Attachment of piliated, Opa- and Opc- gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins.

Attachment of piliated Neisseria gonorrhoeae or Neisseria meningitidis cells to A431, Chang, HEC-1-B, or polarized T(84) cells triggers rearrangements of cortical microfilaments and the accumulation of phosphotyrosine-containing proteins at sites of bacterial contact. Actin stress fibers and the microtubule network remain unaltered in infected cells. The rearrangements reported here are triggered by piliated, Opa- and Opc- strains and also by nonpiliated gonococci (GC) that produce the invasion-associated OpaA protein. Thus, neisserial adhesion via either of at least two different adhesins can trigger cortical rearrangements. In contrast, these rearrangements are not triggered by nonadherent GC or meningococcal strains, by heat-killed or chloramphenicol-treated GC strains, or by Escherichia coli recombinants that adhere to cells via GC OpaA or Opal fusion proteins, suggesting that additional neisserial components are involved. Immunoblotting experiments did not detect consistent increases in the phosphorylation of specific proteins. Possible biological implications of these Neisseria-induced cortical rearrangements are discussed.     

Piliation and colonial morphology among laboratory strains of meningococci.

Colonial morphology and piliation were studied on twelve strains from various serogroups of Neisseria meningitidis. Six different colony types (M1 to M6) were identified. Most strains elaborated only an M1 colonial type, which is similar to gonococcus T4. Several combinations of piliation and colonial morphology were observed: (i) colonial variation in which neither parent nor variant were piliated; (ii) colonial variation involving piliated and nonpiliated cells; (iii) dissociation of piliated from nonpiliated cells with no colonial change; and (iv) colonial variation in which both variants were piliated but with distinctly different pili. Results of this study demonstrate that correlations between piliation and colony morphology within N. meningitidis are exceptions rather than the rule.     

The acute-phase reactant C-reactive protein binds to phosphorylcholine-expressing Neisseria meningitidis and increases uptake by human phagocytes

Neisseria meningitidis is a global cause of meningitis and septicemia. Immunity to N. meningitidis involves both innate and specific mechanisms with killing by serum bactericidal activity and phagocytic cells. C-reactive protein (CRP) is an acute-phase serum protein that has been shown to help protect the host from several bacterial pathogens, which it recognizes by binding to phosphorylcholine (PC) on their surfaces. Pathogenic Neisseria species can exhibit phase-variable PC modification on type 1 and 2 pili. We have shown that CRP can bind to piliated meningococci in a classical calcium-dependent manner. The binding of CRP to the meningococcus was concentration dependent, of low affinity, and specific for PC. CRP appears to act as an opsonin for N. meningitidis, as CRP-opsonized bacteria showed increased uptake by human macrophages and neutrophils. Further investigation into the downstream effects of CRP-bound N. meningitidis may lead us to a better understanding of meningococcal infection and help direct more effective therapeutic interventions.     

Role of Pili in the Virulence of Neisseria gonorrhoeae

Gonococci of the colonial types that are associated with virulence, types 1 and 2, have pili that enable the bacteria both to attach in vitro to human epithelial cells and to resist phagocytosis by polymorphonuclear leukocytes. These piliated gonococci also agglutinate various mammalian and chicken erythrocytes. Gonococci of an avirulent colonial type, i.e., type 4, have no pili and neither attach to epithelial cells or erythrocytes nor resist phagocytosis. Like the type 4 bacteria, mechanically or enzymatically (trypsin) depiliated type 1 gonococci failed to attach to epithelial cells and erythrocytes and were susceptible to phagocytosis. Pili of types 1 and 2 gonococci were antigenically similar. Both type 1 gonococci and pili isolated from them induced in rabbits antibody that (i) precipitated gonococcal pili in immunodiffusion, (ii) reacted with piliated gonococci as tested by indirect immunofluorescent analysis, (iii) inhibited attachment of piliated gonococci to both human epithelial cells and erythrocytes, and (iv) opsonized piliated gonococci.     

