Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophil influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), since Gc cannot make its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea.

Comparison of lipooligosaccharides from human challenge strains of Neisseria gonorrhoeae.

The alarming rise of antibiotic resistance and the emergence of new vaccine technologies have increased the focus on vaccination to control gonorrhea. Neisseria gonorrhoeae strains FA1090 and MS11 have been used in challenge studies in human males. We used negative-ion MALDI-TOF MS to profile intact lipooligosaccharide (LOS) from strains MS11mkA, MS11mkC, FA1090 A23a, and FA1090 1-81-S2. The MS11mkC and 1-81-S2 variants were isolated from male volunteers infected with MS11mkA and A23a, respectively. LOS profiles were obtained after purification using the classical phenol water extraction method and by microwave-enhanced enzymatic digestion, which is more amenable for small-scale work. Despite detecting some differences in the LOS profiles, the same major species were observed, indicating that microwave-enhanced enzymatic digestion is appropriate for MS studies. The compositions determined for MS11mkA and mkC LOS were consistent with previous reports. FA1090 is strongly recognized by mAb 2C7, an antibody-binding LOS with both α- and ß-chains if the latter is a lactosyl group. The spectra of the A23a and 1-81-S2 FA1090 LOS were similar to each other and consistent with the expression of α-chain lacto-N-neotetraose and ß-chain lactosyl moieties that can both be acceptor sites for sialic acid substitution. 1-81-S2 LOS was analyzed after culture with and without media supplemented with cytidine-5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which N. gonorrhoeae needs to sialylate its LOS. LOS sialylation reduces the infectivity of gonococci in men, although it induces serum resistance in serum-sensitive strains and reduces killing by neutrophils and antimicrobial peptides. The infectivity of FA1090 in men is much lower than that of MS11mkC, but the reason for this difference is unclear. Interestingly, some peaks in the spectra of 1-81-S2 LOS after bacterial culture with CMP-Neu5Ac were consistent with disialylation of the LOS, which could be relevant to the reduced infectivity of FA1090 in men and could have implications regarding the phase variation of the LOS and the natural history of infection.

Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase.

OBJECTIVES: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response. METHODS: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested. RESULTS: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases. CONCLUSIONS: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages.

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

Among the virulence factors in Neisseria 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-MS3). 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.

Cationic cell-penetrating peptide is bactericidal against Neisseria gonorrhoeae.

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.

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence.

The pathogenicity of Campylobacter concisus, 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 C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus 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 C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella 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 C. concisus.

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

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.

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

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.

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

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 Neisseria meningitidis, 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.

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

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.

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

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.

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

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.

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

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-1ß 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.

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

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.

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

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-1ß 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-1ß. The rats were euthanized 12h following IL-1ß 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.

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

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.

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

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.

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

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.

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

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.