Molecular Microbiology (1997) 25(4), 639–647

Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria Helena Ka¨llstro¨m,1 M. Kathryn Liszewski,2 John P. Atkinson2 and Ann-Beth Jonsson1* 1 Microbiology and Tumorbiology Center, Karolinska Institute, S-171 77 Stockholm, Sweden. 2 Department of Medicine, Washington University School of Medicine, St Louis, MO 63 110, USA.

cell layer and disseminate into the bloodstream (McGee et al ., 1983). Pili of N . gonorrhoeae and N . meningitidis belong to the type IV class of pili and are also expressed by Pseudomonas aeruginosa, Dichelobacter nodosus , Vibrio cholerae and Moraxella bovis . Type IV pili are, in addition to colonization of the eukaryotic host, involved in the modulation of target specificity, twitching motility, bacteriophage absorption, pilus retraction and DNA uptake (for review, see Strom et al ., 1994). Pili of pathogenic Neisseria are composed of major pilus subunits (PilE proteins or pilins), a minor pilus-associated protein (PilC) and possibly as yet unidentified components. Most strains carry only one locus for the pilin structural gene (pilE ) but have several copies of silent pil loci (pilS ) that lack the 58 end and promoter region of pilE (for review, see Meyer et al ., 1990). Structural variation of pili arises predominantly from recombination events between silent and expressed pilin gene sequences. Strains of N . meningitidis produce one of two types of pili, which have been termed class I and II (Perry et al ., 1987). The vast majority of isolates possess class I pili, which are structurally similar to those produced by N . gonorrhoeae and carry a pilE gene sequence highly homologous to the gonococcal pilE gene (Olafson et al ., 1985; Perry et al ., 1988; Potts and Saunders, 1988; Virji et al ., 1989). Sequence alterations in PilE of pathogenic Neisseria have profound effects on host and tissue tropism (Virji et al ., 1992; 1993; Nassif et al ., 1993; Jonsson et al ., 1994). PilC, a 110-kDa pilus-associated protein, was shown to alter pilus-mediated adhesion to target cells (Rudel et al ., 1992). PilC is essential for pilus biogenesis (Jonsson et al ., 1991) and acts as an adhesin at the tip of the pilus fibre (Rudel et al ., 1995a). There are two copies of pilC , pilC1 and pilC2 , in most strains. In N . gonorrhoeae strain MS11, piliated bacteria that express either PilC1 or PilC2 both adhere to target cells (Jonsson et al ., 1994; Rudel et al ., 1995b), whereas in N . meningitidis 8013 piliated pilC1 mutants, expressing only PilC2, do not bind to host cells (Nassif et al ., 1994). A number of bacterial pili have been established as having lectin-like properties, including type IV pili of P . aeruginosa, which bind to bGalNAc(1–4)bGal of glycosphingolipids (Lee et al ., 1994; Sheth et al ., 1994), the type 1 fimbriae of Escherichia coli , which bind to mannose residues (Salit and Gotschlich, 1977; Rivier and Darekar,

Summary Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cellsurface receptors. We found that purified pili bound to a 55- to 60-kDa doublet band on SDS–PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria . We proved this hypothesis by demonstrating that piliated, but not non-piliated, gonococci bound to CHO cells transfected with human MCP-cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria . Finally, recombinant MCP protein produced in E . coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell-surface receptor for piliated pathogenic Neisseria . Introduction The genus Neisseria includes two human-specific pathogens, N . gonorrhoeae and N . meningitidis, which cause gonorrhoea/pelvic inflammatory disease and bacterial meningitis/sepsis respectively. The first step in the process of establishing infection is adherence of the bacteria to human epithelial cell receptors. Attachment is mediated by pili, long hair-like structures that extend from the bacterial cell surface (Swanson, 1973; Watt and Ward, 1980). Following the initial pilus-mediated interaction, the bacteria adhere more tightly in order to invade the epithelial Received 30 May, 1997; revised 9 June, 1997; accepted 11 June, 1997. *For correspondence. E-mail [email protected]; Tel. (8) 728 71 74; Fax (8) 34 26 51. Q 1997 Blackwell Science Ltd