Lipooligosaccharide-deficient Neisseria meningitidis shows altered pilus-associated characteristics

Molecular interaction between host mucosal surfaces and outer membrane components of microbes is crucial in the infection process. The outer membrane of pathogenic Neisseria contains surface molecules such as pili, PilC, and Opa and a monolayer of lipooligosaccharide (LOS), all of which are involved in the interaction with host cells. Pili mediate the initial attachment to human epithelial cells, which is followed by tight contact between bacteria and the eucaryotic cells, leading to bacterial invasion. To further examine the basis for bacterium-host cell contact, we constructed an LOS-deficient Neisseria meningitidis serogroup C mutant. LOS deficiency was without exception accompanied by altered colony opacity and morphology, which most likely represented an "on" switch for Opa540 expression, and by reduced levels of the iron-regulated proteins FetA and FbpA. We show here that LOS is essential for pilus-associated adherence but dispensable for fiber formation and twitching motility. The absence of attachment to epithelial cells could not be attributed to altered levels of piliation or defects in the pilus adhesion phenotype. Further, LOS mutants do not invade host cells and have lost the natural competence for genetic transformation.     

The Amino Acid Sequence of Neisseria lactamica PorB Surface-Exposed Loops Influences Toll-Like Receptor 2-Dependent Cell Activation

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.     

Escherichia coli adhesion to Saccharomyces cerevisiae and mammalian cells: role of piliation and surface hydrophobicity.

A number of Escherichia coli strains isolated from patients with urinary tract infections, bacteremia, or diarrhea were studied with respect to their (i) capacity to agglutinate human AB, bovine, and guinea pig erythrocytes as well as yeast (Saccharomyces cerevisiae) cells; (ii) adhesion to monolayers of cells from human intestine (intestine 407; ATCC CCL6), monkey kidney (Vero; ATCC CCL81), feline embryo (Flow no. 05-552), and porcine kidney (PK1; ATCC CRL1392) and of primary rat kidney cell cultures; and (iii) surface hydrophobicity as measured by hydrophobic interaction chromatography. No correlation could be found between the capacity of the bacteria to adhere to the different cultured mammalian cells and their agglutination patterns. The results indicated not only a complexity of bacterial receptors on the eucaryotic cells, but also a multiplicity of bacterial adhesions as expressed by the selectivity of bacterial binding. Binding of bacteria was found to be attributed to the presence of pili on the bacterial surface. It was observed that the bacteria were differently piliated: some had only common type I or related pili which gave rise to mannose-sensitive (MS) adhesion or agglutination (MS pili), some had only pili which gave rise to mannose-resistant (MR) adhesion or agglutination (MR pili), and some had both MS and MR pili. Bacteria with MS pili were more hydrophobic than those with MR pili or with none at all.     

Differential Attachment by Piliated and Nonpiliated Neisseria gonorrhoeae to Human Sperm

Piliated type 1 Neisseria gonorrhoeae attached to 50% of human sperm after incubation of mixtures in vitro for 15 min at 35 C. In contrast, nonpiliated type 4 N. gonorrhoeae attached to only 23% of sperm. Similar results were obtained with three different strains of N. gonorrhoeae. Treatment with heat or formaldehyde to kill bacteria did not affect the amount of attachment by piliated or nonpiliated types. Escherichia coli and N. subflava, other species of piliated bacteria, attached to about 40% of sperm, and the nonpiliated species N. meningitidis and N. catarrhalis attached to a comparable number of sperm, as did type 4 N. gonorrhoeae. Prior incubation of type 1 N. gonorrhoeae with purified antibody prepared against gonococcal pili reduced the percentage of sperm with attached bacteria to the same level as that for nonpiliated type 4 gonococci. Similar treatment of other piliated organisms or of nonpiliated Neisseria did not affect the attachment of the bacteria to sperm.     

Neisseria spp. and AIDS.

Neisseria meningitidis from various serogroups and two commensal neisseriae (N. sicca and N. perflava) were isolated from 15 patients at various stages of human immunodeficiency virus infection in this clinical and bacteriological study. The cases were grouped into the following three classes: (i) infections with an N. meningitidis strain of a serogroup known to be pathogenic (A, B, or C) and apparently independent of the human immunodeficiency virus infection, (ii) infections with a N. meningitidis strain of a serogroup which is normally either commensal or poorly pathogenic (serogroups Y, X, Z, and Z,29E), (iii) pulmonary and disseminated infections occurring in the course of the clinical evolutionary stage of AIDS, in two cases of which commensal neisseriae (N. sicca and N. perflava) were isolated from blood cultures.     

