016 Apr 7.

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121, USA.

Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source "prompt" fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1383-3DOI Listing
July 2016

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

PLoS One 2016 25;11(1):e0147637. Epub 2016 Jan 25.

Department of Pharmacology, Rutgers University Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America.

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147637PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726613PMC
July 2016

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage.

J Biol Chem 2016 Feb 11;291(7):3224-38. Epub 2015 Dec 11.

From the Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, California 94121, the Departments of Laboratory Medicine and

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.
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http://dx.doi.org/10.1074/jbc.M115.666214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751370PMC
February 2016

Induction of endotoxin tolerance by pathogenic Neisseria is correlated with the inflammatory potential of lipooligosaccharides and regulated by microRNA-146a.

J Immunol 2014 Feb 17;192(4):1768-77. Epub 2014 Jan 17.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, CA, 94121.

In this article, we report that retreatment of human monocytic THP-1 cells and primary monocytes with pathogenic Neisseria or with purified lipooligosaccharides (LOS) after previous exposure to LOS induced immune tolerance, as evidenced by reduced TNF-α and IL-1β cytokine expression. LOS that we have previously shown to vary in their potential to activate TLR4 signaling, which was correlated with differences in levels of lipid A phosphorylation, had similarly variable ability to induce tolerance. Efficacy for induction of tolerance was proportional to the level of lipid A phosphorylation, as LOS from meningococcal strain 89I with the highest degree of phosphorylation was the most tolerogenic following retreatment with LOS or whole bacteria, compared with LOS from gonococcal strains 1291 and GC56 with reduced levels of phosphorylation. Hydrogen fluoride treatment of 89I LOS to remove phosphates rendered the LOS nontolerogenic. Tolerance induced by the more highly inflammatory meningococcal LOS was correlated with significantly greater downregulation of p38 activation, greater induction of the expression of A20 and of microRNA-146a, and greater reductions in IL-1R-associated kinase 1 and TRAF6 levels following LOS retreatment of cells. The role of miR-146a in regulation of induction of TNF-α was confirmed by transfecting cells with an inhibitor and a mimic of miR-146a. Our results provide a mechanistic framework for understanding the variable pathophysiology of meningococcal and gonococcal infections given that after an initial exposure, greater upregulation of microRNA-146a by more highly inflammatory LOS conversely leads to the suppression of immune responses, which would be expected to facilitate bacterial survival and dissemination.
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http://dx.doi.org/10.4049/jimmunol.1301648DOI Listing
February 2014

Campylobacter jejuni lipooligosaccharide sialylation, phosphorylation, and amide/ester linkage modifications fine-tune human Toll-like receptor 4 activation.

J Biol Chem 2013 Jul 29;288(27):19661-72. Epub 2013 Apr 29.

Infectious Diseases and Microbiology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom.

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.
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http://dx.doi.org/10.1074/jbc.M113.468298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707672PMC
July 2013

Post-injury conditioning with lipopolysaccharide or lipooligosaccharide reduces inflammation in the brain.

J Neuroimmunol 2013 Mar 17;256(1-2):28-37. Epub 2013 Jan 17.

Center for Immunochemistry, 4150 Clement Street, Veterans Affairs Medical Center, San Francisco, CA 94121, USA.

Background: Traumatic brain injury (TBI) is a leading cause of mortality and disability in the Western world. The first stage of TBI results from the mechanical damage from an impact or blast. A second stage occurs as an inflammatory response to the primary injury and presents an opportunity for clinical intervention. In this study, we investigated the effect of pre- and post-injury treatment with lipopolysaccharide (LPS) from Escherichia coli and lipooligosaccharide (LOS) from Neisseria meningitidis on levels of cerebral inflammatory cells, circulating blood cells, and pro- and anti-inflammatory cytokine levels in a rat model of neuroinflammation induced by intrastriatal injection of IL-1β to mimic the second stage of TBI.

Methods: LPS or LOS was administered intravenously (IV) or intranasally (IN) 2h pre- or post-injection of IL-1β. The rats were euthanized 12h following IL-1β injection. Brain sections were immunostained with antibody to ED-1, a microglia cell marker. Cells in whole blood were assessed with a VetScan HM2 analyzer, and cytokine levels in sera were analyzed with a Bio-Plex system.

