Assessment of attachment of Neisseria gonorrhoeae to HeLa cells by double radiolabeling.

Attachment of Neisseria gonorrhoeae to HeLa cells was assessed by a technique using double radioisotopic labeling. Piliated, virulent bacteria from colony type 2 attached to HeLa cells to a greater extent than nonpiliated, avirulent bacteria from colony type 4. Maximal attachment rates for bacteria from both colony types occurred during the early incubation periods at 37 degrees C, and the HeLa cells appeared saturated at 4 h. Attachment was maximum at pH 6.5 and dependent upon the multiplicity of infection. Treatment of the HeLa cells with trypsin diminished the degree of attachment, but this effect substantially disappeared by 24 h after trypsin treatment. Scanning electron microscopy revealed bacteria of colony types 2 and 4 adhered to the HeLa cell surface. Thin-section transmission electron microscopy showed that bacteria were associated with the surface of the HeLa cell but not ingested.     

Type IV pili of Neisseria gonorrhoeae influence the activation of human CD4+ T cells

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea, and infection with this organism is typically associated with an intense inflammatory response. In many individuals, however, the infection is asymptomatic and can progress to serious secondary complications. The type IV pili of Neisseria gonorrhoeae mediate binding of the bacteria to host cells and are involved in cellular signal transduction. In these studies we have demonstrated that gonococcal pili influence human CD4+ T cells by using isogenic strains of N. gonorrhoeae with piliated and nonpiliated phenotypes. To determine the impact of piliation on the cellular status, we examined the expression of activation markers, cellular proliferation, and the production of cytokines after infection. The activation marker CD69 showed significantly increased expression on cells infected with the piliated strain, and this expression was dependent on costimulation of the T-cell receptor. Infection with piliated gonococci also altered T-cell proliferation and influenced the production of the regulatory cytokine interleukin-10. PilC, the putative pilus adhesin, was also observed to influence cellular activation but had no impact on the proliferation of cells further indicating that pilus-mediated adhesion is important in gonococcal stimulation of CD4+ T cells. These results show that the piliation status of gonococci influences CD4+ T-cell activation and that the adhesion mediated by pilus components aids in the regulation of the T-cell response to N. gonorrhoeae.     

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.     

Gonococci-human polymorphonuclear leukocyte interactions: metabolic studies associated with attachment and ingestion

Utilizing monolayers of human polymorphonuclear leukocytes, optimal conditions for attachment and ingestion of Neisseria gonorrhoeae were determined. Both attachment and ingestion were optimal at 36 degrees C when a bacteria-leukocyte ratio of 100:1 was employed. After 30 min of incubation, log-phase viable type 2 gonococci were attached to 90% of leukocytes, whereas log-phase viable type 4 gonococci were ingested by 80 to 90% of cells. Respiratory inhibitors had no effect on attachment or ingestion, whereas glycolytic inhibitors blocked ingestion but did not affect attachment of gonocci to the leukocyte surface. Inhibition was dose dependent and partially reversible. The oxidative metabolism of leukocytes with gonococci attached or ingested was also examined. Attachment of log-phase type 2 gonococci stimulated a minimal increase in glucose oxidation and oxygen consumption by leukocytes in contrast to marked increases by leukocytes that had ingested viable type 4 or heat-killed typed 2 organisms. These results demonstrate that attachment of log-phase type 2 gonococci to the surface membrane does not stimulate significant leukocyte oxidative metabolism nor initiate the phagocytic process.     

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.     

Transcellular Passage of Neisseria gonorrhoeae Involves Pilus Phase Variation

Piliated and nonpiliated Neisseria gonorrhoeae organisms were added on top of confluent layers of HEC-1-B cells, each maintained on a microporous Transwell-COL membrane. The bacteria released into the lower chamber were characterized with respect to the following virulence determinants: pili, which mediate adherence to target host cells; PilE, the major pilus subunit protein; and PilC, which is involved in pilus biogenesis and adherence. Even if >99% of the added bacteria of N. gonorrhoeae MS11 were piliated, bacteria recovered on the other side of the cell layer were predominantly nonpiliated. The recovered clones still expressed unassembled PilE protein, but 50% had lost PilC production. Nonpiliated gonococci, in which the 5′ end of pilE had been deleted, were released in reduced numbers, and piliated recA bacteria added to the cell layer were not released at all, at time points when piliated recA⁺ clones were found at high numbers in the lower chamber. Our data indicate that bacteria producing unassembled PilE protein are selected for during passage through an epithelial cell layer. The finding that the pilE gene sequence was altered in the transmigrants suggests that pilin sequence variation is involved in the transcellular passage of N. gonorrhoeae.     

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.     

