Molecular Microbiology (1992) 6(19), 2785-2795

Expression of the Ope protein correiates with invasion of epitheiiai and endotheiial ceiis by Neisseria meningitidis Mumtaz Virji,^* Katherine Makepeace,^ David J. P. Ferguson,^ Mark Achtman,^ Jasmine Sarkari^ and E. Richard Moxon^ 'Department of Paediatrics, University of Oxford, John Radctiffe Hospital, Oxford OX3 9DU. UK. ^Nuffietd Department of Pathotogy. John Radctiffe Hospitai Oxford 0X3 9DU, UK. ^Max-Ptanck-tnstitut fur Motekutare Genetit729 >729 >729 >729 81 >729 >729 >729 >729

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a.. Phenotypic characterization of variants by immunoblotting and electron microscopy was carried out as described in the Experimental procedures Capsule titres were determined by immuno dot blotting using whole cell lysates of bacteria and anti capsule mAb at 1:2000 dilution ot ascitic fluid. Titres are expressed as the reciprocal of the end point dilution of the antigen. b. Opa proteins were designated Opa A, B or D according to their migration on SDS-PAGE and reactivity with mAbs (Achtman ef al.. 1988:1992; onginal designation: a, b, d), c. Bacterial association with Huvecs was determined 3 h after inoculation with 5 x 1 0 ' bacteria per monolayer. Mean numbers of viable bacteria associated per Huvec in two separate experiments are shown.

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bacteria, like the parent bacteria, did not express assembled pili and gave rise to non-iridescent colonies. Bacteriai interactions with Huvecs are modulated by Ope but not pilin In order to investigate further the role of Ope in the interaction of Nm with Huvecs, several variants were obtained by isolation of single colonies. Variants were analysed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting and several variants were selected which either lacked or expressed Ope and pilin (Fig. 3). Pilin* and pilin" variants were compared since many of the isolated variants of this strain produced pilin which was incorporated in the outer membrane. It was therefore of interest to establish if pilin in these variants contributed to bacterial interactions. Variants b and d were both Ope*, pilin' but expressed pilins of different subunit H . Variant i,1 was Opc~ but expressed OpaA and reacted with mAbs B33 (against Opa) and P322 (against OpaA). Those variants expressing Ope bound to Huvees whether they

variants produced mucoid iridescent colonies with no discernible differences in opaeity when examined using a dissecting microscope with oblique substage lighting. Blake et al. (1989) have shown that eolony opacity is a marker for Opa expression by Nm when grown at 30*^0 but that opaeity variants are not deteetabie when grown at 37"C. We have found that with variants of strain C751 expressing diminished levels of capsular polysaceharide, colony iridescence was lost and transparent and opaque colonies could be readily distinguished as for Ng. even when bacteria were grown at 37'^C.

Opc-expressing bacteria adhere to endothelial cells Variant A7, through phase-variation, gives rise to opaque, transparent or sectored colonies. This phenotype instability corresponded to changes in the levels of Ope expressed {by immunobtotting). Bacteria from a sectored eolony were used to inoculate Huvecs. We compared the opacity and Ope expression in colonies arising from Huvee-assoeiated bacteria and the total population of baeteria in eulture wells. During a 3 h incubation, the proportion of baeteria produeing opaque colonies remained between 40% and 50%. However, the fraction of bacteria that associated with Huvecs during this period produced >99% opaque colonies. These opaque colonies were also Ope* by immunoblotting with mAb to Ope (Fig. 2). Thus from a mixture of bacteria, adherence to Huvecs selected for bacteria that were Ope*. These adherent

Fig. 2. Immunoblotting of colonies of A7 using anti-Opc mAb B306. Colonies arising from bacteria used in inoculation (a), present at the end ot incubation (b), and associated with Huvec monolayers in a 3 h experiment (c). Dark staining Ope' colonies were present in the proportions 47%, 42% and 99% respectively.