Results: Pre- and post-injury IV administration of LPS or LOS significantly reduced microglia in the brain, and IN pre-treatment with LPS or LOS showed a statistical trend towards reducing microglia. Pre- and post-treatment IV with LOS increased circulating levels of IL-2 and IL-4, whereas IN post-treatment with LPS reduced levels of the inflammatory cytokines, TNF-α and IFN-γ.

Conclusions: The findings strongly support continued investigation of post-conditioning with LPS or LOS as potential neuroprotective treatments for neuroinflammation from TBI.
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http://dx.doi.org/10.1016/j.jneuroim.2012.12.009DOI Listing
March 2013

Lack of lipid A pyrophosphorylation and functional lptA reduces inflammation by Neisseria commensals.

Infect Immun 2012 Nov 4;80(11):4014-26. Epub 2012 Sep 4.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, California, USA.

The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.
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http://dx.doi.org/10.1128/IAI.00506-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486066PMC
November 2012

The association between body mass index and severe biliary infections: a multivariate analysis.

Am J Surg 2012 Nov 11;204(5):574-9. Epub 2012 Aug 11.

Department of Surgery, University of California San Francisco, San Francisco, CA 94121, USA.

Background: Obesity has been associated with worse infectious disease outcomes. It is a risk factor for cholesterol gallstones, but little is known about associations between body mass index (BMI) and biliary infections. We studied this using factors associated with biliary infections.

Methods: A total of 427 patients with gallstones were studied. Gallstones, bile, and blood (as applicable) were cultured. Illness severity was classified as follows: none (no infection or inflammation), systemic inflammatory response syndrome (fever, leukocytosis), severe (abscess, cholangitis, empyema), or multi-organ dysfunction syndrome (bacteremia, hypotension, organ failure). Associations between BMI and biliary bacteria, bacteremia, gallstone type, and illness severity were examined using bivariate and multivariate analysis.

Results: BMI inversely correlated with pigment stones, biliary bacteria, bacteremia, and increased illness severity on bivariate and multivariate analysis.

Conclusions: Obesity correlated with less severe biliary infections. BMI inversely correlated with pigment stones and biliary bacteria; multivariate analysis showed an independent correlation between lower BMI and illness severity. Most patients with severe biliary infections had a normal BMI, suggesting that obesity may be protective in biliary infections. This study examined the correlation between BMI and biliary infection severity.
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http://dx.doi.org/10.1016/j.amjsurg.2012.07.002DOI Listing
November 2012

Secretory leukocyte protease inhibitor binds to Neisseria gonorrhoeae outer membrane opacity protein and is bactericidal.

Am J Reprod Immunol 2012 Aug 26;68(2):116-27. Epub 2012 Apr 26.

Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University, Springfield, IL, USA.

Problem: Secretory leukocyte protease inhibitor (SLPI) is an innate immune peptide present on the genitourinary tract mucosa that has antimicrobial activity. In this study, we investigated the interaction of SLPI with Neisseria gonorrhoeae.

Method Of Study: ELISA and far-Western blots were used to analyze binding of SLPI to gonococci. The binding site for SLPI was identified by tryptic digests and mass spectrometry. Antimicrobial activity of SLPI for gonococci was determined using bactericidal assays. SLPI protein levels in cell supernatants were measured by ELISA, and SLPI mRNA levels were assessed by quantitative RT-PCR.

Results: SLPI bound directly to the gonococcal Opa protein and was bactericidal. Epithelial cells from the reproductive tract constitutively expressed SLPI at different levels. Gonococcal infection of cells did not affect SLPI expression.

Conclusion: We conclude that SLPI is bactericidal for gonococci and is expressed by reproductive tract epithelial cells and thus is likely to play a role in the pathogenesis of gonococcal infection.
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http://dx.doi.org/10.1111/j.1600-0897.2012.01149.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395761PMC
August 2012

Modulation of HIV transmission by Neisseria gonorrhoeae: molecular and immunological aspects.

Curr HIV Res 2012 Apr;10(3):211-7

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, CA 94121, USA.