m

640 H. Ka¨llstro¨m, M. K. Liszewski, J. P. Atkinson and A.-B. Jonsson 1975), and the E . coli pap-pili with binding specificity to aGal(1–4)b disaccharides (Bock et al ., 1985). We have previously investigated the nature of the gonococcal host cell receptor and found that the pilus-mediated attachment of the bacteria to human tissue is mediated by a eukaryotic receptor with protein characteristics (Jonsson et al ., 1994). In this study, we present evidence that the complement regulatory protein membrane cofactor protein (MCP or CD46) is a human cell-surface receptor for piliated N . gonorrhoeae and N . meningitidis. Piliated, but not nonpiliated, gonococci bound to CHO cells expressing human MCP. Attachment of the bacteria to epithelial cells could be blocked by monoclonal antibodies against MCP and by recombinant MCP protein produced in E . coli . We could also demonstrate a direct interaction between recombinant MCP and piliated Neisseria .

Results

Purified pili bind to a protein of the same size as membrane cofactor protein (MCP) In order to find a pilus receptor candidate, we screened extracts of the human epithelial cervical cell line ME180 with highly purified pili. Whole-cell extracts of ME180 were separated by SDS–PAGE and blotted onto nitrocellulose membranes. The membranes were overlaid with digoxigenin (DIG)-labelled pili of N . gonorrhoeae MS11mk and N . meningitidis FAM20, purified by repeated crystallization and solubilization. Bound pili were detected with antibodies directed against DIG or pili. Both gonococcal and meningococcal pili bound two bands of 55–60 kDa (Fig. 1, lanes 1 and 2). The DIG antibody did not crossreact with ME180 components in the size range of 50– 60 kDa (data not shown). From our search of possible proteins with the unusual migration pattern of a 55– 60 kDa doublet band, only the membrane cofactor protein (MCP or CD46) produced proteins of this magnitude. MCP is a human transmembrane glycoprotein involved in complement regulation (Liszewski et al ., 1991). The protein is expressed as four isoforms that arise by alternative splicing, giving rise to two classes of glycoproteins with molecular masses of 51–58 kDa and 59–68 kDa. Consequently, we probed the SDS–PAGE-separated ME180 whole-cell lysates with a polyclonal MCP antiserum. The MCP antibodies labelled two bands of 55–60 kDa with the same pattern and size compared with the doublet band obtained with pili (Fig. 1, lane 3). A cDNA-encoding MCP has previously been isolated, cloned into expression vectors and transfected into CHO cells (Liszewski et al ., 1991). The untransfected CHO cells did not react with MCP antiserum (Fig. 1, lane 4), whereas the MCP-expressing CHO cells displayed a band of the same size as the

pilus-binding band of ME180 cells (Fig. 1, lane 5). The band in lane 5 was not as strong as the signal obtained with ME180 cells, indicating that the transfected cell line may express lower amounts of MCP compared with human target cells. These data argue that MCP is a pilusbinding protein.

Monoclonal and polyclonal antibodies directed against MCP inhibit bacterial binding to target cells As a number of antibodies directed against MCP were available, we studied whether polyclonal antiserum and some monoclonal antibodies could interfere with adherence of pathogenic Neisseria to target cells. If MCP is a pilus receptor, the antibodies should be capable of preventing adherence of the bacteria. The MCP structure is composed of four extracellular complement control protein repeats (CCPR I–IV) of about 60 amino acids each (Liszewski et al ., 1991). Polyclonal antiserum against MCP, purified on a protein A column, or the monoclonal antibody GB24, specific for the CCPR III and IV region of MCP, blocked binding of the piliated N . gonorrhoeae MS11 and N . meningitidis FAM20 to ME180 cells (Fig. 2). The monoclonal antibody TRA-2-10, specific for CCPR I of MCP, and the monoclonal antibody J4-48 had no effect on adherence of the bacteria to cells. It has been shown previously that GB24 interferes with C3b/C4b binding (Cho et al ., 1991), indicating that the binding site used for neisserial pili on MCP is close to that used by C3b/ C4b. In control experiments, ME180 cells were pretreated with inactivated normal rabbit serum (NRS) or ascites fluid, which did not reduce bacterial binding. All antibodies

Fig. 1. Purified gonococcal and meningococcal pili bind to a protein of the same size as MCP. SDS–PAGE of whole-cell lysates blotted onto a nitrocellulose membrane. Lane 1: ME180 cells overlaid with DIG-labelled MS11mk -u pili, detected with alkaline phosphatase (AP)-conjugated DIG antibodies. Lane 2: ME180 cells overlaid with FAM20 pili, detected with pili antiserum and APconjugated IgG antibodies. Lane 3, 4 and 5 show separated ME180, CHO and MCP-expressing CHO cells (CHO-BC1) respectively. These cell lines were overlaid with MCP antiserum and AP-conjugated IgG antibodies. Only one MCP band was detected in lane 5. In addition to the 55- to 60-kDa doublet band, a 90-kDa band is visible in lane 1. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