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Variant Fig. 3. Derivation of variants, some phenotypic characteristics and their interactions with Huvecs. a. Summary of variant derivation, ©, opaque; O, transparent colony phenotype, *. variant produced diminished levels of Opc (as shown in c), b, c. Western blot analysis of whole-cell lysates of variants using ami pilus mAb SMI and anti-Opa mAb B33 in initial probing (b) and with anti-Opc mAb B306 (c) in subsequent probing of the same blot, d. Western blot ot whole-cell lysates ot variants a, b and i using mAbs SMI, B33 and B306. e. Association of bacteria with Huvecs in a 3 h experiment using 5 x 10' bactena per monolayer. Ranges represent mean values of triplicate estimations from two experiments.

expressed pilin or not (Fig, 3e). Bacteria producing low levelsof Opc (variant d,1) or lacking Opc (variants a and i) failed to adhere. In addition, variant i,1 expressing OpaA also did not Increase bacterial interactions with Huvecs significantly when compared with Opc"^ variants. There was nevertheless a small increase in adherence compared with Opc", Opa~ bacteria (Fig, 3). The colony variants resembled their parent (A7) in the lack of expression of assembled pili. reactivity with a panel of five anti-LPS mAbs. diminished capsule production and were sensitive to bactericidal action of serum (10%) from a human donor: >99% capsule-deficient variants were killed in contrast to capsulate variant Al 0 which was resistant (99% survival) to this serum concentration.

Opa and Opc expression by the variants used is shown in Eig, 3. Opc increases interaction with several cell lines of different origins In order to investigate the tissue- and host-specificity of bacterial interactions mediated by Opc, we investigated association of the variants expressing (variant b) or lacking (variant i) Opc with bovine aorta endothelial cells (BAOEC), two human epithelial cell lines (Chang and Hep-2), and one dog kidney epithelial cell line (MDCK), In all cases examined. Opc* bacteria associated in larger numbers compared to Opc" bacteria (Fig. 4a). However a

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Fig, 4, a. Proportion of bacteria associated with monolayers ot cells of different origins from an inoculum ot 5 x 10^ bacteria per monolayer in a 3 h experiment. Ranges represent mean per cent association obtained from two separate experiments. Shaded coiumns represent Ope*, and blank columns represent Ope' bacteria. Ratio: per cent association of Ope' : Ope" bacteria, b, Internafization ot Ope* [shaded) and Ope" (unshaded) bacteria by eukaryotic cells as determined by estimation of survival in the presence of gentamicin. Ranges represent mean numbers ot viable bacteria per monolayer from triplicate estimations in two experiments,

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when they expressed lesser amounts of capsular polysaccharide than normal because of undefined mechanisms. Opc-expressing bacteria, like Opa-expressing bacteria, exhibited increased agglutination. Although this may contribute to the level of adherence of bacteria, the facts that there are host-dependent differences in adherence and that mAbs to Ope inhibit bacterial interactions are inconsistent with the notion that agglutination is a primary determinant of increased bacterial interactions. In addition to mAbs against Ope, an antiserum raised against Nm which contained anti-Opc antibodies also inhibited bacterial association with Huvecs. Inhibition by the rabbit antiserum was completely abolished if the antiserum was adsorbed with Ope* bacteria and was reduced when adsorbed with Ope" baeteria. Thus Ope appears to be an important determinant in baeterial interaetions but the possibility that other baeterial eomponents also contribute to interactions cannot be ruled out. Olyhoeke/a/. (1991) have eloned the Ope protein in E. coli. We found that this Ope protein, which is expressed on the surface of £. coli as observed by immunogold labelling and electron mieroseopy, failed to increase baeterial adherence to eukaryotie eells (data not shown). It is possible that the eloned protein laeks the native protein configuration required for interaetions with the eukaryotie eells. Alternatively, Ope, although required, may not be suffieient for mediating baeterial interactions with host eells. These possibilities are being investigated. One outcome of Opc-mediated baeterial interaction was that baeteria were internalized by the endothelial and epithelial eells in large numbers. More baeteria were internalized by endothelial eells than by epithelial cells. This may reflect the greater phagoeytie aetivity of endothelial cells (Ryan, 1988) relative to that of epithelial eells. Thus, whereas eapsulate baeteria did not adhere and were not internalized, eapsule-defieient baeteria were internalized when they expressed Ope. Increased adherence of eapsule-defieient baeteria resulting in increased internalization by eells has been observed previously. Piliated gonoeocei, even when they did not express the Opa proteins, adhered in large numbers to Huvecs; large numbers of extracellular and intraeellular baeteria were observed by eleetron microscopy (Virji etai, 1991e). In Nm also, pili (projeeting beyond the capsule) inerease baeterial interaetions with eells, although organisms are not internalized in appreeiable numbers (Virji etai, 1991e). Thus, pilusfacilitated adherenee in capsule-deficient bacteria, but not eapsulate bacteria, may result in internalization. Studies on other baeteria (Isberg, 1991) and on strains of Ng (Makino et ai, 1991) suggesf that pili mediate adherenee but may not result in ingestion by eells. Rather, other surface struetures may eause bacterial entry into eells for which the Yersinia invasin protein has beeome a paradigm, ft is interesting to note that Ope