Neisseria gonorrhoeae (GC), a major cause of pelvic inflammatory disease, can facilitate HIV transmission. In response to GC infection, genital epithelial cells can produce cytokines, chemokines and defensins to modulate HIV infection and infectivity. GC can also induce the production of cytokines and chemokines in monocytes and modulate T cell activation. In vivo, an increase in the number of endocervical CD4+ T cells has been found in GC-infected women. Additionally, GC appears to modulate HIV-specific immune responses in HIV-exposed sex workers. Interestingly, in vitro, GC exhibits HIV enhancing or inhibitory effects depending on the HIV target cells. This review summarizes molecular and immunological aspects of the modulation of HIV infection and transmission by GC. Future studies using a multi-cellular system or in animal models will offer insight into the mechanisms by which GC increases HIV transmission.
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http://dx.doi.org/10.2174/157016212800618138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149178PMC
April 2012

Human lipooligosaccharide IGG that prevents endemic meningococcal disease recognizes an internal lacto-N-neotetraose structure.

J Biol Chem 2011 Dec 25;286(51):43622-43633. Epub 2011 Oct 25.

Department of Veterans Affairs Medical Center, San Francisco, California 94121; Department of Laboratory Medicine, University of California, San Francisco, California 94121. Electronic address:

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.
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http://dx.doi.org/10.1074/jbc.M111.291583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243514PMC
December 2011

Post-injury treatment with lipopolysaccharide or lipooligosaccharide protects rat neuronal and glial cell cultures.

Brain Res Bull 2011 Jul 7;85(6):403-9. Epub 2011 May 7.

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA.

Traumatic brain injury (TBI) is a major cause of disability in civilians and military personnel worldwide that is caused by the acceleration force of a primary shockwave, blast wind or the force of a direct contact. Following the primary injury, secondary injury is caused by activation of the immune response due to an influx of neuro-inflammatory cells, increased production of inflammatory cytokines, and edema. In ischemia models pre-conditioning with lipopolysaccharide (LPS) has been shown to be neuroprotective, and post-injury conditioning with LPS was found to be protective in a spinal cord and an acute brain injury model. In this study, we utilized an in vitro scratch model of TBI to assess the effect of post-injury treatment with Escherichia coli LPS and Neisseria meningitidis lipooligosaccharide (LOS) on cell death and cytokine induction by assessing the level of lactate dehydrogenase released from cells and rat multiplex cytokine assays. Our results showed that post-injury treatment of C6 glioma cells with either the LPS or the LOS reduced cell death when compared to scratched controls treated with media only. Post-injury treatment of the primary mixed neuronal cultures with LPS reduced cell death and resulted in a significant up-regulation in IL-10 when compared to controls. With LOS post-scratch treatment of the primary cell cultures, we found that IL-1α, IL-1β, IL-6, and TNF-α were significantly upregulated in addition to IL-10 compared to the media-only controls. The results strongly support additional testing of the neuroprotective ability of post-injury treatment with LPS or LOS in models of TBI.
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http://dx.doi.org/10.1016/j.brainresbull.2011.04.007DOI Listing
July 2011

Phosphoryl moieties of lipid A from Neisseria meningitidis and N. gonorrhoeae lipooligosaccharides play an important role in activation of both MyD88- and TRIF-dependent TLR4-MD-2 signaling pathways.

J Immunol 2010 Dec 29;185(11):6974-84. Epub 2010 Oct 29.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, CA, 94121, USA.

We have previously shown that the lipooligosaccharide (LOS) from Neisseria meningitidis and N. gonorrhoeae engages the TLR4-MD-2 complex. In this study, we report that LOS from different meningococcal and gonococcal strains have different potencies to activate NF-κB through TLR4-MD-2 and that the relative activation can be correlated with ion abundances in MALDI-TOF mass spectrometry that are indicative of the number of phosphoryl substituents on the lipid A (LA) component of the LOS. The LOSs from three of the strains, meningococcal strain 89I and gonococcal strains 1291 and GC56, representing high, intermediate, and low potency on NF-κB activation, respectively, differently activated cytokine expression through the TLR4-MD-2 pathway in monocytes. In addition to induction of typical inflammatory cytokines such as TNF-α, IL-1β, and IL-6, MIP-1α and MIP-1β also were significantly higher in cells treated with 89I LOS, which had the most phosphoryl substitutions on the LA compared with 1291 LOS and GC56 LOS. We found that LOS activated both the MyD88- and TRIF-dependent pathways through NF-κB and IFN regulatory factor 3 transcription factors, respectively. Moreover, LOS induced the expression of costimulatory molecule CD80 on the surfaces of monocytes via upregulation of IFN regulatory factor 1. These results suggest that phosphoryl moieties of LA from N. meningitidis and N. gonorrhoeae LOSs play an important role in activation of both the MyD88- and TRIF-dependent pathways. Our findings are consistent with the concept that bacteria modulate pathogen-associated molecular patterns by expression of phosphoryl moieties on the LA to optimize interactions with the host.
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http://dx.doi.org/10.4049/jimmunol.1000953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059745PMC
December 2010

Neisseria gonorrhoeae enhances HIV-1 infection of primary resting CD4+ T cells through TLR2 activation.

