Cytosolic proteins contribute to surface plasminogen recruitment of Neisseria meningitidis

Abstract

Plasminogen recruitment is a common strategy of pathogenic bacteria and results in a broad-spectrum surface-associated protease activity. Neisseria meningitidis has previously been shown to bind plasminogen. In this study, we show by several complementary approaches that endolase, DnaK, and peroxiredoxin, which are usually intracellular proteins, can also be located in the outer membrane and act as plasminogen receptors. Internal binding motifs, rather than C-terminal lysine residues, are responsible for plasminogen binding of the N. meningitidis receptors. Recombinant receptor proteins inhibit plasminogen association with N. meningitidis in a concentration-dependent manner. Besides binding purified plasminogen, N. meningitidis can also acquire plasminogen from human serum. Activation of N. meningitidis-associated plasminogen by urokinase results in functional activity and allows the bacteria to degrade fibrinogen. Furthermore, plasmin bound to N. meningitidis is protected against inactivation by alpha(2)-antiplasmin.

FIG. 1.

Plasminogen binds to several meningococcal proteins. A representative plasminogen overlay assay is shown for strain MC58. Lane 1, silver staining of the total protein extract; lane 2, plasminogen overlay assay; lane 3, negative control without the addition of plasminogen; lane 4, recombinant meningococcal enolase; lane 5, recombinant meningococcal DnaK; lane 6, recombinant meningococcal peroxiredoxin. At least six meningococcal proteins binding to plasminogen can be identified in the overlay assay. Three of them could be identified by N-terminal sequencing as enolase, DnaK, and peroxiredoxin. The other bands have not yet been identified unequivocally.

FIG. 2.

Binding of plasminogen does not depend on C-terminal lysine residues. The recombinant proteins His-6-enolase (Eno), His-6-peroxiredoxin (Perox), and His-6-DnaK (DnaK) (A) and the respective Lys-negative variants (B) were dotted directly on a membrane to assess plasminogen binding of the native proteins by overlay assays. Three, 1, and 0.3 μg of each protein were applied for the dot blot. In this native binding assay, no difference in binding capacity could be detected between the recombinant receptors (A) and their C-terminal lysine-depleted variants (B), indicating that internal binding motifs are responsible for plasminogen recognition. No significant signal could be detected for albumin (A).

FIG. 3.

Soluble recombinant receptor proteins competitively inhibit plasminogen binding to N. meningitides. Microtiter wells were coated with 4 × 10⁶ bacteria (strain MC58 ΔsiaD), and plasminogen (11 pM) was added to individual wells with increasing amounts of the recombinant receptor proteins enolase (E), DnaK (D), and peroxiredoxin (P) for 30 min at 37°C. The recombinant proteins were used in 50-fold (550 pM), 100-fold (1.1 nM), and 200-fold (2.2 nM) molar excess over plasminogen. Plasminogen binding to bacteria without inhibitor was set to 100%. Albumin (B), which displays nonspecific binding capacities to a large number of proteins, was used as a negative control. *, P < 0.001. The error bars indicate standard deviations.

FIG. 4.

Flow cytometric analysis shows that meningococcal enolase (eno), DnaK, and peroxiredoxin (perox) are present at the bacterial surface. Bacteria were labeled with the IgG fraction of the rabbit antisera raised against the three proteins. IgG fractions from preimmune sera were used as negative controls. Significantly higher signals were detected with IgG fractions from sera than with IgG fractions from preimmune sera (P < 0.01). The values are mean fluorescence intensity times the percentage of labeled bacteria from three independent experiments ± the standard deviation. Signals measured on Δperox show background levels detected by the anti-peroxiredoxin IgG fraction. Opc served as a control for an abundant meningococcal surface protein. Significantly more peroxiredoxin than enolase or DnaK was detected (P < 0.01).

FIG. 5.

Transmission electron micrographs of N. meningitidis strain MC58 ΔsiaD demonstrate that enolase (A), DnaK (B), and peroxiredoxin (C) are present at the surface of N. meningitidis. Magnification of single gold particles (right) clearly shows association with the outer membrane (OM), which has been dissociated from the inner membrane by osmotic swelling. Enolase, DnaK, and peroxiredoxin were detected with rabbit antisera and a gold-labeled anti-rabbit mouse monoclonal antibody. No signals were detected with the preimmune sera. The arrows indicate gold particles in the lower magnification.

FIG. 6.

Enolase (A), DnaK (B), and peroxiredoxin (C) were detected on the surfaces of meningococci preincubated with plasminogen. Receptor molecules were detected by the respective rabbit antisera and a tetramethyl rhodamine isothiocyanate-labeled anti-rabbit antibody (magenta). Plasminogen was detected with a goat anti-plasminogen antibody (1:1,000) and a FITC-labeled rabbit-anti-goat antibody (1:500) (blue). The arrows indicate overlapping of receptor and plasminogen staining (light magenta).

FIG. 7.

(A) N. meningitidis can recruit plasminogen activity directly from human serum. MC58 ΔsiaD was incubated with either purified plasminogen (PLG), 30% human serum (HS) (41) diluted in PBS, or 100% human serum washed and activated with uPA. Plasmin activity was measured at 405 nm by degradation of the chromogenic substrate S-2251. The activity detected after incubation with purified plasminogen was set to 100%. (B) N. meningitidis-associated plasminogen can cleave fibrinogen after activation by uPA. Purified fibrinogen was incubated with either N. meningitidis alone (lane 1), plasmin (lanes 2 and 3), plasminogen-coated meningococci (lane 4), or plasminogen-coated meningococci that were subsequently activated with uPA (lanes 5 to 7). Fibrinogen degradation was detected in an immunoblot at different time points (indicated in minutes at the bottom of each lane). (C) N. meningitidis-associated plasmin activity is protected against inactivation by α₂-antiplasmin. Plasmin activity was measured by S-2251 degradation in the absence or presence of α₂-antiplasmin. Lane 1, plasmin; lane 2, plasmin plus α₂-antiplasmin (activity in relation to lane 1); lane 3, N. meningitidis-associated plasmin; lane 4, N. meningitidis-associated plasmin plus α₂-antiplasmin (activity in relation to lane 3), *, P < 0,001. The error bars indicate standard deviations.