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Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcer disease that facilitates the transmission of human immunodeficiency virus (17). Initiation of H. ducreyi infection requires trauma to the skin (13), indicating that adherence targets may include subsurface skin components, such as the extracellular matrix (ECM). Many bacterial pathogens adhere to ECM proteins via surface structures such as fimbriae and lipopolysaccharide (LPS), as well as afimbrial surface proteins (9, 16, 18). To characterize the binding of H. ducreyi to ECM proteins, we used an enzyme-linked immunosorbent assay (ELISA)-based assay with ECM proteins found in adult human skin, including fibronectin, laminin, several collagens, and elastin. We chose H. ducreyi 35000HP for these studies. Strain 35000HP is a human-passaged variant of 35000 and is fully virulent in the human challenge model of chancroid (2). We also examined the roles of two H. ducreyi surface structures, the fine tangled pili and lipooligosaccharide (LOS), in ECM adherence.

ECM protein binding assay. Microtiter plates (Immulon 4 HBX; Dynex Technologies, Chantilly, Va.) were coated with ECM proteins by incubating 100 µl of the appropriate protein, diluted in coating buffer (0.25 M NaHCO₃, 0.25 M Na₂CO₃ [pH 9.6]) to 20 µg of protein per ml or the concentrations indicated below, in assay wells overnight at 4°C (for collagens) or 35°C (for fibronectin and laminin) (8, 12). Wells were then washed three times with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 0.69 mM KH₂PO₄, 6.4 mM Na₂HPO₄ [pH 7.5]) containing 0.05% Tween 20 (PBST), blocked with 300 µl of 10% fetal calf serum in PBST for 2 h at 35°C, and washed three times in PBS. H. ducreyi 35000HP (2) was grown in broth with fetal calf serum to mid-log phase as described previously (6), harvested by centrifugation, and washed and suspended in PBS to the desired optical density at a wavelength of 660 nm (OD₆₆₀). Staphylococcus aureus Phillips and PH100 (gift of Joseph Patti) (10) were grown overnight on Luria-Bertani agar (11), harvested, washed, and suspended in PBS. PH100 was supplemented with gentamicin (10 µg/ml). Bacteria were added to each well, and the plates were incubated at 35°C in 5% CO₂. For bacterial dose-response assays, bacteria were serially diluted twofold in PBS and tested at concentrations ranging from an OD₆₆₀ of 0.8 to 0.003. For ECM dose-response assays, bacteria were added to all wells at an OD₆₆₀ of 0.2.

Deposition of ECM proteins and detection of ECM-bound bacteria. To confirm that ECM proteins had bound to the ELISA plates, we probed wells coated with fibronectin, fetuin, and type I, III, IV, V, and VI collagen (all of human origin; Southern Biotechnology Associates, Inc., Birmingham, Ala., or Sigma Chemicals) with S. aureus Phillips and its isogenic cna mutant PH100. Phillips, known to bind to type I, II, and III collagen (4, 5), bound to all proteins tested except type VI collagen (Fig. 1A) and fetuin (Fig. 1B). This binding was dependent on the amount of ECM protein used to coat the wells (data not shown). PH100, in which the S. aureus collagen adhesin gene, cna, is insertionally inactivated (10), showed greatly reduced binding to the panel of collagens (Fig. 1A). These data indicated that the plates were coated with the ECM proteins and that the assay was able to distinguish between strains on the basis of their ability to bind to ECM proteins.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   S. aureus strains binding to ECM proteins. Error bars indicate standard deviations of triplicate wells. Data shown are representative of at least three independent assays. (A) S. aureus Phillips (open symbols) and PH100 (closed symbols) binding to collagens. Symbols represent binding to type I (squares), type III (circles), type IV (triangles), type V (diamonds), and type VI (inverted triangles) collagen. (B) S. aureus Phillips binding to fibronectin and fetuin.

H. ducreyi binds to specific ECM proteins. Utilizing the ECM adherence assay, we examined the ability of H. ducreyi 35000HP to bind to human type I, III, IV, V, and VI collagen, with BSA as a negative control. Strain 35000HP bound efficiently to type I and III collagen (Fig. 2). This binding varied with the amount of bacteria (Fig. 2A) and with the amount of collagen (Fig. 2B) used in the assay. Levels of binding to type IV, V, and VI collagen varied somewhat, but they were usually in the range of levels of binding to BSA and were consistently lower than levels of binding to type I and III collagen (Fig. 2).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   H. ducreyi 35000HP binding to collagens. Error bars indicate standard deviations of triplicate wells. Data shown are representative of at least three independent assays. (A) Bacterial dose-response assays; (B) protein dose-response assays.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   H. ducreyi 35000HP binding to ECM proteins. Error bars indicate standard deviations of triplicate wells. Data shown are representative of at least three independent assays. (A) Bacterial dose-response assays; (B) protein dose-response assays.

ECM binding is not mediated by FtpA. H. ducreyi expresses a fine tangled pilus, the major subunit of which is FtpA (3). Pili of several bacterial species have been shown to bind specifically to ECM proteins (7, 15, 19), and one report indicated a correlation between piliation of H. ducreyi and binding to laminin (1). To examine the role of the H. ducreyi pilus in ECM binding, we tested the ability of 35000HP-SMS1 [an isogenic mutant of 35000HP in which ftpA, which encodes the major subunit of the fine tangled pili, is insertionally inactivated by mTn3(Cm)] to bind to fibronectin, laminin, and type I and III collagen. Strain 35000HP-SMS1 bound as well as 35000HP to each of these proteins (data not shown). Neither strain bound to type IV collagen or fetuin. The kinetics of ECM binding were also unaffected by the mutation in ftpA (data not shown). These data indicate that FtpA is not required for adherence to ECM proteins, although we cannot rule out a contributory role for FtpA in conjunction with other adhesins. These data confirm those of Brentjens et al., who reported that both strain 35000 and an isogenic ftpA derivative bind to laminin (3), and contradict those of Abeck et al., who reported that strain 35000 does not bind to laminin and that piliation is required for laminin binding (1). These discrepancies could be due to differences in the assays or in the strains tested.

Full-length LOS is not required for ECM binding. Another surface structure that can mediate ECM binding is LPS. Helicobacter pylori LPS binds specifically to laminin (16). This activity is most likely mediated by the core sugars, although the specific saccharides involved are unknown. We examined the role of full-length H. ducreyi LOS in ECM binding by testing 35000HP-RSM2 (gift of Robert S. Munson, Jr.), an isogenic losB mutant of 35000HP, for binding to the panel of ECM proteins. The major LOS saccharide chain produced by 35000HP-RSM2 consists of a single glucose attached to a heptose trisaccharide core and 2-keto-deoxyoctulosonic acid and cannot be sialylated. The mutation in losB in 35000HP-RSM2 had no effect on binding to collagens, fibronectin, or laminin (data not shown). Thus, neither the terminal oligosaccharide nor sialylation is required for the observed ECM binding by H. ducreyi. However, these data do not rule out a role for the remaining core sugars expressed in the LOS moiety of the losB mutant.