Institut Pasteur de Lille, INSERM U447, Mécanismes moléculaires de la pathogénie microbienne, 1 Rue du Professeur Calmette, 59019 Lille Cedex, France1
Institut National de la Recherche Agronomique, Laboratoire de Génie des Procédés et Technologie Alimentaires, 369 Rue Jules Guesde, 59651 Villeneuve dAscq, France2
Author for correspondence: Franco D. Menozzi. Tel: +33 320871154. Fax: +33 320871158. e-mail: franco.menozzi{at}pasteur-lille.fr
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ABSTRACT |
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Keywords: cytoadherence, purification, virulence
Abbreviations: CHO, chinese hamster ovary; O-glycosidase, glycopeptide -N-acetylgalactosaminidase; PTX, pertussis toxin; FHA, filamentous haemagglutinin; THP, TammHorsfall glycoprotein
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INTRODUCTION |
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Although the physiological function of THP remains uncertain, several observations suggest that it could be involved in urothelial defences against infection; for example Orskov et al. (1980) showed that Escherichia coli with type I fimbriae are trapped by THP. The binding of THP to E. coli type I pili depends on the mannose content of THP (Kuriyama & Silverblatt, 1986
); more recently, in vitro cytoadherence assays showed that THP also inhibits the binding of S- and P-fimbriated E. coli to human renal tubular epithelial cells (Leeker et al., 1997
). Additionally, THP may stimulate polymorphonuclear leukocytes in the course of an inflammatory response (Horton et al., 1990
). Although THP distribution in humans is limited to the kidney, immunoreactive THP-related proteins have been detected at other mucosal surfaces of several vertebrates (Howie et al., 1993
). These observations suggest that THP-like proteins could also be involved in the defence(s) against infectious agents at other mucosal sites, by impeding their cytoadherence. To test the capability of THP to reduce the epithelial adherence of non-uropathogenic bacteria, we investigated the interaction of THP with Bordetella pertussis, the aetiological agent of whooping cough, which colonizes the human upper-respiratory tract. In this study, we show that besides its anti-adherence activity, THP is also able to trap pertussis toxin (PTX).
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METHODS |
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PTX was purified from B. pertussis BPSM culture supernatant using Affi-Gel Blue chromatography (Bio-Rad) followed by fetuin-Sepharose (Sigma) affinity chromatography, as described by Sekura et al. (1983) . Purified PTX was dialysed against PBS and stored in 500 µl aliquots at -30 °C.
Production of antisera.
Five hundred microlitres of purified PTX or THP antigen [100 µg (ml PBS)-1] were mixed with 500 µl of a monophosphoryl lipid A solution (MPL+TDM Adjuvant System; Sigma) prepared according to the manufacturers recommendations. BALB/c mice were immunized with 300 µl of the antigen suspension in the following way. For each animal, 200 µl of the antigen suspension was given intraperitoneally, followed by two subcutaneous injections of 50 µl each. The animals were boosted the same way with the same amounts of antigen 4 and 6 weeks later; the antisera were collected 3 weeks after the last boost.
Enzymic deglycosylation of THP.
Purified human THP at 5 mg ml-1 in PBS was treated for up to 15 h at 37 °C with 0·5 U N-glycosidase F (EC 3.5.1.52; Roche) per mg THP or with 1 mU glycopeptide -N-acetylgalactosaminidase [O-glycosidase; EC 3.2 . 1.97; Roche] per mg THP.
Covalent coupling of THP to Sepharose.
Native or deglycosylated human THP was covalently coupled to CNBr-activated Sepharose 4B (Pharmacia) as follows. Three grams of CNBr-activated Sepharose 4B was first washed with 50 ml ice-cold 1 mM HCl followed by 150 ml PBS. The gel was then mixed with 10 ml of THP (5 mg ml-1) in PBS and incubated for 5 h at room temperature. After blocking of the remaining active groups by incubation for 1 h at room temperature in 25 ml 0·2 M glycine, pH 8·0, the THP-Sepharose gel was stored at 4 °C in PBS containing 20% (v/v) ethanol. For both the native and the deglycosylated forms of human THP, coupling efficiencies were between 95 and 100%.
Chromatography of B. pertussis culture supernatants onto THP-Sepharose.
Late-exponential phase supernatants from 800 ml B. pertussis cultures were collected by centrifugation at 10000 g for 15 min at 4 °C and were then loaded at 2 ml min-1 onto 5 ml PBS-equilibrated THP-Sepharose packed in a 1 cm diameter column. After extensive washing, retained material was step-wise eluted using successively PBS+3 M NaCl, PBS+0·5 M methyl -D-mannopyranoside, PBS+3 M urea and PBS+6 M urea. Eluted material was collected in 1 ml fractions and stored at -30 °C until further use.
