CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells

Neisseria gonorrhoeae is a gram-negative bacterial pathogen which infects the human mucosal epithelium. An early critical event in neisserial infection is the type IV pilus-mediated adherence to the host cell. The PilC protein, located on the pilus tip, has earlier been identified as the major pilus adhesin. Previous studies suggested that the cell surface protein CD46 is a pilus receptor for Neisseria. We investigated the role of CD46 in pilus-mediated gonococcal infection of epithelial cells. Differences in binding efficiencies of piliated gonococci as well as purified pilus adhesin PilC2 on human epithelial cell lines did not correlate to the level of surface-expressed CD46. Additionally, no binding of piliated gonococci or PilC2 protein was observed on CD46-transfected CHO and MDCK cells. Furthermore, specific down-regulation of CD46 expression in human epithelial cell lines by RNA interference did not alter the binding efficiency of piliated gonococci or purified PilC2 protein, although other CD46-dependent processes, such as measles virus infection and C3b cleavage, were significantly reduced. These data support the notion that pilus-mediated gonococcal infection of epithelial cells can occur in a CD46-independent manner, thus questioning the function of CD46 as an essential pilus receptor for pathogenic neisseriae.     

Experimental infection of native human ureteral tissue with Neisseria gonorrhoeae: adhesion, invasion, intracellular fate, exocytosis, and passage through a stratified epithelium.

The exact mechanisms by which Neisseria gonorrhoeae invades the mucosal lining to cause local and disseminated infections are still not fully understood. The ability of gonococci to infect the human ureter and the mechanism of gonococcal infection in a stratified epithelium were investigated by using distal ureters excised from healthy adult kidney donors. In morphological terms, this tissue closely resembles parts of the urethral proximal epithelium, a site of natural gonococcal infection. Using piliated and nonpiliated variants of N. gonorrhoeae MS11, we demonstrated the importance of pili in the attachment of gonococci to native epithelial cells as well as their association with epithelial damage. By electron microscopy we elucidated the different mechanisms of colonization and invasion of a stratified epithelium, including adherence to surface cells, invasion and eventual release from infected cells, disintegration of intercellular connections followed by paracellular tissue infiltration, invasion of deeper cells, and initiation of cellular destruction and exfoliation resulting in thinning of the mucosa.     

Differential response of human monocytes to Neisseria gonorrhoeae variants expressing pili and opacity proteins.

Experiments in vitro suggest that Neisseria gonorrhoeae surface variation plays a key role in gonococcal pathogenesis by providing the appropriate bacterial phenotypes to go through different stages of the infection. Here we report on the effects of phase and antigen variation of two major gonococcal adhesins, pili and opacity (Opa) outer membrane proteins, on the interaction of the gonococci with human monocytes. Using a set of recombinants of gonococcus strain MS11 that each express 1 of 11 genetically defined Opa proteins or a defined type of pilus, we found that both Opa proteins and pili promote bacterial phagocytosis by monocytes in the absence of serum and that this feature largely depends on the type of protein that is expressed. One of the Opa proteins (Opa[50]) strongly promoted uptake by monocytes but had little effect on the interaction with polymorphonuclear leukocytes under the conditions employed. Similarly, the phagocytosis-promoting effect of the pili was much more pronounced in monocytes than in neutrophils (4-fold versus 22-fold stimulation of uptake, respectively). Only a subpopulation of both types of phagocytes actively ingested bacteria, as has been observed during natural infections. Measurements of luminol-enhanced chemiluminescence demonstrated that phagocytosis of opaque but not piliated gonococci was accompanied by an increase in oxygen-reactive metabolites. These findings demonstrate that the monocyte response towards gonococci is highly dependent on the bacterial phenotype and differs from the neutrophil response. This diversity in bacterial behavior towards various types of human phagocytic cells underlines the biological impact of gonococcal surface variation and may explain previous contradictory results on this subject.     