Pilus receptor for pathogenic Neisseria 641

Fig. 2. Antibodies directed against MCP inhibit adherence of N . gonorrhoeae MS11mk (P þ )-u and N . meningitidis FAM20(P þ ). The monoclonal antibodies are GB24, directed against CCPR III–IV, TRA-2-10, directed against CCPR I, and J4-48. Polyclonal antiserum (pAb) was raised in rabbits. Ascites fluid and normal rabbit serum (NRS) were used as controls. ME180 cells preincubated with antibodies were overlaid with bacteria for 1 h. After washing, the layer was treated with saponin and spread onto GCB plates. The experiments were repeated on three separate occasions. Inhibition was determined in relation to controls without antibodies. The concentration of functional antibody was 50 mg ml¹1.

bound to the ME180 cells at equivalent levels, as evaluated by visual inspection after detection with fluorescence-conjugated anti-IgG antibodies (data not shown). Since antibodies against MCP prevented attachment of piliated pathogenic Neisseria to target cells, we concluded that MCP is most probably acting as a receptor for these bacteria.

Piliated Neisseria bind to CHO cells expressing MCP To prove directly that MCP is a receptor for piliated Neisseria , we tested whether MCP-expressing CHO cells could support binding of the bacteria. The MCP gene produces different mRNAs through alternative splicing, which give rise to the four different isoforms of MCP: MCP-BC1, MCP-BC2, MCP-C1 and MCP-C2 (Post et al ., 1991; Purcell et al ., 1991). These isoforms produce the same four extracellular CCPRs, a transmembrane region, a cytoplasmic anchor and a cytoplasmic tail. However, they differ in the length and composition of an extracellular serine– threonine–proline (STP)-rich area adjacent to the membrane and also in their cytoplasmic tails. The four isoforms of MCP have previously been characterized, transfected and expressed in CHO cells (Liszewski and Atkinson, 1996). To determine the level of MCP expression, all cells were analysed by flow cytometry using MCP antiserum. The four CHO-MCP isoforms, but not CHO-R cells, containing the MCP-cDNA fragment inserted in the opposite direction, expressed similar levels of MCP (Fig. 3A) (see also Liszewski et al ., 1996). However, the Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

ME180 cell line showed an additional peak with a higher fluorescence intensity, probably representing a subpopulation of cells, with more MCP expressed. Adherence of piliated N . gonorrhoeae MS11mk -u to the MCP-expressing CHO cells was investigated by immunofluorescence microscopy. These studies showed that piliated gonococci adhered to the CHO-BC1 cells by an average of 10 bacteria per cell, and to the CHO-BC2 cells with an average of five bacteria per cell (Fig. 3B). The number of bound bacteria varied from cell to cell. The CHO-C1 and CHO-C2 cells did not bind bacteria at significant levels, i.e. the adherence was less than 0.5 bacteria per cell. The two isoforms, BC1 and BC2, contain 15 additional amino acids in the STP region compared with C1 and C2 (Liszewski et al ., 1991), which could explain the difference in adherence between the CHO-BC and CHO-C cells. Untransfected CHO cells and CHO-R cells did not bind piliated gonococci (Fig. 3B). Further, non-piliated MS11(P ¹n), lacking the 58 end of the pilE gene, did not adhere to CHO cells expressing any of the MCP isoforms. The adherence of gonococci to CHO-BC1 and CHO-BC2 was more fragile than that seen for ME180 cells and required careful removal of unbound bacteria. The fact that CHO cells expressing human MCP bound piliated gonococci is a strong indication that MCP acts as a pilus receptor for pathogenic Neisseria .

Purified MCP inhibits bacterial binding In order to demonstrate an interaction between the MCP protein and piliated Neisseria , we constructed and expressed recombinant MCP-BC in E . coli . The surface-exposed domain of MCP was PCR amplified from a cDNA clone and inserted at the end of the malE gene, which resulted in expression of a maltose binding protein (MBP)–MCP fusion protein. Piliated MS11mk -u and FAM20, pretreated with purified MBP-MCP fusion protein, showed much reduced adherence to ME180 cells, compared with the control in which bacteria were preincubated with only MBP or PBS (Fig. 4).