B. pertussis cytoadherence assays.
Human pulmonary epithelial cells (A549; ATCC CCL-185) and canine kidney epithelial cells (MDCK; ATCC CCL-34) were cultured in 75 cm2 Roux flasks (Costar) using RPMI medium supplemented with 50 µg streptomycin ml-1 (Sigma), 2 mM L-glutamine (Gibco) and 10% (v/v) heat-inactivated fetal bovine serum (Gibco). The day before the adherence assay, the wells of 24-well tissue culture trays (Nunc) were seeded with 2x105 exponentially growing epithelial cells resuspended in 2 ml of serum-supplemented RPMI medium. Before the addition to each well of 500 µl of RPMI medium containing 4x106 35S-labelled B. pertussis cells, corresponding to an m.o.i. of 10, the epithelial cells were washed twice with 1 ml PBS. At the end of 2 h incubation at 37 °C, non-adherent bacteria were removed by three washes with 1 ml PBS and the cells were lysed by adding 500 µl distilled water containing 0·1% (w/v) SDS. The radioactivity associated with the cellular lysates was quantified using a liquid scintillation counter (model LS 6000SC; Beckman). Bacterial adherence was expressed as the percentage of radioactivity associated with the cell lysates relative to that present in the inoculum. The adherence assays were performed in quadruplicate and the results are expressed as the mean±SD.
Flow cytometry analysis of the B. pertussisTHP interaction.
Approximately 108 exponentially growing B. pertussis cells were harvested by low-speed centrifugation and washed twice in 2 ml PBS. Bacteria were then resuspended in 1 ml PBS containing 100 µg purified human THP and incubated for 30 min at room temperature. After three washes with PBS, the cells were incubated for 1 h at room temperature with mouse-anti-human THP antiserum diluted 500-fold in PBS. The bacteria were then washed three times with PBS and incubated for 30 min at room temperature in 1 ml of FITC-conjugated donkey-anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted 500-fold in PBS. After three final washes with PBS, the bacteria were fixed with 500 µl of 4% paraformaldehyde. Cell-associated fluorescence (expressed as the percentage of FITC-labelled B. pertussis) was quantified by flow cytometry using an EPICS Elite cytometer (Coulter); fluorescence associated with bacteria that were not incubated with THP was used for background determination.
Scanning electron microscopy.
B. pertussis BPSM grown for 48 h on BordetGengou agar plates was scraped using an inoculating loop and carefully resuspended at a density of 107 cells ml-1 in PBS or in PBS supplemented with 100 µg THP ml-1. The bacteria were then directly fixed for 5 h at room temperature in a 1·25% (v/v) glutaraldehyde solution prepared in 100 mM sodium cacodylate buffer, pH 7·0. Aliquots containing approximately 106 cells were filtered through a 25 mm diameter/0·2 µm porosity Anodisc (Whatman); the filters were then rinsed five times for 10 min in 25 ml cacodylate buffer. Post-fixation was performed for 3 h in a 1% OsO4 solution prepared in cacodylate buffer, followed by five washes in ultrapure water. The samples underwent progressive dehydration by successive soaking in 50, 70, 95 and 100% ethanol. Soaking in isopentyl acetate was performed before critical-point drying in CO2, using an EMSDCOPE CPD 750 apparatus. The filters were then attached to large scanning-electron-microscopy stubs and coated with gold/palladium by cathodic spreading in a Polaron E 5100 coater. Sample observation and microphotographs were done in a JEOL JSM35CF scanning electron microscope, operating at a voltage of 10 kV.
Other analytical procedures.
Chinese hamster ovary (CHO) cells (CHO-K1; ATCC CRL-9618) were cultured in monolayers using RPMI 1640 medium containing 10% (v/v) fetal bovine serum. The clustering effect of PTX on CHO-K1 cells was monitored in flat-bottom microtitre plates (Dynatech Laboratories) as described by Hewlett et al. (1983) , using purified PTX at concentrations ranging from 2 µg ml-1 to 1 ng ml-1.
SDS-PAGE was performed according to Laemmli (1970) by using a 4% stacking gel and a 15% separating gel. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Merck).
The transfer of proteins from SDS-polyacrylamide gels to nitrocellulose sheets (BA 85; Schleicher & Schuell) was performed as described by Towbin et al. (1979) . Immobilized PTX was probed with a murine-anti-PTX antiserum diluted 1000-fold and developed with alkaline phosphatase-linked goat-anti-mouse immunoglobulin G (ProtoBlot System; Promega).
Protein concentrations were determined with the bicinchoninic acid protein assay (Pierce), using BSA as a standard.
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RESULTS |
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THP binding to virulent and avirulent B. pertussis
To investigate whether the THP ligand is part of the general virulence regulon under the control of the BvgAS two-component system (Uhl & Miller, 1995 ), we compared the binding of THP to virulent B. pertussis BPSM and to avirulent B. pertussis BPLOW, which carries a chromosomal deletion of the bvgAS operon. The binding of THP to B. pertussis BPLOW was reduced by approximately 40% compared to that of virulent B. pertussis BPSM, as evidenced by flow cytometry analysis (data not shown). This indicates that bvgAS-regulated virulence factors are involved in the interaction with THP.