J Immunol 2010 Mar 10;184(6):2814-24. Epub 2010 Feb 10.

Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

Sexually transmitted infections increase the likelihood of HIV-1 transmission. We investigated the effect of Neisseria gonorrheae (gonococcus [GC]) exposure on HIV replication in primary resting CD4(+) T cells, a major HIV target cell during the early stage of sexual transmission of HIV. GC and TLR2 agonists, such as peptidylglycan (PGN), Pam(3)CSK(4), and Pam(3)C-Lip, a GC-derived synthetic lipopeptide, but not TLR4 agonists including LPS or GC lipooligosaccharide enhanced HIV-1 infection of primary resting CD4(+) T cells after viral entry. Pretreatment of CD4(+) cells with PGN also promoted HIV infection. Anti-TLR2 Abs abolished the HIV enhancing effect of GC and Pam(3)C-Lip, indicating that GC-mediated enhancement of HIV infection of resting CD4(+) T cells was through TLR2. IL-2 was required for TLR2-mediated HIV enhancement. PGN and GC induced cell surface expression of T cell activation markers and HIV coreceptors, CCR5 and CXCR4. The maximal postentry HIV enhancing effect was achieved when PGN was added immediately after viral exposure. Kinetic studies and analysis of HIV DNA products indicated that GC exposure and TLR2 activation enhanced HIV infection at the step of nuclear import. We conclude that GC enhanced HIV infection of primary resting CD4(+) T cells through TLR2 activation, which both increased the susceptibility of primary CD4(+) T cells to HIV infection as well as enhanced HIV-infected CD4(+) T cells at the early stage of HIV life cycle after entry. This study provides a molecular mechanism by which nonulcerative sexually transmitted infections mediate enhancement of HIV infection and has implication for HIV prevention and therapeutics.
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http://dx.doi.org/10.4049/jimmunol.0902125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739425PMC
March 2010

Natural phosphoryl and acyl variants of lipid A from Neisseria meningitidis strain 89I differentially induce tumor necrosis factor-alpha in human monocytes.

J Biol Chem 2009 Aug 15;284(32):21515-25. Epub 2009 Jun 15.

Center for Immunochemistry, Veterans Affairs Medical Center, San Francisco, California 94121, USA.

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-alpha by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-alpha induction varied by approximately 2-10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.
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http://dx.doi.org/10.1074/jbc.M109.004887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755876PMC
August 2009

Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome.

J Immunol 2009 May;182(10):6460-9

Department of Medicine, Division of Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1beta and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1beta-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1beta/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1beta production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1beta secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.
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http://dx.doi.org/10.4049/jimmunol.0802696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722440PMC
May 2009

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction.

J Lipid Res 2009 Mar 2;50(3):424-438. Epub 2008 Oct 2.

Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121; Department of Laboratory Medicine, University of California, San Francisco, CA 94143. Electronic address:

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.
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http://dx.doi.org/10.1194/jlr.M800184-JLR200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2638105PMC
March 2009

Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

J Immunol 2008 May;180(9):6176-85

Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.
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http://dx.doi.org/10.4049/jimmunol.180.9.6176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042429PMC
May 2008

Elderly patients have more severe biliary infections: influence of complement-killing and induction of TNFalpha production.

Surgery 2008 Jan 3;143(1):103-12. Epub 2007 Dec 3.

Department of Surgery, University of California, San Francisco, CA 94121, USA.

Background: Biliary bacteria are more common in elderly patients and cause more serious illnesses. The reasons for this are unclear. We noted previously that bacterial serum-sensitivity and induction of TNFalpha production in sera (iTNFsera) were associated with severe biliary infections. We examined the influence of age and these factors on illness severity.

