A Novel Lectin, DltA, Is Required for Expression of a Full Serum Resistance Phenotype inHaemophilus ducreyi

Abstract

Haemophilus ducreyi, the causative agent of chancroid, is highly resistant to the complement-mediated bactericidal activity of normal human serum (NHS). Previously, we identified DsrA (for ducreyi serum resistance A), a major factor required for expression of the serum resistance phenotype in H. ducreyi. We describe here a second outer membrane protein, DltA (for ducreyi lectin A), which also contributes to serum resistance in H. ducreyi. Isogenic dltA mutants, constructed in 35000HP wild-type and FX517 dsrA backgrounds, were more susceptible to the bactericidal effects of NHS than each respective parent, demonstrating the additive effect of the mutations. Furthermore, expression of dltA in H. influenzae strain Rd rendered this highly susceptible strain partially resistant to 5% NHS compared to a vector-control strain. Although primary basic local alignment search tool analysis of the dltA open reading frame revealed no close bacterial homologue, similarity to the ␤-chain of the eukaryotic lectin ricin was noted. DltA shares highly conserved structural motifs with the ricin ␤ chain, such as cysteines and lectin-binding domains. To determine whether dltA was a lectin, ligand blots and affinity chromatography experiments were performed. DltA was affinity purified on immobilized lactose and N-acetylgalactosamine, and N-glycosylated but not glycosidase-treated model glycoproteins bound DltA. These data indicate that DltA is a lectin with specificity for lactose-related carbohydrates (CHO) and is important for H. ducreyi serum resistance.

Haemophilus ducreyi Outer Membrane Determinants, Including DsrA, Define Two Clonal Populations

“…Recently, our laboratory described a novel lipoprotein lectin, termed DltA, found in H. ducreyi strain 35000HP and involved in serum resistance (33). In this study, we determined that all strains tested express DltA and that class II strains possess a variant form of DltA that migrates at a different molecular weight in Western blots than does DltA from class I strains.…”

“…H. ducreyi resistance to 50% normal human serum (NHS) was determined as previously described (13,19,33,41) except that H. ducreyi cultures were grown on chocolate agar plates containing 5% fetal bovine serum. Data are expressed as percent survival in fresh NHS compared to survival in heated NHS (⌬NHS) [(CFU in fresh NHS/CFU in ⌬NHS) ϫ 100].…”

“…The study of chancroid pathogenesis has lead to the identification of a number of H. ducreyi antigens that may be important for the production of disease (4,11,12,18,19,25,31,33,35,39,45,50,54,65,67,71,72). Thus far, few cell surface determinants, including full-length lipooligosaccharide (LOS), have been shown to be essential for H. ducreyi infection in the human model of chancroid (5,9,22,28,56).…”

A Database of Bacterial Lipoproteins (DOLOP) with Functional Assignments to Predicted Lipoproteins

“…The availability of a larger database of experimentally verified lipoproteins has enabled the devising of predictive rules that have been found to be fairly accurate. Reports of identification of putative lipoproteins using this method followed by experimental verification justified the approach (1,24,33,51). Using the currently obtained largest set of 278 distinct lipoproteins, the following predictive rules have been derived.…”

An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding

“…We treated whole cells of H. ducreyi 35000HP with limiting amounts of protease (18) and identified cleaved HgbA peptides in Western blot assays (data not shown). HgbA peptides were subjected to Edman degradation.…”