Localization of the domains of the Haemophilus ducreyi trimeric autotransporter DsrA involved in serum resistance and binding to the extracellular matrix proteins fibronectin and vitronectin

Abstract

Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi.

FIG. 1.

Sequence alignment of selected TAA proteins. The mature protein sequences were aligned using CLUSTALW (http://workbench.sdsc.edu) with default settings. The signal sequences were determined using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). Underlining indicates helix secondary structures, whereas bold type indicates beta strands (obtained using http://bioinf.cs.ucl.ac.uk/psipred/). Boxes indicate residues that are fully conserved or that show conservation of strong or weak groups (determined by CLUSTALW). Domains of YadA1, adapted from the study of Hoiczyk et al. (19), are indicated under the protein sequences by horizontal lines and arrows that show where the domains begin and end. In-frame dsrA deletion mutants, referred to by the designations of the truncated DsrA proteins (Table 1), are indicated by circles that show the first amino acid of the truncated protein. In-frame dsrA mutants were constructed using plasmid pUNCH1260, which expresses full-length dsrA. In-frame mutations fused the first three amino acids of the mature sequence (QQP) to the amino acids indicated by the designations of the truncated DsrA proteins (see Table 1 and Fig. 2 for more details). SR indicates the smallest truncated DsrA protein to confer serum resistance to FX517; FN+VN indicates the smallest truncated DsrA protein that confers FN and VN binding to FX517. The head, neck, and stalk are parts of the passenger (effector) domain; the coiled-coil and membrane anchor encompass the translocator (β) domain.

FIG. 2.

Schematic diagrams of the H. ducreyi full-length and truncated DsrA proteins. (A) Schematic diagrams of the full-length and truncated DsrA proteins. The arrows indicate the positions of the amino acids at the N-terminal ends of the truncated proteins compared to the sequence of the immature (arrows above the boxes) or mature (arrows below the boxes) DsrA_I protein. The striped boxes represent the translocator domain, which includes the coiled-coil (CC) and membrane anchor (MA) regions (see text for details). SS, signal sequence; AA, amino acids. (B) Schematic diagrams of the recombinant proteins used for production of the antibodies used in this study.

FIG. 3.

Cell surface expression and trimer formation of truncated DsrA proteins. (A) Sarkosyl-insoluble OMPs (approximately 1 μg per lane) were solubilized at 100°C (panel 1) and 37°C (panel 2) and subjected to Western blotting with anti-rFL-DsrA_I. (B) (Top panel) Whole-cell binding assay with anti-rNt-DsrA_I, a rabbit polyclonal antibody that binds to the surface of H. ducreyi. (Bottom panel) Total cellular proteins from the indicated strains were solubilized at 100°C and subjected to Western blotting with anti-rNt-DsrA_I. RLU, relative light units.

FIG. 4.

Localization of the domains of DsrA required for serum resistance and ECM protein binding. (A) Survival (gray bars) in 50% NHS of H. ducreyi dsrA mutant FX517 expressing truncated DsrA proteins. The arrow indicates the shortest construct that is serum resistant (≥50% survival in 50% NHS). The open bars indicate the results obtained with the IgM binding assay. An asterisk indicates a value that is statistically different from the value for strain FX517 expressing full-length DsrA (DsrA_I) (P < 0.05). The data are presented using a box plot because statistical analysis determined that some data were not equally distributed. Each box indicates the median and 25th and 75th percentiles, while the error bars indicate the 10th and 90th percentiles. The filled circles indicate data for outliers within the 5th and 95th percentiles. (B) FN and VN binding by the dsrA mutant strain FX517 expressing truncated DsrA proteins. Whole cells of H. ducreyi were mixed with purified FN or NHS (as a source of VN). After binding, cells were washed to remove unbound ligand. Cell pellets were subjected to Western blotting with the indicated antibodies to determine binding to VN and FN. Both FN and VN binding assays were repeated four times with FX517 expressing each truncated DsrA protein. The blots shown are representative Western blots from one of these experiments. The numbers below the Western blots indicate the mean band densities calculated from the results of four experiments. Band density, measured using NIH Image, was adjusted using the value for FX517 expressing full-length dsrA (DsrA_I), defined as 100% binding. An asterisk indicates that a value is statistically different from the value for strain FX517 expressing full-length DsrA (DsrA_I) (P < 0.05).

FIG. 5.

Correlations between the amounts of IgM bound at the surface of FX517 expressing truncated DsrA proteins and the sizes of the DsrA proteins (A) and between IgM binding and the serum resistance of strain FX517 expressing truncated DsrA proteins (B). Data from Fig. 4 were used to determine the correlations using Sigma Plot. For IgM binding and serum resistance, “maximum” refers to the amount of IgM bound at the surface of strain FX517 expressing DsrA_I, which was defined as 100%.