Killing of dsrA mutants of Haemophilus ducreyi by normal human serum occurs via the classical complement pathway and is initiated by immunoglobulin M binding

Abstract

Previously, we showed that serum resistance in Haemophilus ducreyi type strain 35000HP required expression of the outer membrane protein DsrA because the isogenic dsrA mutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptible dsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing of dsrA mutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5- and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited onto dsrA mutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serum-resistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in the dsrA mutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.

FIG. 1.

Bactericidal killing of H. ducreyi parent strains and dsrA mutants. The indicated parent strains and their isogenic dsrA mutants were incubated with NHS for 45 min, and numbers of CFU were determined by plating. Percent survival was calculated by dividing the CFU from fNHS by CFU from hNHS and multiplying by 100. The P values for strains 35000HP (35000), 010-2, and 425 and each of their isogenic mutants were <0.0001, <0.0001, and <0.0001, respectively.

FIG. 2.

NHS killing of dsrA mutants requires Ca²⁺. The indicated dsrA mutants were subjected to bactericidal killing in the presence and absence of EGTA/Mg. P values for FX517 and FX529 (fNHS versus fNHS plus EGTA/Mg) were <0.0001 for both strains.

FIG. 3.

Bactericidal killing of dsrA mutants requires Clq. Bactericidal killing (50% NHS) was performed as described using NHS (fNHS), NHS depleted of C1q (fNHS/C1q×), or NHS depleted of C1q and then reconstituted with physiological concentrations of purified C1q (fNHS/C1q−/C1q+). P values for FX517 and FX529 (fNHS versus fNHS/C1q−) were < 0.0001 and < 0.0001, respectively. P values for both strains (fNHS/C1q− versus. fNHS/C1q−/C1q+) were <0.0001.

FIG. 4.

Binding of immunoglobulins by H. ducreyi. The indicated strains of H. ducreyi (1 × 10⁷ CFU) were mixed with iodinated purified human IgM or IgG (1 × 10⁶ cpm) in a total volume of 100 μl. After 45 min, the H. ducreyi strains were suctioned and washed five times with PBS, and bacteria-associated radioactivity was determined. P values for 35000HP (35000) versus FX517 for IgM depletion and IgG depletion were <0.0001 and <0.0693, respectively.

FIG. 5.

C1q binding to H. ducreyi as measured by immunoblotting. H. ducreyi strains were incubated with fNHS for the indicated time periods. After binding of complement components, bacteria were pelleted and washed by cycles of centrifugation. Cell pellets with bound complement components were subjected to SDS-PAGE and immunoblotting using polyclonal anti-human C1q. C1q is composed of three individual chains, A, B, and C, that migrate at 27.5, 25.2 (doublet), and 23.8 kDa, respectively. Similar results were observed in three other experiments. Normal human serum, 0.1 μl, was loaded in the first lane as a positive control (approximately 7 nanograms of C1q).

FIG. 6.

Binding of C3 and C4 to H. ducreyi. Bacteria were incubated in the absence of NHS or in the presence of hNHS or fNHS. After washing bacteria to remove unbound serum proteins, bound C′ proteins were detected by SDS-PAGE (reducing conditions) using anti-human C3 and C4. NHS, 0.1 μl, was loaded as a positive control. The upper band in the NHS standard lane is the alpha chain, and the lower band is the beta chain for both C4 and C3. FA6564 (serum resistant) and F62 (serum susceptible) are gonococcal control strains. Similar results were obtained in three other experiments.

FIG. 7.

Binding of C6 and C7 by H. ducreyi. Bacteria were incubated in the absence of NHS or in the presence of hNHS or fNHS. After unbound proteins from bacteria were washed, bound proteins were detected using anti-human C6 or C7 in Western blots. Similar results were observed in three other experiments.

FIG. 8.

Binding of terminal MAC complex. H. ducreyi strains were incubated in fNHS or hNHS (10%) for 45 min at 35°C. Bacteria were suctioned and washed with PBS, and an iodinated MAb directed against a C5b-C9 neoepitope was added (1 × 10⁶ cpm). After binding of MAb and removal of unbound MAb by washing, bacterium-associated cpm were determined. Shown are the cpm bound in fNHS minus the cpm bound in hNHS. The P value for 35000HP (35000) versus FX517 was <0.0004.

FIG. 9.

Fresh normal human serum was treated as described here, and the bactericidal activity was determined using a 20% concentration. 1, fNHS heated to 56°C to inactivate the complement (hNHS); 2, fNHS incubated on ice without treatment (fNHS); 3, fNHS incubated with agarose containing no ligand on ice (mock IgM-depleted fNHS); 4, fNHS incubated with anti-human IgM agarose on ice (IgM-depleted fNHS); 5, IgM-depleted fNHS reconstituted with IgM (IgM-depleted fNHS + IgM); 6, IgM-depleted fNHS reconstituted with IgG (IgM-depleted fNHS + IgG); 7, IgM-depleted fNHS reconstituted with hNHS as a source of immunoglobulins (IgM-depleted fNHS + hNHS).

FIG. 10.

Absorption of NHS with FX517 dsrA removes bactericidal IgM. fNHS was absorbed with FX517 dsrA on ice as described in the text. After absorption, fNHS was used in bactericidal assays against FX517. To determine if killing could be restored to absorbed serum, purified IgM equal to the amount present in NHS was added. Alternatively, hNHS was added as a source of immunoglobulins.