shares sequenee homology with the Yersinia invasin (Olyhoek et ai., 1991). Further studies are required to determine the influence of assembled pili and Ope in modulation of interaction of eapsule-defieient baeteria with host eells. Nm isolated from disseminated infeetions invariably express eapsule, but some nasopharyngeal isolates may be relatively eapsule-defieient (Craven et ai, 1980). In addition, Masson and Holbein (1985) have suggested that baeteria from rapidly growing Nm euitures are relatively capsule-deficient compared with bacteria grown in conditions of slower growth, and that pH and iron limitation affect capsulation. It is likely that conditions encountered by baeteria in different niehes in the host may profoundly affect capsulation, thus resulting in modulation of baeterial interactions with the host. The data presented here would indicate that bacteria expressing Ope might eolonize and invade the mueosal epithelium more effieiently than other bacteria if the local eonditions downregulated eapsule produetion. Indeed greater numbers of isolates from the nasopharynx of healthy carriers and patients expressed large amounts of Ope protein relative to isolates from bloodstream or eerebrospinal fluid (Aehtman etai, 1991). Thus, initial adherenee and internalization of Nm strains may oceur in a manner analogous to that postulated for Ng (Watt and Ward, 1977), i.e. bacteria may adhere to epithelial cells by pili and a secondary, more intimate, interaetion may oeeur via Ope or Opa proteins. Onee inside the host, eapsulate meningoeoeei may be seleeted out because of their ability to evade host clearance mechanisms, and expression of Ope may beeome less important. In the present studies, OpaAc75i protein did not inerease meningoeoceal interaetions with any of the host eells signifieantly when eompared with the Ope protein. Other studies have shown that an Opa protein which reacts with the mAb recognizing Opa proteins B, D, E, and G, but not with the mAb against OpaA, also increases baeterial interaetions with Huvees only marginally when compared with the Ope protein (data not shown). These observations do not rule out the possibility that yet other variant Opa proteins may be similar in their effect to the Ope protein in meningoeoceal interactions with eukaryotic cells but are consistent with reports that some Opa proteins may be less effective than others in mediating baeterial adherenee (Virji and Everson, 1981; Belland etai, 1992). In summary, the data presented here show correlation between the expression of an outer-membrane protein and invasion of human eells by Nm. Our studies suggest a potential function for Ope in the pathogenesis of Nm infeetions and indieate that it may represent an important virulence faetor for numerous strains of that speeies.

Invasion of eukaryotic cells Experimental procedures Bacteria, growth conditions and characterization Bacteria were grown on brain-heart intusion agar supplemented with 10% heated horse blood at 37"C in 5% CO2 (v/v) in air. Strain C751 was isolated from the cerebrospinal tluid ot a patient with meningitis during the 1982-1988 epidemic period in ttie Gambia (Olyhoek et ai., 1991). Spontaneous Ope. Opa and capsule-deticient variants were obtained by single-colony isolation. Capsule-deficient variant A7 expressed Ope and produced an opaque colony phenotype but showed a high degree of phase shift (to transparent colonies) during culture on agar. Further Ope variants were obtained by single-colony isolation. Bacteria frozen in 10% glycerol broth were used after a single subculture from liquid nitrogen stocks. Protein and lipopoiysaccharide (LPS) profiles ot the variants were compared by SDS-PAGE and silver staining. The variants were further characterized by immunoblotting and enzyme-linked immunosorbent asay (ELISA) as described previously (Virji etal.. 1991c) using mAbs directed against (i) pili (SMI: reacts with meningococcal Class I pilin; (ii) Opa proteins (mAb B33, which reacts with all Opa proteins; Lammel et al., 1988), mAbs P322 and P514 reacting with OpaA and Opa B, D, E, and 6 of strain 0751 and B306 and A222/5b reacting with Ope (Aehtman ef al., 1992, Olyhoek efa/., 1991); (iii) several LPS epitopes (Virji et al., 1991e); and (iv) group A capsule (mAb against capsular polysaccharide was a generous gift from Dr W. D. Zollinger). The relative amounts of capsule present were also determined by rocket gel electrophoresis (Achtman etal.. 1988). The presence ot assembled pili was investigated by TEM (see below).