Involvement of PTX in the THP-mediated inhibition of B. pertussis adherence
To determine whether any of the known virulence factors, such as filamentous haemagglutinin (FHA) and/or PTX, are involved in the THP-mediated inhibition of B. pertussis adherence, the adherence assay was carried out using BPSM derivatives lacking FHA or PTX. As observed with MDCK cells, THP also induced a saturable and dose-dependent inhibition of B. pertussis BPSM adherence onto A549 pneumocytes (Fig. 1). However, this inhibition was less pronounced than that observed with MDCK cells, since it was limited to approximately 50% in the presence of 10 µg THP ml-1. Although the adherence of B. pertussis BPGR4 (a strain lacking FHA) was reduced by approximately 70% compared to its parental B. pertussis BPSM strain, even in the absence of THP, 10 µg THP ml-1 further reduced B. pertussis BPGR4 adherence by an additional 50%. This finding suggests that FHA is not involved in the THP-mediated inhibition of adherence. In contrast, the adherence of B. pertussis BPRA (a strain deficient for PTX), which is comparable to that of B. pertussis BPSM in the absence of THP, was less sensitive to THP, since 10 µg THP ml-1 induced only a 15% reduction of adherence, suggesting that PTX is implicated in the B. pertussisTHP interaction.
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Glycosylation of THP is required for PTX binding
Since carbohydrates account for approximately 30% of the THP weight (Fletcher et al., 1970 ), we investigated the role of the carbohydrate moiety in the THPPTX interaction. THP was enzymically deglycosylated using N-glycosidase F and O-glycosidase, which hydrolyse N-glycans and O-glycans, respectively. As shown in Fig. 4
, N-glycosidase F treatment reduced the apparent molecular mass of THP from approximately 95 kDa to a doublet of approximately 70 and 66 kDa. In contrast, no apparent shift in molecular mass was observed when THP was treated with O-glycosidase, consistent with the low level of O-glycosylation of THP (Easton et al., 2000
). Since THP contains several disulfide bridges that could interfere with the enzymic deglycosylation, THP was also reduced and carboxymethylated prior to deglycosylation (data not shown). When O- or N-deglycosylated THP was covalently coupled to Sepharose 4B and tested for its ability to bind to PTX produced by B. pertussis BPSM, the toxin was no longer retained by immobilized N-deglycosylated THP (Fig. 3
). In contrast, the PTX-binding capacity of O-deglycosylated THP was similar to that of native THP (not shown). The same results were obtained using deglycosylated reduced THP. These findings indicate that the N-glycans borne by THP are required for PTX binding.
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DISCUSSION |
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The interaction of PTX with immobilized THP has been shown to be particularly strong, since it requires 3 M urea to be completely abolished. This feature allowed us to develop a one-step chromatographic procedure to purify PTX to homogeneity from B. pertussis culture supernatant. This new purification procedure is cheap and may thus constitute an attractive alternative to conventional methods that generally combine two purification steps (Sekura et al., 1983 ). Moreover, the THP-purified PTX totally recovers its conformational structure and toxic activity, as demonstrated by the CHO-cell-clustering assay. The present study has also demonstrated that the THPPTX interaction is essentially mediated by the S2 subunit of the PTX B oligomer and that it depends on the glycosylation of THP. From enzymic deglycosylation experiments, it appears that the N-linked polysaccharides of THP are crucial for the interaction with PTX, whereas the O-linked sugars do not appear to be involved in this interaction. This finding may reflect the predominant and complex N-glycosylation content of THP (Hard et al., 1992
) and its very low O-glycosylation, which is limited to short and simple chains terminated with either sialic acid or fucose (Kumar & Muchmore, 1990
).
Although the receptors for PTX on eukaryotic cells have not yet been identified, it has been shown that CHO cells express a 165 kDa surface glycoprotein that is recognized by PTX or by purified B oligomer (Brennan et al., 1988 ). The sialic acid moiety on the N-linked oligosaccharide of this protein is an important motif of the receptor, since treatment of CHO cells with sialidase abolishes PTX binding. Moreover, some serum sialoglycoproteins, such as haptoglobin and fetuin, have been shown to bind to PTX via their N-glycosylated moieties (Witvliet et al., 1989
). Similar to THP, which also bears sialylated carbohydrate chains (Hard et al., 1992
), the binding of PTX to haptoglobin is mediated by the S2 subunit of the B oligomer (Francotte et al., 1989
). This suggests that the same binding mechanism may mediate the interaction of PTX with THP as mediates binding of PTX with haptoglobin.
The observations reported in the present study certainly open new research perspectives for the elucidation of the physiological role of THP within the urinary tract. We show that the anti-adherence activity of THP is not limited to fimbriated uropathogenic E. coli. Moreover, we have demonstrated that THP may directly interact with a bacterial toxin, PTX. Even if this interaction has no physiological relevance, it can be taken as a model to investigate the interactions of THP with bacterial virulence factors. Because of the large repertoire of sugar motifs borne by THP, it is possible that it may also bind to toxins like the haemolysins secreted by uropathogenic bacteria, such as E. coli or Proteus mirabilis. Investigations are currently in progress in our laboratory to clarify these potential antimicrobial activities of THP.
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ACKNOWLEDGEMENTS |
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Received 22 August 2001;
revised 16 November 2001;
accepted 27 November 2001.
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