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing.

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.     

Adherence of Bacteroides fragilis group species.

The ability of piliated and capsulated Bacteroides fragilis and Bacteroides ovatus to adhere to intestinal cells and mucus was investigated. The adherence of piliated and capsulated strains was at least five times greater than the adherence of their nonpiliated and noncapsulated or capsulated only counterparts. These data illustrate the importance of pili as promoters of adherence of B. fragilis group species to the gastrointestinal mucosa.     

Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin.

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.     

The role of human dendritic cells in meningococcal and listerial meningitis

Few bacteria are capable of causing infections of the central nervous system (CNS), one of the most subtly shielded anatomical structures within the human body. Neisseria meningitidis is an important cause of bacterial meningitis and commonly affects otherwise healthy infants and adolescents. In contrast, Listeria monocytogenes is a cause of septicaemia and meningitis in neonates and immunocompromised adults. Dendritic cells (DCs) provide the physical link between the innate and adaptive immune system and play a crucial role in host defence against invading bacterial pathogens. The mechanisms of interaction of L. monocytogenes and N. meningitidis with DCs are entirely distinct. Whereas L. monocytogenes is readily phagocytosed by DCs by a serum-dependent mechanism, N. meningitidis is largely protected against phagocytotic uptake by its polysaccharide capsule. In addition, the pattern of secreted cytokines induced by L. monocytogenes is dominated by interleukin (IL)-12 and IL-18, capable of initiating a Th-1 response, whereas N. meningitidis induces high levels of proinflammatory cytokines. Therefore, we propose distinct functions of DCs in both types of bacterial meningitis.     

Association of type 1 pili with the ability of livers to clear Salmonella typhimurium

The role of type 1 pili in the adherence of Salmonella typhimurium strain SR-11 to hepatic sinusoidal cells was investigated. An average of 66.7% of piliated organisms was cleared by perfused livers on a single pass. Mannose and alpha-methyl-D-mannoside inhibited such trapping in a dose-dependent manner. Preincubation of the bacteria, but not the liver, with either sugar also inhibited trapping, suggesting that the sugar binds to bacterial, not hepatic, receptors. Significant numbers of previously trapped bacteria could be eluted by adding mannose to the wash medium. Bacteria with reduced piliation, obtained either by growing bacteria on agar or by using a nonpiliated variant of the parent strain, were trapped to a significantly lesser extent than the parent strain. The liver appears to selectively trap heavily piliated organisms since reperfusion of bacteria through a second liver results in significantly less trapping than occurs with the first perfusion. In vivo, the nonpiliated variant strain was cleared much more slowly than the piliated parent strain. Mannose and alpha-methyl-D-mannoside, but not glucose, decreased clearance rates of piliated organisms. Cumulatively, the data suggest that type 1 pili are a major factor in hepatic clearance of S. typhimurium.     

Escherichia coli-induced activation of neutrophil NADPH-oxidase: lipopolysaccharide and formylated peptides act synergistically to induce release of reactive oxygen metabolites.

The prevailing view of neutrophil NADPH-oxidase activation during interaction with bacteria is that the production of toxic oxygen metabolites should be directed into the phagosome containing the engulfed prey. However, in this report we show that a common Escherichia coli strain, HB101, may induce a release of neutrophil oxygen metabolites to the extracellular milieu. This phenomenon is dependent on three factors: (i) the mobilization (upregulation) of neutrophil secretory vesicles prior to interaction with the bacteria, (ii) soluble bacterial factors binding to the formylmethionyl-leucyl-phenylalanine receptor and tentatively identified as formylated peptides, and (iii) a bacterium-associated priming factor identified as lipopolysaccharide.     

Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells.     

The (alpha2-->8)-linked polysialic acid capsule of group B Neisseria meningitidis modifies multiple steps during interaction with human macrophages.

