Cellular Fatty Acids of Pathogenic Neisseria

The cellular fatty acid composition of 20 isolates of Neisseria gonorrhoeae and 21 isolates of N. meningitidis was examined by gas-liquid chromatography. Each isolate of the two species possessed similar fatty acid profiles which were characterized by five major acids, accounting for 80 to 85% of the total. The three most abundant acids in each species were palmitic, palmitoleic, and beta-hydroxylauric acids; lauric and myristic acids were the next most abundant. The presence of large amounts of beta-hydroxylauric acid (20% or greater) and the relative concentrations of the other four major acids appear to be useful markers for distinguishing N. gonorrhoeae and N. meningitidis fatty acids from those of other bacteria.     

Pathogenic consequences of Neisseria gonorrhoeae pilin glycan variation

A Neisseria gonorrhoeae (gonococcus, GC) pilin glycosylation gene, pgtA, can either possess or lack phase-variation ability. Many GC, particularly the disseminated strains, carry a phase-variable pgtA. However, other GC, predominantly the uncomplicated gonorrhea isolates, carry a pgtA lacking phase-variability. These and other results suggest GC pilin glycan's pathogenic involvement.     

Goose barnacles hitchhike on satellite-tracked southern elephant seals

Goose barnacles (Lepas australis) attached to satellite-relay data loggers were carried by three southern elephant seals (Mirounga leonina) from Marion Island. Their movements across the Polar Frontal Zone are presented, providing further evidence that megafauna are potential vectors for the transport of species into the Southern Ocean.     

Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome‐primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome–granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule‐negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule‐negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome–granule fusion. Ectopically increasing primary granule–phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule–phagosome fusion, thus preventing formation of a degradative phagolysosome.     

Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils

Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil‐rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram‐negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild‐type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule‐localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation.     

A New Family of Secreted Toxins in Pathogenic Neisseria Species

The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation.     

Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.     

Effects of taxol on human neutrophils

Taxol, a plant alkaloid, promotes and stabilizes microtubule assembly in cells and cellfree systems. In the present study, the effects of taxol on various functional, morphologic, and biochemical phenomena in human peripheral blood PMN (Hypaque-Ficoll) were examined. Taxol (10(-7) M) inhibited PMN chemotaxis stimulated by N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) or endotoxin-activated serum by more than 60%. The inhibition was not readily reversed by washing, and taxol itself was not a chemoattractant, nor is it a secretagogue. Spontaneous nondirected migration, cell spreading on a glass surface, and orientation of cell organelles in response to a chemoattractant gradient were also inhibited by taxol. Taxol (10(-5) M) decreased killing of Staphylococcus aureus, but did not alter phagocytosis of heat-killed Candida or hexose monophosphate shunt activity in resting or stimulated PMN. Ultrastructural studies showed that PMN incubated in f-met-leu-phe, taxol, or both had increased (p less than 0.001) numbers of centrosome-associated microtubules, and the microtubules of cells incubated in taxol with or without f-met-leu-phe were organized into bundles. Taxol (10(-5) M) markedly inhibited post-translational tyrosinolation of alpha-chains of tubulin in both resting and f-met-leu-phe-stimulated PMN. The data indicate that taxol inhibits PMN locomotion and bacterial killing, supporting a role for microtubules in these processes. The ultrastructural and biochemical data also support the view that taxol mediates its effects on PMN by its effect on microtubules.     

A bacterial siren song: intimate interactions between Neisseria and neutrophils

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that are exquisitely adapted for growth at human mucosal surfaces and for efficient transmission between hosts. One factor that is essential to neisserial pathogenesis is the interaction between the bacteria and neutrophils, which are recruited in high numbers during infection. Although this vigorous host response could simply reflect effective immune recognition of the bacteria, there is mounting evidence that in fact these obligate human pathogens manipulate the innate immune response to promote infectious processes. This Review summarizes the mechanisms used by pathogenic neisseriae to resist and modulate the antimicrobial activities of neutrophils. It also details some of the major outstanding questions about the Neisseria-neutrophil relationship and proposes potential benefits of this relationship for the pathogen.     

Effect of bilirubin on the fungicidal capacity of human neutrophils

The effect of unconjugated bilirubin on the fungicidal capacity of human neutrophils was examined. Per cent Torulopsis glabrata killed in the presence of 1 x 10(-5) and 5 x 10(-5) M bilirubin was 72.7 +/- 2.5 and 40.9 +/- 7.2 respectively, compared to 81.4 +/- 4.1 in control (p less than 0.01 and p less than 0.001), respectively). This effect was not due to a concomitant inhibition of phagocytosis. The results suggest that jaundiced neonates may be more susceptible to fungal infections.     

Physicochemical properties of the N-formyl peptide receptor on human neutrophils

In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.     

Function of neutral endopeptidase on the cell membrane of human neutrophils

Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.     

Dual effects of formylpeptides on the adhesion of endotoxin-primed human neutrophils

Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (> 5 × 10^?8 M ) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10^?10 M to 5 × 10^?9 M ) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (> 10^?7 M ) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (> 10^?9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10^?9 M ) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10^?9 M ) and high (5 × 10^?7 M ) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Role of Glycosylation at Ser63 in Production of Soluble Pilin in Pathogenic Neisseria

Pilus-mediated adhesion is essential in the pathogenesis of Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.