Methods: Three-hundred and forty patients were studied. Gallstones and bile were cultured. Illness was staged as none (no clinical infection or inflammation), SIRS (fever, leukocytosis), severe (cholangitis, abscess, empyema), or MODS (bacteremia, hypotension, organ dysfunction/failure). Bacterial serum-sensitivity and TNFalpha induction were measured. Younger (< 70 years) and elderly (> or = 70 years) patients were compared.

Results: Biliary bacteria were more common in elderly (64% vs 41%, P < .0001). Among patients with biliary bacteria, the elderly had more serious illnesses: none: 44% younger, 19% elderly; SIRS: 16% younger, 22% elderly; severe: 22% younger, 21% elderly; MODS 18% younger, 38% elderly (P = .003). Bacteria from elderly patients induced more TNFalpha (580 vs 310 pg/ml, P = .023). In both groups, serum-sensitive bacteria caused infectious manifestations and induced abundant TNFalpha; however, serum-resistant bacteria from elderly usually (69%) caused infectious manifestations and abundant TNFalpha, while serum-resistant bacteria from younger patients rarely (8%) caused infectious manifestations and minimal TNFalpha. Elderly patients with high iTNFsera bacteria had more severe illnesses.

Conclusions: Biliary bacteria were more common in elderly patients and produced more serious illnesses. Many younger patients with biliary bacteria displayed no infectious manifestations. Elderly patients harbored more virulent bacteria, and had a heightened response to high iTNFsera bacteria, as well as bacteria largely tolerated by younger patients.
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http://dx.doi.org/10.1016/j.surg.2007.06.035DOI Listing
January 2008

Bacteria entombed in the center of cholesterol gallstones induce fewer infectious manifestations than bacteria in the matrix of pigment stones.

J Gastrointest Surg 2007 Oct;11(10):1298-308

Department of Surgery, University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.

Purpose: The clinical significance of bacteria in the pigment centers of cholesterol stones is unknown. We compared the infectious manifestations and characteristics of bacteria from pigment stones and predominantly cholesterol stones.

Methods: Three hundred forty patients were studied. Bile was cultured. Gallstones were cultured and examined with scanning electron microscopy. Level of bacterial immunoglobulin G (bile, serum), complement killing, and tumor necrosis factor-alpha production were determined.

Results: Twenty-three percent of cholesterol stones and 68% of pigment stones contained bacteria (P < 0.0001). Stone culture correlated with scanning electron microscopy results. Pigment stone bacteria were more often present in bile and blood. Cholesterol stone bacteria caused more severe infections (19%) than sterile stones (0%), but less than pigment stone bacteria (57%) (P < 0.0001). Serum and bile from patients with cholesterol stone bacteria had less bacterial-specific immunoglobulin G. Cholesterol stone bacteria produced more slime. Pigment stone bacteria were more often killed by a patient's serum. Tumor necrosis factor-alpha production of the groups was similar.

Conclusions: Bacteria are readily cultured from cholesterol stones with pigment centers, allowing for analysis of their virulence factors. Bacteria sequestered in cholesterol stones cause infectious manifestations, but less than bacteria in pigment stones. Possibly because of their isolation, cholesterol stone bacteria were less often present in bile and blood, induced less immunoglobulin G, were less often killed by a patient's serum, and demonstrated fewer infectious manifestations than pigment stone bacteria. This is the first study to analyze the clinical relevance of bacteria within cholesterol gallstones.
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http://dx.doi.org/10.1007/s11605-007-0173-4DOI Listing
October 2007

Gallstones containing bacteria are biofilms: bacterial slime production and ability to form pigment solids determines infection severity and bacteremia.

J Gastrointest Surg 2007 Aug;11(8):977-83; discussion 983-4

Department of Surgery (112), University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.

Objective: Gallstone bacteria provide a reservoir for biliary infections. Slime production facilitates adherence, whereas beta-glucuronidase and phospholipase generate colonization surface. These factors facilitate gallstone formation, but their influence on infection severity is unknown.

Methods: Two hundred ninety-two patients were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for beta-glucuronidase/phospholipase production and quantitative slime production. Infection severity was correlated with bacterial factors.

