Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA

Abstract

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

FIG. 1.

Variability of H. ducreyi DsrA proteins. (A) Schematic depiction of DsrA_I protein showing typical architecture of TAAs. Also indicated are the shortest truncated DsrA_I proteins to render a dsrA mutant strain capable of serum resistance (Sr) and binding to Fn and Vn (Fn/Vn+) and the amino acid sequence involved in binding to Fg (Fg+). The numbers below refer to the last amino acid of the particular domain, in reference to immature DsrA_I. (B) Amino acid alignment of predicted amino acid sequences of DsrA protein from selected H. ducreyi strains, including representative isolates from both classes of H. ducreyi strains and two non-genital/cutaneous chancroid isolates (BE3145 and SB5756). Sequences were aligned using ClustalW (http://workbench.sdsc.edu) using default settings. Boxes indicate identical residues, while underlined amino acids share homology.

FIG. 2.

Anti-DsrA MAbs 1.82 and 6.95 recognize monomers and multimers of class I and class I-like DsrA proteins, but not DsrA_II. (A) Reactivity of MAbs to DsrA proteins in Western blots under reducing and denaturing conditions. Rabbit polyclonal anti-rFL-DsrA_I serves as a control for DsrA expression since it recognizes both classes of DsrA proteins.⁽¹³⁾ Anti-rD15 polyclonal Ab serves as a control for well loading. FX517 is the isogenic dsrA mutant of prototypical class I H. ducreyi strain 35000HP (35000HPΔdsrA). (B) Reactivity of anti-DsrA_I MAbs to multimers of DsrA was assessed using concurrently developed Western blots. Shown are representative blots from two experiments with identical results.

FIG. 3.

Surface binding of MAbs 1.82 and 6.95 to viable bacteria varies among different classes of H. ducreyi strains. Whole cell binding of anti-rDsrA_I MAbs to bacteria grown either in CAP (A) or heme plates (B). Whole, viable H. ducreyi were probed with 1 μg/mL of MAbs. Isogenic dsrA mutant strain FX517 (35000HPΔdsrA) serves as negative control for expression of DsrA. Shown are means±standard deviations of at least five independent experiments. *p<0.05 using a paired t-test.

FIG. 4.

Anti-DsrA MAb 1.82 partially inhibits binding of DsrA to Fn, Vn, and Fg. Whole, viable, class I H. ducreyi strain 35000HP was incubated with the indicated IgG prior to incubation with either Fn (A), DIG-Vn (B), or Fg (C–E), as described in the Materials and Methods section. (A–C) Fn, DIG-Vn, and Fg binding to the surface of viable H. ducreyi in the presence of competitor IgG was measured using Western blot. Shown are representative blots from at least three independent experiments performed with each Fn, DIG-Vn, or Fg. Densitometry carried out on appropriate bands from each blot is shown in the graphs below the blots as means+/- standard error of Fn, DIG-Vn, or Fg binding compared to no treatment (no addition of IgG), defined as 100% binding (line). Western blot with anti-rFLDsrA_I⁽¹³⁾ shows equal loading of wells. (D–E) Fg binding to the surface of viable H. ducreyi, in the presence or absence of competitor IgG, was measured using flow cytometry. (D) Representative histogram of three independent experiments. (E) Means+/− standard deviations of three independent experiments where FITC-Fg binding to H. ducreyi was compared to a no treatment control (no addition of IgG), defined as 100% binding (line). *p<0.05 using a single sample t-test.

FIG. 5.

Shortest nominal peptide of DsrA bound by MAb 1.82 is MEQNTHNINKLS. (A) Total cellular proteins (from ∼1×10⁷ CFU) from dsrA mutant strain expressing truncated DsrA proteins⁽²¹⁾ were subjected to 4–12% gradient SDS-PAGE, followed by Western blot with 0.5 μg/mL MAb 1.82. Shown is a representative blot from two identical replicate studies. (B) Reactivity of anti-rDsrA_I MAb 1.82 to a peptide library representing full-length class I DsrA protein from H. ducreyi class I strain 35000HP. Below the chart are amino acid sequences of reactive peptides and their residue numbers in reference to immature DsrA. *Peptide charges were obtained using the online calculator at Innovagen website (www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp). (C) Representative titration binding curves of 1.82 Fab with peptides 48 (left), 49 (middle), and 50 (right) from two replicate studies. (D) Schematic representation of residues in the N-terminal section of the translocator (grey) domain of DsrA_I. The smallest nominal epitope of MAb 1.82 determined by SPR is shown in bold.