The Haemophilus ducreyi serum resistance antigen DsrA confers attachment to human keratinocytes

Abstract

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. H. ducreyi serum resistance protein A (DsrA) is a member of a family of multifunctional outer membrane proteins that are involved in resistance to killing by human serum complement. The members of this family include YadA of Yersinia species, the UspA proteins of Moraxella catarrhalis, and the Eib proteins of Escherichia coli. The role of YadA, UspA1, and UspA2H as eukaryotic cell adhesins and the function of UspA2 as a vitronectin binder led to our investigation of the cell adhesion and vitronectin binding properties of DsrA. We found that DsrA was a keratinocyte-specific adhesin as it was necessary and sufficient for attachment to HaCaT cells, a keratinocyte cell line, but was not required for attachment to HS27 cells, a fibroblast cell line. We also found that DsrA was specifically responsible for the ability of H. ducreyi to bind vitronectin. We then theorized that DsrA might use vitronectin as a bridge to bind to human cells, but this hypothesis proved to be untrue as eliminating HaCaT cell binding of vitronectin with a monoclonal antibody specific to integrin alpha(v)beta(5) did not affect the attachment of H. ducreyi to HaCaT cells. Finally, we wanted to examine the importance of keratinocyte adhesion in chancroid pathogenesis so we tested the wild-type and dsrA mutant strains of H. ducreyi in our swine models of chancroid pathogenesis. The dsrA mutant was less virulent than the wild type in both the normal and immune cell-depleted swine models of chancroid infection.

FIG. 1.

Effect of DsrA expression on H. ducreyi attachment to HaCaT cells. In each assay, approximately 10⁶ CFU of each strain was incubated with 10⁵ HaCaT cells for 2 h. The percentage adherence was determined as follows: (number of adherent CFU after 2 h of incubation/total number of CFU after 2 h of incubation) × 100. The data are presented in a box plot due to unequal variance. Each box indicates the median, 25th, and 75th percentiles, and the error bars indicate the 10th and 90th percentiles.

FIG. 2.

Effect of DsrA expression on H. ducreyi attachment to HS27 cells. In each assay, approximately 10⁶ CFU of each strain was incubated with 10⁵ HS27 cells for 2 h. The percentage of adherence was determined as follows: (number of adherent CFU after 2 h of incubation/total number of CFU after 2 h of incubation) × 100. The data are means and standard deviations.

FIG. 3.

rDsrA enhanced the attachment of H. influenzae to HaCaT cells. rDsrA confers an attachment phenotype to H. influenzae in the presence and in the absence of FBS. The attachment data are presented in a box plot due to unequal variance. Each box indicates the median, 25th, and 75th percentiles, and the error bars indicate the 10th and 90th percentiles.

FIG. 4.

DsrA is responsible for the binding of vitronectin by H. ducreyi. (A) Western blot of DsrA-expressing and non-DsrA-expressing strains that were exposed to NHS, solublized, transferred, and probed with anti-human vitronectin antibody. (B) Autoradiograph of affinity-purified vitronectin binding proteins from iodinated H. ducreyi strains 35000 and FX517. Lanes 1, iodinated H. ducreyi whole cells; lanes 2 to 5, affinity purification using human vitronectin, recombinant human vitronectin, bovine vitronectin, and no vitronectin, respectively. The position of DsrA is indicated by the arrow. HuVn, human vitronectin.

FIG. 5.

Effect of a blocking α_vβ₅ monoclonal antibody on HaCaT cells binding to vitronectin. Symbols: •, percentage of HaCaT cell attachment in the presence of various concentrations of the α_vβ₅ antibody; ▴, percentage of HaCaT cell attachment in the presence of the isotype control. The data are normal and are means and standard deviations.

FIG. 6.

Effect of a blocking α_vβ₅ monoclonal antibody on H. ducreyi attachment to HaCaT cells. The concentrations of the α_vβ₅ antibody are indicated. The data are normal and are means and standard deviations.

FIG. 7.

Percentages of day 7 lesion biopsies from normal and CPA-treated, immune cell-depleted pigs that were culture positive. The percentages were determined as follows: (number of culture-positive lesion biopsies/total number of lesion biopsies) × 100.

FIG. 8.

Average histology scores from day 2 and 7 lesion biopsies from normal pigs. The data are normal and are means and standard deviations.