Piliated Neisseria co-aggregate with Staphylococcus aureus coated with recombinant MCP The interaction between pili and MCP was evaluated by incubating piliated Neisseria and Staphylococcus aureus coated with the MBP–MCP fusion protein. S . aureus strain Cowan 1, expressing protein A on the surface, was incubated with MBP antisera and then coated with purified MBP–MCP fusion protein. By mixing the coated S . aureus with piliated N . gonorrhoeae MS11mk -u or N . meningitidis FAM20 on a glass slide, we observed co-aggregation of the bacteria after 3 and 4 min respectively (Table 1). Non-piliated bacteria and MBP–MCP-coated S . aureus ,

642 H. Ka¨llstro¨m, M. K. Liszewski, J. P. Atkinson and A.-B. Jonsson

Fig. 3. Flow cytometry assay and bacterial adherence to CHO cell lines that express human MCP. A. FACS analysing histogram showing the level of MCP expression (10 000 cells per analysis). B. Adherence of piliated MS11mk (P þ )-u to CHO cells expressing the different isoforms of MCP. Bound bacteria were detected with B2 antisera and FITC-conjugated antibodies against IgG. The average number of bacteria per cell was counted on 100 cells by visual inspection in a fluorescence microscope. All experiments were repeated at least three times. The adherence of MS11mk (P þ )-u to CHO-BC1 is also demonstrated by an immunofluorescence picture. The piliated bacteria do not bind to the CHO-R control cells.

piliated bacteria and S . aureus coated with only MBP, or piliated Neisseria and uncoated S . aureus , did not coaggregate. These data indicate that piliated Neisseria and MCP have affinity for each other.

Discussion

N . gonorrhoeae and N . meningitidis are two human-specific pathogens. Pili of these organisms play a central role in

Table 1. Co-aggregation of Neisseria and MCP-coated S. aureus.

S. aureus a MBP b Incubation time

MCP-coated S. aureus Incubation time

Relevant phenotype Strain

Pili

PilE

2 min

3 min

4 min

5 min

5 min

5 min

MS11 (P þ ) MS11 (P ¹ n) FAM20 (P þ )

Yes No Yes

Yes No Yes

¹ ¹ ¹

þ ¹ ¹

þ ¹ þþ

þþ ¹ þþ

¹ ¹ ¹

¹ ¹ ¹

¹, No co-aggregation; þ, clear co-aggregation; þþ, strong co-aggregation. a. The strain was mixed with uncoated S. aureus. b. The strain was mixed with S. aureus coated with pure MBP. These data represent the average of three independent experiments. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

Pilus receptor for pathogenic Neisseria 643

Fig. 4. Recombinant MCP inhibit adherence of (A) MS11mk(P þ )-u and (B) FAM20(P þ ) to ME180 cells. Bacteria preincubated with various amounts of MBP–MCP fusion protein (black columns) or MBP protein (hatched columns) were added to ME180 cells for 1 h. After washing, the layer was treated with saponin and spread onto GCB plates. Percentage bacterial adherence was calculated as follows: 100× colony-forming units (cfu) per well/cfu per well with no protein added. The experiments were repeated three times.

the colonization of epithelial and endothelial cells (Kellogg et al ., 1968; Virji et al ., 1992). The adherence to epithelial cells is dependent on a pilus adhesin, called PilC, and on variations in the major pilus subunit, PilE (Virji et al ., 1992; 1993; Nassif et al ., 1993; Jonsson et al ., 1994; Rudel et al ., 1992; 1995a,b). Our previous studies have shown that piliated N . gonorrhoeae bind to a wide variety of human tissues and cell types and that the receptor is sensitive to proteolysis (Jonsson et al ., 1994). This receptor probably determines the host range of wild-type piliated Neisseria . In this paper, we have identified membrane cofactor protein (MCP) as a cellular receptor for piliated N . gonorrhoeae and N . meningitidis. MCP has previously been shown to be a receptor for measles virus (Do¨rig et al ., 1993; Naniche et al ., 1993; Manchester et al ., 1995) and Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