Multiple protein differences exist between Neisseria gonorrhoeae type 1 and type 4.

Neisseria gonorrhoeae undergoes a spontaneous conversion from a form which is virulent, competent for DNA-mediated transformation, and piliated (type 1) to a form which is avirulent and neither piliated nor competent (type 4). This phase variation has become thought of as simply a conversion from piliated to nonpiliated. Using the techniques of cell fractionation, two-dimensional electrophoresis, and nonequilibrium pH gradient gel electrophoresis, we identified differences in the expression levels of multiple proteins between type 1 and type 4 cells. A total of 26 type 1-specific (T1S) and 23 type 4-specific (T4S) cytoplasmic or cytoplasmic membrane proteins were identified in O'Farrell two-dimensional gels. Using nonequilibrium pH gradient gel electrophoresis, we detected a minimum of eight T1S outer membrane proteins and four T4S outer membrane proteins which were not detected in the O'Farrell gels. Thus, the conversion from type 1 to type 4 is a complex event involving many different proteins of all cellular locations.     

Comparative virulence of opacity variants of Neisseria gonorrhoeae strain P9.

The invasive properties of nine variants of Neisseria gonorrhoeae strain P9 known to vary in their surface composition have been investigated. Relative virulence was evaluated by their cytotoxic effect on Chang epithelial cell monolayers. Piliated variants P9-2 (with alpha pili) and P9-20 (with beta pili plus protein II) showed increased ability to kill the target cells compared with the prototype P9-1 (lacking pili and additional outer membrane proteins). Two nonpiliated variants, P9-11 (with proteins IIa and IId) and P9-19 (with proteins II and IIc), were also relatively more virulent compared with P9-1. Enhanced attachment was exhibited by both piliated and some nonpiliated variants: beta-piliated P9-20 (with protein II; molecular weight, 29,000) and nonpiliated P9-16 (with protein IIb; molecular weight, 28,000) were the most effective in adherence to the target monolayers.     

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.     

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.     

Analysis of damage to human ciliated nasopharyngeal epithelium by Neisseria meningitidis.

We used an in vitro model of human nasopharyngeal tissue in organ culture to evaluate the effects of Neisseria meningitidis on human cilia and ciliary function. Encapsulated, viable meningococci damaged ciliated epithelium of nasopharyngeal organ cultures, whereas Neisseria subflava, a commensal species, did not. Meningococcus-induced ciliary damage was due to loss of ciliated cells to which meningococci were not attached. Damage was seen with piliated and nonpiliated meningococci and did not appear to require the presence of other specific meningococcal surface proteins. Meningococcal viability was a requirement for both ciliary damage and interactions of meningococci with microvilli of nonciliated epithelial cells. That is, filter-sterilized supernatants from meningococcus-infected organ cultures, heat-killed meningococci at high inoculum, and purified meningococcal or gonococcal lipopolysaccharide at concentrations of 100 micrograms/ml did not damage ciliary activity of nasopharyngeal organ cultures. In contrast, meningococcal lipopolysaccharide at 10 micrograms/ml markedly damaged ciliary activity of human fallopian tube organ cultures, suggesting a selective toxicity of lipopolysaccharide for specific human ciliated cells. Damage to nasopharyngeal ciliated epithelium by N. meningitidis may be an important first step in meningococcal colonization of the human nasopharynx, but meningococcal lipopolysaccharide does not appear to be directly responsible for this toxicity.     

Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells.

An important step in the pathogenesis of Neisseria meningitidis is the crossing of two cellular barriers, one in the nasopharynx and one in the brain. To approach the mechanisms by which this bacterium can achieve these goals, we studied the interactions between N. meningitidis and a monolayer of polarized tight junction-forming T84 cells grown on filter units. A capsulated, piliated, Opa-, and Opc- N. meningitidis strain is shown to be capable of adhering to and crossing this monolayer several orders of magnitude more efficiently than an isogenic nonpiliated derivative. This bacterial interaction does not affect the barrier function of tight junctions, as assessed by (i) the absence of modification of the transepithelial resistance, (ii) the lack of increase of [3H]inulin penetration across the monolayer, and (iii) the absence of delocalization of ZO-1, a tight junction protein. Electron microscopy studies and confocal examinations demonstrated that N. meningitidis (i) induces cytoskeletal rearrangements with actin polymerization beneath adherent bacteria, (ii) is intimately attached to the apical membrane of the cells, and (iii) can be internalized inside cells. Immunofluorescent staining with antipilus antibodies showed evidence that meningococcal piliation was dramatically reduced at later time points of bacterial cell interaction compared to the early phase of this interaction. In addition, adhesive bacteria recovered from an infected monolayer are piliated, capsulated, Opa-, and Opc-, a phenotype similar to that of the parental strain. Taken together, these data demonstrate that following pilus-mediated adhesion, N. meningitidis is involved in an intimate attachment which requires a bacterial component different from Opa and Opc and that meningococci cross a monolayer of tight-junction-forming epithelial cells by using a transcellular pathway rather than a paracellular route.     