Group B Neisseria meningitidis causes systemic disease, including meningitis, after initial colonization and subsequent penetration of nasopharyngeal mucosa, a tissue which is richly populated by macrophages. In an initial effort to characterize the interaction of N. meningitidis and mature human macrophages, the influence of the alpha2-->8) -linked polysialic acid capsule on the interaction of N. meningitidis with human monocyte-derived macrophages was investigated with a capsulate case isolate and an isogenic Tn916-derived noncapsulate transformant. The capsulate strain was fourfold less adherent to the macrophage surface after cold incubation, although adherence of both strains was significantly increased after opsonization with nonimmune C5-depleted serum. When opsonized inocula were adjusted so that they adhered to macrophages in equal numbers, the two strains were internalized at equivalent rates and both entered membrane-bound compartments (phagosomes). Colocalization of bacteria with the late endosomal and lysosomal marker lysosome-associated membrane protein revealed that fusion of lysosomes with phagosomes containing the capsulate organism was significantly reduced 10 and 30 min after entry, but by 1 h, no difference between the strains was observed. Once internalized, meningococci were effectively killed, although more rapid killing of the capsulate strain was observed over the first 3 h. These results indicate that the (alpha2-->8)-linked polysialic acid capsule modifies the interaction of meningococci with human macrophages at multiple steps, including adherence to the macrophage surface and phagosome-lysosome fusion. Moreover, the discordance between the kinetics of phagosome- lysosome fusion and bacterial killing suggests that a nonlysosomal mechanism may be responsible for a significant fraction of macrophage killing of N. meningitidis.     

Pseudomonas aeruginosa selective adherence to and entry into human endothelial cells.

The pathogenesis of Pseudomonas aeruginosa disseminated infections depends on bacterial interaction with blood vessels. We have hypothesized that in order to traverse the endothelial barrier, bacteria would have to adhere to and damage endothelial cells. To test this hypothesis, we studied the adherence to human endothelial cells in primary culture of the piliated P. aeruginosa strain PAK and of two isogenic nonpiliated strains: PAK/p-, which carries a mutation in the pilin structural gene, and PAK-N1, a mutant defective in the regulatory rpoN gene. PAK adhered significantly more than did the pilus-lacking strains. P. aeruginosa was also taken up by endothelial cells, as determined by quantitative bacteriologic assays and by transmission electron microscopy. This internalization of P. aeruginosa seems to be a selective process, since the piliated strain was taken up significantly more than the nonpiliated bacteria and the avirulent Escherichia coli DH5 alpha, even following bacterial centrifugation onto the cell monolayers. A significant fraction of the internalized P. aeruginosa PAK was recovered in a viable form after 6 h of residence within endothelial cells. Progressive endothelial cell damage resulted from PAK intracellular harboring, as indicated by the release of lactate dehydrogenase. An increasing concentration of PAK cells was recovered from the extracellular medium with time, suggesting that ingested bacteria were released from endothelial cells and multiplied freely. We speculate that in vivo the ability of some P. aeruginosa strains to resist intracellular residence would afford protection from host defenses and antibiotics and that the release of viable bacteria into bloodstream may represent a central feature of the pathogenesis of bacteremia in compromised patients.     

The Neisseria lipooligosaccharide-specific alpha-2,3-sialyltransferase is a surface-exposed outer membrane protein

Neisseria gonorrhoeae and Neisseria meningitidis express an approximately 43-kDa alpha-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all N. gonorrhoeae strains and some N. meningitidis serogroups) or endogenous (in other N. meningitidis serogroups) sources of 5'-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect N. gonorrhoeae and N. meningitidis from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both N. gonorrhoeae and N. meningitidis. Western immunoblot analysis of subcellular fractions of N. gonorrhoeae strain F62 and N. meningitidis strain MC58 not subset 3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an approximately 43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of N. gonorrhoeae were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic lst insertion knockout mutant of N. gonorrhoeae F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of N. meningitidis MC58 not subset 3 and wild-type N. gonorrhoeae F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole N. gonorrhoeae cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable Neisseria cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.     

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.