for the M protein of Streptococcus pyogenes (Okada et al ., 1995). MCP, also called CD46, has been found in virtually every human cell/tissue with the exception of erythrocytes. Among others, it has been demonstrated in endometrium, cervical mucosa, fallopian tubes (Jensen et al ., 1995), respiratory tract (Varsano et al ., 1995) and as a secreted soluble form of 68 kDa in the blood (Seya et al ., 1994). The role of soluble MCP in the blood and its eventual function for neisserial pathogenesis is still unknown. It is well established that piliated Neisseria bind erythrocytes. Two binding functions have been associated with the gonococcal pilus, one for erythrocytes and one for epithelial cells (Rudel et al ., 1992). Therefore, another pilus receptor exists on erythrocytes. MCP is a C3b/C4b-binding transmembrane glycoprotein and a member of the regulators of complement activation (RCA) family (Liszewski et al ., 1991). The MCP-encoding gene is located on the human chromosome number 1 (q3.2), together with the other genes in the RCA family (Liszewski et al ., 1991). Proteins in this family protect the host from attack by its own complement system by regulating the activation state and deposition on host cells of complement components. The MCP structure contains, starting at the N-terminus, four complement control protein repeats (CCPRs) of about 60 amino acids each, a Ser– Thr–Pro (STP)-rich region near the membrane, an area of undefined function, a transmembrane hydrophobic domain, a cytoplasmic anchor and a cytoplasmic tail. The CCPR I, II and IV are N-linked glycosylated, and the STP region is often heavily O-linked glycosylated (Liszewski et al ., 1991). The length and amino acid content of MCP varies owing to alternative splicing of the STP region. The cytoplasmic tails: cyt 1 (16 amino acids) is expressed by isoforms BC1 and C1; and cyt 2 (23 amino acids) is expressed by isoforms BC2 and C2. The bacteria bound less to CHO-BC1 (10 bacteria per cell) than to ME180 (60 bacteria per cell). It is possible that there is an additional factor(s) needed for efficient adherence to human cells. It could be some sort of coreceptor or a variation in carbohydrates that facilitates neisserial binding. The MCP expressed in CHO cells may be differently glycosylated, which could affect pilus binding simply by sterical hindrance. We found that careful washing procedures were necessary in order to detect the binding of MCP-expressing CHO cells. Piliated MS11mk -u did not adhere to CHO-C1 or CHO-C2 cells. It is possible that the 15 amino acids present in the STP-rich region of BC1 and BC2, but absent in C1 and C2, are involved in the molecular recognition of the pilus molecule. There are also functional differences between BC and C isoforms. BC isoforms bind C4b more efficiently than the C isoforms and also provide more protection against the classical pathway-mediated system (Liszewski and Atkinson, 1996). The STP domain of the BC isoforms might

644 H. Ka¨llstro¨m, M. K. Liszewski, J. P. Atkinson and A.-B. Jonsson facilitate the interaction with pili just as it facilitates the interaction with C4b. We found that both the monoclonal antibody GB24 directed against the CCPR III and IV of MCP and the purified recombinant MCP inhibited attachment of piliated Neisseria to target cells. Similar results were obtained for N . gonorrhoeae FA1090 (data not shown). Piliated MS11mk -u or FAM20, co-aggregated with S . aureus coated with recombinant MCP, clearly showed an interaction between the MCP protein and the pili. Since the recombinant MCP was produced in E . coli , the protein is not likely to be glycosylated. Therefore, it is likely that a protein domain of MCP interacts with pili. The correlation between neisserial pathogenesis and deficiencies in complements is well known. Individuals with deficiencies in complement C3 experience severe infection in 70% of cases, often early in life and caused particularly by N . meningitidis. Furthermore, individuals with deficiencies in complement factors C5–C9 have 50–60% increased incidence of a meningococcal disease (Cunliffe et al ., 1995). Patients with properdin deficiency (PD) have approximately 50% elevated incidence of meningococcal disease and characteristically develop fulminating septicaemia. Chronic meningococcaemia is also recognized as occurring in association with PD (Figueroa and Densen, 1991). Properdin works against the effects of negative regulators in the complement pathway (such as MCP) by, for example, inhibiting the cleavage of C3b by factor I (Liszewski et al ., 1996). This is an example of an area in which the complement system is involved in the neisserial pathogenesis. Now, it is also clear that the bacteria use MCP as a receptor for pilus-mediated adherence. Receptors for the invasion-associated opacity (Opa) outer membrane proteins of meningococci and gonococci have been reported. Recently, Virji et al . (1996a,b) have shown that an immunoglobulin superfamily cell adhesion molecule, belonging to the carcinoembryonic antigen (CEA, CD66) family, is a receptor for the majority of the meningococcal and gonococcal Opa proteins. Also, a small portion of gonococcal Opa proteins interact with cell surface-associated heparan sulphate proteoglycan receptors (Chen et al ., 1995; van Putten and Paul, 1995). The initial attachment between pili and MCP may be a short event, simply leading to transduction of signals inducing bacteria–cell attraction and internalization. Since MCP is a transmembrane protein with a cytoplasmic tail, it may transduce signals from the bacteria into the eukaryotic cell. Investigations are currently in progress concerning the identification of the responsible pilus component(s) that act as a ligand for MCP. In the future, it would be informative to see whether transgenic mice, expressing MCP, are capable of developing disease.

