Background The DsrA protein ofHaemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, is a multifunctional outer membrane trimeric autotransporter (TA) and a virulence factor. MostH ducreyi strains, which are grouped in two classes (I and II), express a DsrA protein. Although DsrA proteins from both classes ofH ducreyi strains share little identity in their N-terminal functional passenger domain, both are involved in serum resistance and function as adhesins to the extracellular matrix (ECM) proteins fibronectin (Fn) and vitronectin (Vn), as well as to fibrinogen (Fbg) and HaCat keratinocytes.
Methods Monoclonal antibodies (mAbs) to DsrA were developed by immunising mice with full-length recombinant DsrA from class I strain 35 000HP (DsrAI). Western blots were used to determine the specificity of the mAbs and their ability to recognise multimers of DsrA. Whole-cell binding ELISAs were used to examine the capacity of the mAbs to bind the surface ofH ducreyi. The domains and the shortest nominal peptides of DsrA recognised by those mAbs were identified using a library of truncated DsrA proteins expressed in a dsrA mutant, a peptide library representing full-length DsrAI, as well as Surface Plasmon Resonance and mutagenesis studies. mAbs were tested for bactericidal activity and their ability to block binding of Fn, Vn and Fbg byH ducreyi using bactericidal assays and whole cell blocking assays, respectively.
Results Anti-DsrAI mAbs bound monomers and dimers of the DsrA protein from several class IH ducreyi strains but not the DsrAII protein. mAb 1.82 bound native DsrAI protein at the surface of a panel of class IH ducreyi isolates, but not the H .ducreyi strains that cause cutaneous chancroid. The DsrA protein from these strains was recognised by mAb 4.79, along with the DsrA protein from strain 35 000HP. Both 1.82 and 4.79 bound with high affinity to peptides in the N-terminal region of the DsrA translocator domain and the shortest nominal epitope for 1.82 is MEQNTHNINKLS. In whole cell blocking assays, mAb 1.82 partially blocked binding of Fn and Fbg by class IH ducreyi strain 35 000HP.
Conclusions 1.82 and 4.79 are DsrAI-specific mAbs that recognise multimers of DsrA and bind the surface of intactH ducreyi. Both mAbs bind an epitope in the N-terminal region of the translocator domain of DsrAI with high affinity. mAb 1.82 has modest Fn and Fbg blocking activity.
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