Serum-sensitivity Capsule-deficiency in the variants of strain C751 was recognized by reduced iridescence of the colonies as well as quantification ot capsule as described above. In addition, since capsule-deficient strains are more sensitive to the bactericidal action of normal serum (f\^asson and Holbein, 1985), this property was examined. Sensitivity of the variants to bactericidal action of 10% human serum was determined as previously described (Virji and Heckels, 1985).

2793

fetal bovine serum (FBS), heparin, penicillin and streptomycin. For a given experiment, cells trom passages 1-4 were seeded in gelatin-coated culture plates and used 3-4 d after reaching confluency. Bovine aortic endothelial cells prepared by coliagenase treatment of isolated bovine aorta were a gift from Dr A. Hussein. The procedure for their isolation and culture has been reported (Hussein etal, 1988). Cells (passage 7) were seeded from a frozen stock and used 2-3 d after reaching confluency. Growth medium was similar to those used for Huvecs except that 10% Ryan growth supplement and 5% FBS replaced FBS and ECGS supplements used for Huvees. Epithelial celt lines. Chang conjunctiva epithelial cells, Hep-2 larynx carcinoma cells and MDCK dog kidney epithelial eells were obtained from Flow Laboratories (UK). Chang and Hep-2 cell lines were cultured in Medium 199 supplemented with 10% heat-inactivated FBS. MDCK eells were cultured in medium MEM supplemented with 1 % non-essential amino acids (Sigma) and 10% FBS. Cells were seeded in tissue culture plates at confluency and used the next day. Growth medium was replaced with medium 199 containing 2% FBS during incubations with bacteria.

Determination of bacteriat association with host cells Endothelial cells were washed prior to infection and the growth medium was replaced with medium without ECGS and antibiotics. Bacterial suspensions were prepared (Virji et al., 1991 e) and added to endothelial or epitheiiai monolayers at the required density. In a standard experiment, 5 x 1 0 ' ' bacteria were added to a monolayer and incubated at 37°C in 5% CO2 (v/v) in air for a period of 3 h, Unassociated bacteria were then washed away and remaining bacteria were enumerated after saponin treatment of the host cells. Released bacteria were serially diluted and plated on agar to determine the colonyforming units. Specific association with host celis was determined after correction for adherence to the exposed well area as described previously (Virji ef al., 1991c). All bacteria were equally resistant to treatment with 1 % saponin.

Determination of internalized bacteria Eiectrophoresis and immunochemicai studies SDS-PAGE for analysis of Ope variants was carried out as described previously (Virji etat.. 1990) using whole-eell lysates and 15% polyacrylamide gels. Gels were silver stained aecording to the Amersham protoeol (RPN.17). Conditions for immunoblotting were as previously described (Virji ef at.. 1991c) and used alkaline-phosphatase-conjugated seeond antibodies with nitroblue tetrazolium and 5-bromo-4-chloro-3indoiylphosphate (Sigma) as substrates.

Cell monolayers were incubated with 5 xiO'' bacteria for 3 h and washed twice to remove excess non-adherent bacteria. A predetermined concentration of gentamicin {200 \ig mP') was added for a period of 1.5 h to eliminate all extracellular bacteria. Cytochalasin D (1-2 Mg ml'^) was used to inhibit phagocytic internalization of baeteria as described previously (Virji ef al.. 1991c). The variants used in these studies were equally sensitive to gentamicin (minimum inhibitory eoneentration 6 ^g ml"^). MIC was determined as described previously (Virji etat., 1991b). Cytochalasin D had no effect on the viability of bacteria.

Tissue culture Endotheliai cetts. Human umbilical vein endothelial cells were isolated and propagated as previously described (Virji ef at.. 1991b). Briefly, endotheliat cells were obtained from umbilical veins by coilagenase treatment and cultured in medium 199 supplemented with endothelial growth supplement (ECGS),

Preadsorption of antibodies Antiserum raised against meningococci was preadsorbed using Ope* and Ope" bacteria. Baeterial suspensions (1O'° bacteria per ml) were incubated with diluted antiserum at 4°C

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with gentle mixing for 1 h. Bacteria were removed by repeated centifugations and antibodies were used in experiments fo investigate their effect on association of Ope"" variants with Huvecs.

Statistical anatysis All estimations were done in triplicate. Relative associafion of different variants was compared in the same experiment. Results from two independent experiments are shown in most cases.