Experimental procedures

Bacterial strains and growth conditions N . gonorrhoeae MS11mk(P þ ) and MS11mk(P ¹n) (Swanson et al ., 1986) were obtained from Dr M. Koomey. The MS11mk(P þ ) strain sample used in our studies is designated MS11mk (P þ )u. Piliated and non-piliated variants were distinguished by colony morphology under a binocular microscope. Meningococcal strain FAM20 (Moore and Sparling, 1995), expressing class I pili, was obtained from Dr Janne Cannon. Bacteria were grown on GCB-agar plates, containing Kellogg’s supplement (Kellogg et al ., 1968) at 378C in 5% CO2 , and passaged daily. E . coli strain TB1 (Biolabs) and Staphylococcus aureus Cowan 1 (Rankin et al ., 1992) have been described previously.

Cell lines and growth conditions ME180 (ATCC HTB33), an epithelial-like human cell line from cervical carcinoma, was maintained in McCoy’s 5A medium supplemented with 10% inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. CHO, a Chinese hamster ovary cell line, was maintained in nutrient mixture (F12) supplemented with 10% FBS and 2 mM L-glutamine. The transfected CHOMCP cells were maintained in nutrient mixture (F12) supplemented with FBS, L-glutamine and G418 (Geneticin). All cell lines were grown at 378C, 5% CO2 and occasionally grown in penicillin/streptomycin (PEST) containing medium to prevent contamination. All the experiments were carried out free from FBS, antibiotics and L-glutamine. Media and growth supplements were purchased from Life Technologies. Cell culture materials were purchased from Costar, except the chamber slides, which were from Nunc. Flow cytometry analysis of cell lines was carried out according to McClelland et al . (1987). Briefly, cells were labelled with polyclonal antisera against MCP (diluted 1:100) and FITC-conjugated antibodies against IgG (diluted 1:100). Cells were excited on FACScan with a 15-mW laser at 488 nm. Results were presented as a histogram of the number of cells (10 000 per analysis) vs. the logarithmic scale of fluorescence intensity.

Immunoblots and antibodies Confluent layers of cells were washed in medium, scraped off from the plastic bottle, washed twice in PBS, pH 7.4, and thoroughly resuspended in PBS. The whole-cell extracts were electrophoresed on 12% SDS–polyacrylamide gels for the detection of MCP (Laemmli, 1970). All samples were boiled at 1008C before electrophoresis. Proteins were transferred onto nitro-cellulose membranes (Towbin et al ., 1979) and overlaid with DIG-labelled highly purified pili (10 mg ml¹1 ), or unlabelled pili, and with polyclonal antiserum directed against MCP (diluted 1:1000) for 1 h at room temperature. Bound pili or MCP antibodies were detected with alkaline phosphatase-conjugated monoclonal antibodies against DIG (diluted 1:1000; Boehringer Mannheim) or IgG (Bio-Rad) respectively. Pili preparations of MS11mk -u and FAM20 were performed according to Brinton et al . (1978). The pili preparation used in overlays was crystallized and solubilized five times and contains small amounts of minor proteins detected in Coomassie blue-stained gels (shown in Jonsson et al ., Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

Pilus receptor for pathogenic Neisseria 645 1991). Pili antisera were raised in rabbits as described before (Jonsson et al ., 1991). The monoclonal antibodies GB24 and TRA-2-10, directed against MCP, and a polyclonal antiserum against MCP have been described (Seya et al ., 1988; Cho et al ., 1991) and showed no cross-reactivity with other cellular epitopes. The monoclonal antibody J4-48 was purchased from Serotec. The protein A column-purified antibodies were stored at ¹208C. Ascites fluid, NRS and alkaline phosphatate-conjugated or FITC-conjugated goat anti-rabbit IgG was obtained from Sigma. The B2 antiserum directed against non-piliated pathogenic Neisseria has been described previously (Jonsson et al ., 1991).

were washed three times and finally resuspended in 200 ml of PBS (pH 7.4). To each co-aggregation assay, 10 ml of coated Cowan 1 bacteria was mixed with 10 ml of a fresh gonococcal suspension of OD600 ¼ 0.5, poured on a glass slide and gently moved in circles. The co-aggregation was visualized by eye inspection and under an inverted light microscope.