Hen Fluorescein-Labeled Gonococcal Lipopolysaccharide Antibody in the Delayed Fluorescent Antibody Technique for the Confirmation of Neisseria gonorrhoeae

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.     

Inhibition of Neisseria gonorrhoeae attachment to HeLa cells with monoclonal antibody directed against a protein II.

This study showed that a protein II (PII) of Neisseria gonorrhoeae FA1090 appeared to act as a mediator of attachment to HeLa cells. Two colony variants of FA1090 were selected. Both gonococcal variants were nonpiliated, but one contained a PII and the other did not. A monoclonal antibody (1090-10.1), which was directed against the PII, inhibited the apparent PII-mediated attachment to HeLa cells. Antibodies produced from clone 1035-4, which had no PII specificity, did not inhibit the attachment and were used as controls. Inhibition of gonococcal attachment by the 1090-10.1 monoclonal antibodies was demonstrated by fluorescent microscopy analysis. Monoclonal antibody 1090-10.1 appeared to cause agglutination of the PII-containing organism. To block the clumping caused by the PII-specific monoclonal antibodies, Fab fragments of goat anti-mouse IgG were incubated with gonococci and the 1090-10.1 monoclonal antibodies. The results showed that the goat anti-mouse IgG Fab fragments partially blocked the agglutination caused by the PII-specific monoclonal antibody. The effect of the 1090-10.1 antibodies on attachment was also determined by monitoring the HeLa cells with attached iodinated gonococci. The monoclonal antibody appeared to inhibit the PII-mediated attachment.     

A Borrelia-specific monoclonal antibody binds to a flagellar epitope.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.     

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.     

Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.

Phase variation of type 1 pili (fimbriae) was studied during the in vivo growth of Escherichia coli in two animal models. In the first, a heavily piliated urinary tract isolate (strain 149) was placed in 1-cm polypropylene chambers sealed with 0.22-micron-pore-size filters. The chambers were surgically implanted intraperitoneally in mice and recovered at various times. Piliation, as determined by electron microscopy and by measuring the minimum number of bacteria needed to produce mannose-sensitive hemagglutination, gradually decreased, and by day 5, most of the organisms were nonpiliated. In the second model, piliated and nonpiliated E. coli phase variants were inoculated into the bladders of BALB/c mice via urinary catheters, and their fate in the lower urinary tract was studied. Viable counts of bladder homogenates revealed that piliated phase variants were significantly more effective in colonizing the bladder urothelium than were their nonpiliated counterparts. Specific antibody to type 1 pili prevented colonization by the piliated organisms. After inoculation of piliated variants, the bladder-associated bacteria gave rise to approximately 80% mannose-sensitive hemagglutination-positive colonies, and immunocytochemistry of bladder lavages revealed large numbers of type 1 piliated bacteria adhering to the bladder transitional cells. Electron microscopy confirmed the presence of piliated bacteria in association with the bladder urothelium. The urine of these mice, whose bladders were colonized with piliated bacteria, frequently showed no growth, and when bacteria were present, strain 149 yielded less than 30% hemagglutination-positive colonies. The results suggest that for some E. coli strains, phase variation may be a factor in determining the fate of the E. coli in the urinary tract and that the urine may not necessarily reflect the bacteriologic state of the bladder mucosa.     

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.     

Comparative genomics identifies the genetic islands that distinguish Neisseria meningitidis,the agent of cerebrospinal meningitis,from other Neisseria species

Neisseria meningitidis colonizes the nasopharynx and, unlike commensal Neisseria species, is capable of entering the bloodstream, crossing the blood-brain barrier, and invading the meninges. The other pathogenic Neisseria species, Neisseria gonorrhoeae, generally causes an infection which is localized to the genitourinary tract. In order to investigate the genetic basis of this difference in disease profiles, we used a strategy of genomic comparison. We used DNA arrays to compare the genome of N. meningitidis with those of N. gonorrhoeae and Neisseria lactamica, a commensal of the nasopharynx. We thus identified sequences conserved among a representative set of virulent strains which are either specific to N. meningitidis or shared with N. gonorrhoeae but absent from N. lactamica. Though these bacteria express dramatically different pathogenicities, these meningococcal sequences were limited and, in contrast to what has been found in other pathogenic bacterial species, they are not organized in large chromosomal islands.