Scanning and transmission etectron microscopy (SEM. TEM) Detailed procedures have been described previously (Virji et ai, 1991b), Briefly, the cells for SEM were grown on 13 mm coverslips and those for TEM were grown on polycarbonate filters (Transwells, Costar), Coverslips and filters used for endothelial cells were precoated with gelatin. In both cases, samples were fixed in giutaraldehyde, post-fixed in osmium tetroxide, and dehydrated. The samples for SEM were criticalpoint dried and sputter-coated prior to examination. For TEM, samples were treated with propylene oxide and embedded in epoxy resin. Thin sections were cut and stained with uranyl acetate and lead citrate prior to examination with Jeoi 100CX TEM. Ruthenium Red staining was as previously described (Virji eM/,, 1991b), Negative staining. Agar-grown bacteria! cultures were suspended in phosphate-buffered saline and transferred to Formvar-coaled grids. Grids were then floated successively on drops of 4% giutaraldehyde in 0-1 M phosphate buffer, distilled water and 1 % methyl tungstate and dried after removal of excess liquid with filter paper. Grids were then examined by TEM. tmmunogotd etectron microscopy. The locations of pilin and Ope on variants of strain C751 were demonstrated by incubation of bacteria on grids with specific antibodies (either SMI for pilin or B306 for Ope) after blocking of non-specific sites with 1 % bovine serum albumin in phosphate-buffered saline. The antibody binding was detected with either Protein A conjugated to 5 nm gold particles (Biocell) or rabbit anti-mouse immunoglobulins conjugated to lOnm gold particles (Biocell), Grids were subsequently negatively stained prior to examination by TEM.

Acknowledgements This work was supported by programme grants from the Wellcome Trust, the Meningitis Trust and the MRC (Grant No. 8325352, E,R,M,) and by Grant AC 36/4-6 from the Deutsche Forschungsgemeinschaft (M.A,), We are grateful to Mrs Carol Alexandrescu tor technical assistance, Dr C, J, Lammel for mAb B33. and to Dr W. D. Zollinger for mAb against capsular polysaccharide.

References Achtman, M. (1990) Molecular epidemiology of epidemic bacterial meningitis. Rev Med Microbioi ^: 29-38,

Achtman, M. (1991) Clonal properties of meningococci from epidemic meningitis. Trans R Soc Trop t\Aed hlyg 85: Supplement 1, 24-31. Achtman, M., Neibert, M,, Crowe, B,A., Strittmatter, W,, Kusecek, B., Weyse, E,, Walsh, M,J., Slawig, B,, Morelli, G., Moll, A,, and Blake, M, (1988) Purification and characterization of eight Class 5 outermembrane protein variants from a clone of Neisseria meningitidis serogroup A, J Exp Med 168: 507525, Achtman, M., Wall, R.A., Bopp, M,, Kusecek, B,. Morelli, G,, Saken, E,, and Hassan-King, M, (1991) Variation in Class 5 protein expression by serogroup A meningococci during a meningitis epidemic, JtnfDis^%A: 375-382. Achtman, M,, Kusecek, B,, Morelli, G,, Eickmann, K,, Wang, J., Crowe, B,, Wall, R.A,. Hassan-King, M,, Moore. P.S,, and Zollinger, W, (1992) A Comparison of the variable antigens expressed by clone IV-1 and subgroup III of Neisseria meningitidis serogroup A. J tnfDis^G5: 53-68. Belland, R,J,, Chen, T., Swanson, J,, and Fischer, S.H. (1992) Human neutrophil response to recombinant neisseriai Opa proteins, tvtot Microbiol6: 1729-1737. Blake, M,S,, MacDonald, C-M,, and Klugman, K,P, (1989) Colony morphology of piiiated Neisseria meningitidis. J Exp Wed 170:1727-1736, Craven, D.E,. Peppier, M,S,, Frasch, CE,, Mocca, L,F,, and McGrath, P,P. (1980) The role of Neisseria meningitidis surface structures in the adherence of case and carrier isolates. In Genetics and tmmunobiotogy oi Pathogenic Neisseria. Proceedings of an EMBO Workshop, Hemavan, Sweden. Danielsson, D,, and Normark, S. (eds). pp. 189-193. Crowe, B,A., Wall, R,A,, Kusecek, B,, Neumann, B,, Olyhoek, T., Abdillahi, G., Hassan-King, M,, Greenwood, B,M,, Poolman, J,T., and Achtman, M, (1989) Clonal and variable properties of Neisseria meningitidis isolated from cases and carriers during and after an epidemic in the Gambia, West Africa, J tnf Dis 159: 686-700, Fischer, S,H,, and Rest, R,F, (1988) Gonococci possessing only certain P,ll outer membrane proteins Interact with human neutrophils. infect Immun 5B: 1574-1579, Hitchcock, P,J, (1989) Unified nomenclature for pathogenic Neisseria species, Ctin Microbiol Rev 2: S64-S65, Hussein, A,, Meyrick, B,, Graber, S,, Berry, Jr, L and Brigham, K,L, (1988) Attenuation of endotoxin-induced cytotoxicity and prostacyclin production in cultured bovine pulmonary artery endothelial cells by phosphodiesterase inhibition. Exp Lung Res-1^:637-654. Isberg, R.R, (1991) Discrimination between intracellular uptake and surface adhesion of bacterial pathogens. Science 252: 934-938, Jahn, S,. Schwab, J,, Hansen, A,, Heider, H,. Schroeder. C , Lukowsky, A,. Achtman, M,, Matthes, H,. Kiessig, S,T,, Volk, H,D,, Kruger, D., and von Baehr. R, (1991) Human hybridomas derived from CD-5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM(Kappa) antibodies, Clin Exp tmmunot 83: 413^17. Lammel, C.J,, Karu, A,E,, and Brooks, G,F, (1988) Antigenic specificity and biological activity of a monoclonal antibody that is broadly cross reactive with gonococcal protein Us, In Gonococci and Meningococci Poolman, J.T, ef at. (eds), Dordrecht; Kluwer Academic Publishers, pp, 737-743, Makino. S,, van Putten, J,P,M,, and Meyer, T,F, (1991) Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells. EMBO J10:1307-1315.