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.     

Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes

Infection with Neisseria gonorrhoeae can trigger an intense inflammatory response, yet there is little specific immune response or development of immune memory. In addition, gonorrhea typically correlates with a transient reduction in T lymphocyte counts in blood, and these populations recover when gonococcal infection is resolved. Such observations suggest that the gonococci have a suppressive effect on the host immune response. We report here that N. gonorrhoeae Opa proteins were able to bind CEACAM1 expressed by primary CD4+ T lymphocytes and suppress their activation and proliferation. CEACAM1 bound by gonococcal Opa52 associated with the tyrosine phosphatases SHP-1 and SHP-2, which implicates the receptor's ITIM (immunoreceptor tyrosine-based inhibitory motif) in this effect.     

Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells

In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.     

Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated.     

Roles of PilC and PilE Proteins in Pilus-Mediated Adherence of Neisseria gonorrhoeae and Neisseria meningitidis to Human Erythrocytes and Endothelial and Epithelial Cells

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.     

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.     

Conservation of Neisseria gonorrhoeae pilus expression regulatory genes pilA and pilB in the genus Neisseria.

The pili of Neisseria gonorrhoeae mediate bacterial adhesion to the host-susceptible tissues. We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression. Besides this regulatory function, pilA participates in an essential function for bacterial viability. Here we show that pilA and pilB homologs are also present in a variety of other members of the Neisseriaceae family of bacteria in contrast to the gonococcal pilin gene which hybridizes only to the pathogenic Neisseria species.     

Gonococci possessing only certain P.II outer membrane proteins interact with human neutrophils.

We investigated the role of the protein II (P.II) family of gonococcal outer membrane proteins in the interaction of seven single P.II variants of Neisseria gonorrhoeae FA1090 with human neutrophils in vitro. The abilities of nonpiliated gonococci to adhere to and be killed by neutrophils and to stimulate luminol-dependent chemiluminescence (CL) depended on the possession of at least one P.II. Gonococci lacking P.II (i.e., P.II-) adhered poorly to and were not killed by neutrophils and induced only minimal CL. Although most P.II-containing (i.e., P.II+) variants adhered to, stimulated, and were readily killed by neutrophils, one variant, containing P.IIa, possessed none of these characteristics; it acted just like a P.II- variant. No correlation was found between the colony opacity phenotype and the interaction of gonococci with neutrophils. Data from CL experiments suggest that the stimulatory effect of P.II was dominant over that of pili; i.e., piliated P.II+ gonococci were much more stimulatory than piliated P.II- gonococci. The results indicate that most but not all P.II proteins mediate, in part or in full, the interaction of N. gonorrhoeae with human neutrophils, including adherence, stimulation of the neutrophil respiratory burst, and phagocytic killing.     

Interactions of gonococci with HeLa cells: attachment, detachment, replication, penetration, and the role of protein II.

Colony variants of Neisseria gonorrhoeae differ in their interactions with eucaryotic cells. When gonococci were cultivated with HeLa cell monolayers, the opacity phenotype (Op) became increasingly dominant in the subpopulation of organisms which adhered to the HeLa cells. Once bound, Op organisms displayed very low levels of detachment. Adherent Op gonococci exhibited generation times up to threefold greater than cultures containing gonococci in the absence of HeLa cells. In addition, the progeny of adherent Op organisms remained bound to the HeLa cell monolayer. Both piliated (P+) and transparent (Tr) colony types attached to HeLa cells, but their progeny were retained less efficiently. Gonococci bound to HeLa cells were subjected to the bactericidal action of fresh rat serum and approximately 0.5 to 2.5% survived, irrespective of their opacity or piliation phenotype. Incubation with gentamicin resulted in a 10- to 50-fold further reduction in viability. Pretreatment of HeLa cell monolayers with the microfilament-disrupting agent cytochalasin b diminished gonococcal survival in either serum or gentamicin by up to eightfold. In contrast, cytochalasin b treatment did not decrease survival of the commensal organism N. sicca. The data suggest that very few gonococci are completely interiorized and a small proportion of adherent gonococci are partially protected from the soluble-phase environment by HeLa cells.     