Inhibition with antibodies or MBP–MCP fusion protein

The cells were maintained in 4-well chamber slides for 2– 3 days until each chamber contained 1 × 105 cells. The monolayers were carefully washed three times in medium, prewarmed to 378C. Bacteria grown for 18–20 h were suspended in medium, added to the cell layer (2 × 107 bacteria/well) in a volume of 500 ml and incubated for 90 min at 378C. The infected cell layers were washed for 3 × 5 min, overlaid with B2 antisera (diluted 1:500) and incubated at 378C for 1 h. The cells were washed as above, incubated for 1 h at 378C with a FITC-conjugated monoclonal anti-IgG (diluted 1:1000) and finally washed for 3 × 5 min. Bound bacteria were counted by visual inspection in a fluorescence microscope.

For the inhibition of bacterial binding, antibodies were diluted in medium to a volume of 50 ml and added to a monolayer of cells in 48-well plates at 378C, 5% CO2 for 1 h. A suspension of piliated bacteria, grown for 18–20 h, was added and again incubated for 1 h, allowing competition between the antibodies and the bacteria for binding to MCP. The cell layers were washed three times in medium, treated with 1% saponin for 5 min and spread onto GCB plates. In inhibition assays with recombinant MCP protein, various concentrations of MBP–MCP fusion protein (0–10 mg) or MBP protein in a volume of 100 ml were mixed with 50 ml of bacterial suspension (OD600 ¼ 0.5) and incubated at 378C for 30 min. The whole mixture (150 ml) was added to monolayers of cells maintained in 24-well plates, 350 ml of medium was added and incubation was carried out at 378C for 1 h. The cell layers were washed three times in medium, treated with 1% saponin for 5 min and spread onto GCB plates. The inhibition experiments were repeated three times in triplicate.

Construction of the fusion protein of MBP–MCP

Acknowledgements

A cDNA clone encoding MCP-BC1 has been isolated and characterized previously (Liszewski et al ., 1991). Primers used for the amplification of the complete extracellular region of MCP from the cDNA clone were 58-CGGAATTCCTGTGAGGAGCCACCAACATTTG-38 (including a restriction site for Eco RI) and 58-CCGCTCGAGTCCAAACTGTCAAGTATTCCTTC-38 (including a restriction site for Xho I). Dynazyme DNA polymerase was used in the following polymerase chain reaction (PCR) cycles: one cycle of 958C for 5 min, 30 cycles of 948C for 1 min, 458C for 2 min and 728C for 3 min and finally one cycle of 728C for 10 min. The MBP–MCP fusion protein was constructed in pMAL-c2 vector and produced according to the manual (BioLabs). Briefly, the MBP– MCP fusion protein was induced in E . coli TB1 host cells with 0.3 mM IPTG at 378C for 3 h. Cells were harvested, sonicated and centrifuged at 9000 × g for 30 min. The crude extract was purified over an amylose column. The column was washed and the protein was eluted with 10 mM maltose.

This work was supported by grants from the Swedish Medical Research Council (Dnr 10846), Swedish Society of Medicine, ˚ ke Wibergs Stiftelse, Anders Magnus Bergvalls Stiftelse, A Otto Sva¨rds Stiftelse and Sven och Dagmar Salens Stiftelse.

Binding of bacteria to epithelial cells

Co-aggregation of MCP-coated S. aureus Staphylococcus aureus strain Cowan 1 was grown at 378C overnight on horse blood agar plates. Bacteria were scraped off, washed twice in PBS (pH 7.4) and resuspended to OD600 ¼ 1. Equal amounts (500 ml) of Cowan 1 suspension and antisera against MBP (diluted 1:100) (Biolabs) were mixed and left at 48C overnight, washed three times in PBS (pH 7.4), mixed with 20 mg of MBP–MCP fusion protein (300 ml) and incubated at 48C for 4 h. The coated bacteria Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 639–647

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