Invasion of eukaryotic cells Masson, L,, and Holbein, B,E, (1985) Influence of environmental conditions on serogroup B Neisseria meningitidis capsular polysaccharide levels. In The Pathogenic NeisseriaeSchoolnik G.K. et al. (eds). Washington, D.C.: American Society for Microbiology, pp. 571-578. Olyhoek, A.J.M., Sarkari, J,, Bopp, M., Morelli, G-, and Achtman, M. (1991) Cloning and expression in Escherichia coli of opc. the gene for an unusual Class 5 outer membrane protein from Neisseria meningitidis. Microb Pathogen 11: 249257. Ryan, U.S. (1988) Macrophage-like properties of endothelial cells. News Physio!SciZ: 93-96. Virji, M., and Everson, J.S. (1981) Comparative virulence of opacity variants of Neisseria gonorrhoeae strain P9, Infect /mmivn 31: 965-970. Virji, M., and Heckels, J.E. (1985) Role of anti-pilus antibodies in host defense against gonococcal infection studied with monoclonal anti-pilus antibodies. Infect Immun 49: 621 -628Virji, M., and Heckels, J-E, (1986) The effect of protein II and pili on the interaction of Neisseria gonorrhoeae with human polymorphonuclear leucocytes, J Gen Microbioi 132: 503512, Virji, M., Weiser, J,N,, Lindberg, A,A., and Moxon, E,R, (1990)

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Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells, Microb Pathogen^: 441-450, Virji, M,, Kayhty, H,, Ferguson, D,J,P,, Alexandrescu, C , Heckels, J,E., and Moxon. E,R, (1991a) Interactions of pathogenic Neisseria wWU human endothelial cells in vitro. In Neisseriae 1990. Achtman. M, etal. (eds), Berlin: Walter de Gruyter. pp, 645-650, Virji. M,, Kayhty, H,. Ferguson, D,J.P,. Alexandrescu, C , and Moxon, E,R, (1991b) Interactions of Haemophilus infiuenzae with cultured human endothelial cells. Microb Pathogen 10: 231-245, Virji, M,, Kayhty, H,, Ferguson, D,J,P,, Alexandrescu, C , Heckels, J,E, and Moxon, E,R, (1991c) The role of piii in the interactions of pathogenic Neisseria with cultured human endothelial cells, Moi Microbiol 5:1831-1841, Virji, M,, Alexandrescu. C , Ferguson. D.J,P,, Saunders, J,R., and Moxon, E.R. (1992) Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells, Moi MicrobiolB: 1271-1279. Watt, P.J.. and Ward, M.E, (1977) The interaction of gonococci with human epithelial cells. In The Gonococcus. Roberts. R.B, (ed,). New York: John Wiley & Sons. Inc, pp. 355-368,