The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Abstract

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis.

Fig. 1

SDS-PAGE of the OMPs of the Bacteroides fargilis MC2 strain passed through an affinity column with LMN-1. Lane 1 – molecular weight standards; lane 2 – OMPs of the B. fragilis MC2 strain; lanes 3, 4, 5 and 6 – elutions of the proteins with 0.25, 0.5, 1.0 and 2M NaCl. The arrow (→) indicates the purified fraction.

Fig. 2

PCR for detection of the gene bfp60 in Bacteroides fragilis MC2 strain. Lane 1 – 1-kb ladder; lane 2 – the amplicon of c.1700bp.

Fig. 3

Purification of the recombinant Bfp60 protein in SDS-PAGE. Lane 1 – molecular weight standards; lane 2 – whole cell lysates proteins of the Escherichia coli containing the bfp60 gene without the isopropyl β-d-1-thiogalactopyranoside (IPTG) induction; lane 3 – whole cell lysate proteins of E. coli containing the bfp60 gene IPTG-induced; lane 4 – recombinant Bfp60 protein (Bfp60-HP) after purification with Ni-resin. The arrow (→) indicates the size of the recombinant Bfp60-HP protein of 60 kDa.

Fig. 4

Adhesion and inhibition of Bacteroides fragilis MC2 strain to LMN-1. 1 – negative control (2% BSA); 2 – adhesion assay of the strain MC2 to LMN-1; 3, 4 and 5 – inhibition assay of the B. fragilis MC2 strain incubated with LMN-1 (10 μg mL⁻¹), plasminogen (10 μg mL⁻¹) and Bfp60 protein (10 μg mL⁻¹), respectively. All tests were performed in triplicate.

Fig. 5

Adhesion and inhibition assay of Bacteroides fragilis MC2 strain to plasminogen. 1 – negative control (2% BSA); 2 – adhesion assay of the B. fragilis MC2 strain to plasminogen; 3 and 4, inhibition assay of the MC2 strain incubated with 10 μg mL⁻¹ of plasminogen and 10 μg mL⁻¹ of Bfp60-HP, respectively. All tests were performed in triplicate.

Fig. 6

Enhancement of plasminogen activation in the presence of Bfp60-HP. Plasmin formation was measured with S-2251 at a Bfp60-HP concentration of 10 μg mL⁻¹. Plasmin with the chromogenic substrate (▲); Bfp60-HP incubated with plasminogen (□); plasminogen in the presence streptokinase (■); negative control plasminogen with HEPES buffer and substrate (●). All tests were performed in duplicate.

Fig. 7

Plasminogen activation by the Bacteroides fragilis MC2 strain. Plasmin formation by whole cells was measured with S-2251 at a concentration of 10⁸ CFU mL⁻¹. Plasmin with the chromogenic substrate (▲); B. fragilis MC2 strain incubated with plasminogen (□); plasminogen in the presence of streptokinase (■); plasminogen incubated with Staphylococcus aureus ATCC 968 strain (*); negative control plasminogen with HEPES buffer and substrate (●). All tests were performed in duplicate.