Evidence for functionally distinct pili expressed by Neisseria meningitidis.

In order to investigate possible functional consequences of phase and antigenic variation of meningococci, the attachment of 15 strains of Neisseria meningitidis to human erythrocytes was studied by a nitrocellulose hemadsorption assay. This assay allows the study of individual meningococcal colonies with respect to erythrocyte attachment. Of the 15 strains studied, 7 demonstrated binding of human erythrocytes (HA+). Among these seven strains, the percentage of colonies that were HA+ ranged from 0.2 to 97%. Meningococcal colonies that did not produce pilin (the major structural subunit of pili) did not demonstrate erythrocyte binding (HA-). The HA+ colony phenotype was correlated with assembly of pilin into pili and expression of pili on the meningococcal surface. However, only some piliated colonies bound human erythrocytes. This could not be explained by differences between piliated HA+ and HA- colonies in the amount of pilin produced or by differences in number of pili expressed per diplococcus. Pili of five of the meningococcal strains with HA+ colonies were antigenically related to gonococcal pili (class I meningococcal pili), but HA+ colonies were also seen in two meningococcal strains expressing class II meningococcal pili. Changes from HA+ to HA- and from HA- to HA+, in the presence of continuing pilin production and pilus assembly, occurred at frequencies of up to 10(-2)/CFU per generation. Such frequencies resemble those of phase and antigenic variation described previously for Neisseria species pilin. These studies indicate that phase variation influences the ability of meningococci to attach to human cells and suggest that meningococci may express functionally different pili.     

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.     

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.     

Escherichia coli pili as possible mediators of attachment to human urinary tract epithelial cells.

Presence of pili of fimbriae on Escherichia coli bacteria isolated from the urine of patients with urinary tract infection was related to the ability of the bacteria to attach to human uroepithelial cells. Piliated E. coli strains agglutinated guinea pig erythrocytes. D-Mannose and alpha-methyl-D-mannopyranoside inhibited this agglutination with all but one of the 12 strains tested. D-Mannose, D-galactose, alpha-methyl-D-mannopyranoside, and L-fucose did not afect attachment of piliated strains to uroepithelial cells. Heating as well as washing of piliated strains caused a parallel decrease of piliation and adhesive ability. Growth in glucose-enriched medium increased capsule formation but decreased piliation and adhesion. Capsulated strains retained their adhesive ability provided that pili extended outside the capsule.     

Pili as a mediator of the attachment of gonococci to human erythrocytes.

Isolated pure gonococcal pili were found capable of producing direct agglutination of human erythrocytes. Four different strains of gonococci were compared, and hemagglutination was produced by isolated pili or piliated gonococci but not by nonpiliated gonococci of each strain. Pili from each of the four strains were antigenically distinguishable using antisera specific for pili to agglutinate piliated gonococci, form precipitin lines in Ouchterlony immunodiffusion, or inhibit hemagglutination caused by purified pili or piliated gonococci. However, these tests also demonstrate some shared antigenicity among pili. Shared antigens among the four pili types were quantitated at less than or equal to 2.5% by radioimmunoassay. Inhibition of hemagglutination was most marked with antiserum to the homologous pili type. Inhibition of hemagglutination by antiserum to heterologous pili suggested that shared antigens on pili from B and 2686 strains of gonococci are located near the erythrocyte attachment moiety of B strain pili and removed from the attachment moiety of 2686 strain pili. These results suggest that antigenic heterogeneity of pili will prove an important factor in any efforts to use pili as a vaccine for gonorrhea.     

Model for invasion of human tissue culture cells by Neisseria gonorrhoeae.