Results: Bacteria were present in 43% of cases, 13% with bacteremia. Severe infections correlated directly with beta-glucuronidase/phospholipase (55% with vs 13% without, P < 0.0001), but inversely with slime production (55 vs 8%, slime <75 or >75, P = 0.008). Low slime production and beta-glucuronidase/phospholipase production were additive: Severe infections were present in 76% with both, but 10% with either or none (P < 0.0001). beta-Glucuronidase/phospholipase production facilitated bactibilia (86% with vs 62% without, P = 0.03). Slime production was 19 (+/-8) vs 50 (+/-10) for bacteria that did or did not cause bacteremia (P = 0.004). No bacteria with slime >75 demonstrated bacteremia.

Conclusions: Bacteria-laden gallstones are biofilms whose characteristics influence illness severity. Factors creating colonization surface (beta-glucuronidase/phospholipase) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. This shows how properties of the gallstone biofilm determine the severity of the associated illness.
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http://dx.doi.org/10.1007/s11605-007-0168-1DOI Listing
August 2007

Affinity-purified human immunoglobulin G that binds a lacto-N-neotetraose-dependent lipooligosaccharide structure is bactericidal for serogroup B Neisseria meningitidis.

Infect Immun 2007 Feb 13;75(2):1025-33. Epub 2006 Nov 13.

Department of Pediatrics, Laboratory Medicine, University of California at San Francisco, San Francisco, California 94121, USA.

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.
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http://dx.doi.org/10.1128/IAI.00882-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828497PMC
February 2007

Biliary bacterial factors determine the path of gallstone formation.

Am J Surg 2006 Nov;192(5):598-603

Department of Surgery, University of California San Francisco, San Francisco, CA 94121, USA.

Background: Bacteria cause pigment gallstones and can act as a nidus for cholesterol gallstone formation. Bacterial factors that facilitate gallstone formation include beta-glucuronidase (bG), phospholipase (PhL), and slime. The current study sought to determine whether bacterial factors influence the path of gallstone formation.

Methods: A total of 382 gallstones were cultured and/or examined using scanning electron microscopy (SEM). Bacteria were tested for bG and slime production. Gallstone composition was determined using infrared spectrography. Ca-palmitate presence documented bacterial PhL production. Groups were identified based upon bacterial factors present: slime and bGPhL (slime/bGPhL), bGPhL only, and slime only. Influence of bacterial stone-forming factors on gallstone composition and morphology was analyzed.

Results: Bacteria were present in 75% of pigment, 76% of mixed, and 20% of cholesterol stones. Gallstones with bGPhL producing bacteria contained more pigment (71% vs. 26%, P < .0001). The slime/bGPhL group was associated (79%) with pigment stones, bGPhL was associated (56%) with mixed stones, while slime (or none) only was associated (67%) with cholesterol stones (P < .031, all comparisons).

Conclusions: Bacterial properties determined the path of gallstone formation. Bacteria that produced all stone-forming factors promoted pigment stone formation, while those that produced only bGPhL promoted mixed stone formation. Bacteria that only produced slime lacked the ability to generate pigment solids, and consequently were more common in the centers of cholesterol stones. This shows how bacterial characteristics may govern the process of gallstone formation.
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http://dx.doi.org/10.1016/j.amjsurg.2006.08.001DOI Listing
November 2006

Gonococcal lipooligosaccharide suppresses HIV infection in human primary macrophages through induction of innate immunity.

J Infect Dis 2006 Sep 8;194(6):751-9. Epub 2006 Aug 8.

Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

Gonorrhea often occurs as a coinfection with human immunodeficiency virus (HIV). Lipooligosaccharide (LOS) is a component of the gonococcal outer membrane that induces innate immunity through engagement of Toll-like receptor 4 (TLR4). We investigated the effects that LOS from 5 different strains of Neisseria gonorrhoeae have on HIV infection and on HIV provirus in primary human macrophages. LOS-treated human primary macrophages developed resistance to new HIV infection as well as to HIV provirus. Gonococcal LOS from the 5 strains and lipopolysaccharide (LPS) from Escherichia coli showed no significant difference in their anti-HIV activities. Suppression of HIV provirus resulted from the induction of interferon (IFN)-beta and subsequent activation of signal transducer and activator of transcription 1. Neutralization of IFN-beta , but not IFN-alpha , via antibody significantly reduced the anti-HIV activity induced by LOS and LPS. We conclude that LOS expressed by various strains of N. gonorrhoeae induce specific innate immune responses through TLR4 signaling, resulting in anti-HIV activity in human primary macrophages in vitro.
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http://dx.doi.org/10.1086/506360DOI Listing
September 2006
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