A tissue culture model has been developed for studying the ability of Neisseria gonorrhoeae to invade eucaryotic cells. The cell line HecIB, a human adenocarcinoma endometrial cell line, was found to support gonococcal invasion. The bactericidal antibiotic gentamicin was used to kill those bacteria that had not entered the HecIB cells, allowing us to quantitate internalized bacteria. Kinetic studies showed an increase in the titer of gentamicin-protected gonococci at 4 h postinfection followed by a decrease; a second increase occurred after 6 h. The state of piliation did not affect the degree of invasion when the bacteria were spun down onto the monolayer. Gonococcal invasion was inhibited when the HecIB cells were preincubated with cytochalasin D before bacterial infection. N. lactamica was used as a negative control. No internalized N. lactamica cells were observed by electron microscopy. Electron microscopy documented the intracellular location of the gonococci in HecIB cells and the eventual destruction of the invaded HecIB cells. After 24 h, clusters of gonococci encased in a matrix of cell debris were observed.     

Relationship of pili to colonial morphology among pathogenic and nonpathogenic species of Neisseria.

Growth in colonies with type 1 morphology and the presence of pili are characteristics that have been associated with virulence of gonococci for humans. To determine whether the presence of pili per se might be responsible for colony type 1 morphology, the relationship of pili to colony type was examined in various species of Neisseria. Short pili (175 to 210 nm in length) were seen only on nonpathogenic neisseria, whereas long pili (up to 4,300 nm) were seen on organisms of both nonpathogenic and pathogenic species. Although long pili, similar to those found on organisms from high-domed, type 1 colonies of gonococci, were observed on organisms from high-domed, type 1 colonies of nonpathogenic Neisseria species, they were also observed on low-convex, type 4 colonies of meningococci and nonpathogenic neisseria. Among meningococci there was no difference in the morphology of colonies consisting of organisms with many long pili and colonies consisting of organisms that completely lacked pili. Thus, there was no consistent relationship of pili to colonial morphology. Unless the pili of N. gonorrhoeae are unique among Neisseria species in their influence on colonial morphology, it is likely that factors other than pili determine colony type 1 morphology of gonococci. Whether these same factors, either alone or in conjunction with pili, are also responsible for gonococcal virulence warrants further investigation.     

Role of anti-pilus antibodies in host defense against gonococcal infection studied with monoclonal anti-pilus antibodies.

Several monoclonal antibodies directed against gonococcal pili have been used to investigate the potential contribution of anti-pilus antibodies to host defense against gonococcal infection. Included were two antibodies (SM1 and SM2) which reacted with conserved determinants present on pili from all strains tested and others which exhibited antigenic specificity. Immunoblotting experiments revealed that antibodies SM1 and SM2 recognize epitopes on two different peptides derived by CNBr cleavage of alpha-pili from Neisseria gonorrhoeae P9-2. All antibodies used were capable of activating complement, as shown by their ability to bind Clq, and one type-specific antibody was effective in complement-mediated bactericidal killing. Antibodies directed against at least some pilus epitopes may therefore contribute to bactericidal activity during the course of natural infection. The opsonic effect of type-specific antibodies was demonstrated by their ability to stimulate luminol-dependent chemiluminescence of human polymorphonuclear leukocytes and promote phagocytic killing of variant P9-2. Phagocytic killing in the presence of each monoclonal antibody paralleled the increase in chemiluminescence, suggesting that for this variant killing was an inevitable consequence of the interaction of polymorphonuclear leukocytes with gonococci opsonized with anti-pilus antibodies. Antibody-mediated chemiluminescence of polymorphonuclear leukocytes was enhanced in the presence of human complement, and a weak opsonic effect was detected with one of the cross-reacting antibodies (SM1) when this system was used. Although cross-reacting antibody SM1 and type-specific antibody SM13 showed considerable differences in biological properties, they were of the same isotype and bound to native pili on intact gonococci in similar numbers and with similar avidity.