Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1

Abstract

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Figure 1

Evaluation of bovine holo-lactoferrin (BholoLf) as a sole iron source in Mannheimia haemolytica strains (MhF and MhR). Moraxella bovis was used as positive control and Actinobacillus pleuropneumoniae as a negative control of use of BholoLf. The bacterial growth was determined by OD_595nm, at 24 h of incubation at 37 °C, in agitation (200 rpm) in different conditions: BHI, Brain heart infusion broth; CB, BHI chelated with dipyridyl; CB + FC, chelated BHI plus ferric chloride [80 μM]; CB + BholoLf, chelated BHI plus BholoLf [80 μM of iron]. The results are shown as the mean ± SD, *p < 0.05 versus CB and CB + BholoLf.

Figure 2

Bovine lactoferrin (BapoLf and BholoLf) binding to M. haemolytica OMP. A 12% SDS-PAGE of M. haemolytica OMP extracted with sarcosyl, stained with Coomassie blue. B OMP overlay, incubated with BapoLf coupled to horseradish peroxidase (HRP-BapoLf), the arrow shows the 40 kDa BapoLf binding protein; Lane 2, MhF (field strain); lane 3, MhR (reference strain). C Competence between BapoLf and HRP-BholoLf (MhF), OMP transferred were incubated with BapoLf and afterwards with HRP-BholoLf, the arrow shows the 40 kDa BLf binding protein. D OMP overlay (MhF), the membrane transferred was incubated with HRP-lactoferricin B (HRP-LfcinB), in the right line the membrane was incubated with BapoLf (without HRP) and afterwards with HRP-LfcinB; the arrow shows the 40 kDa BLfcin binding protein.

Figure 3

Separation of the M. haemolytica outer membrane proteins that bind to bovine apolactoferrin (BapoLf) using 2-D gel electrophoresis. A 2-D gel electrophoresis of the M. haemolytica OMP, stained with Coomassie blue. B Overlay of M. haemolytica OMP, from 2-D gel electrophoresis; the nitrocellulose membrane was incubated with HRP-BapoLf. The arrows show the spots of BapoLf binding proteins (A) and their MW and estimated IP (B), which were further identified by mass spectrometry.

Figure 4

Alignment between MhHM [UniProtKB: Q6XAY2] and MhMP sequences [UniProtKB: S9YBF1]. The prediction of the secondary structure performed with PSS PRED is shown in the top and the bottom of each sequence; the arrows show the β-sheets and the spiral α-helix structure. The red boxes show amino acid identity and the yellow boxes similarity [40, 41].

Figure 5

Prediction in Pred-TMBB of the protein localization at the membrane of MhHM and MhMP. In the sequence, amino acids in green represent the inner localization; amino acids in red, the transmembrane localization; amino acids in blue, the outside localization [44].

Figure 6

3-D model for MhHM in I-Tasser server with a C-score of −3.56. A Lateral view in rainbow ribbon diagram. B Top view of the rainbow ribbon diagram. C Alignment between the close structure OmpA transmembrane domain of E. coli, which is represented by purple lines, and MhHM, in the rainbow ribbon diagram. D Prediction of the ligand binding site; the consensus binding residues of OmpA-like domain of A. baumannii are represented by blue sticks and MhHM in gray color diagram (before as rainbow ribbon).

Figure 7

3-D model for MhMP in I-Tasser server with a C-score of 1.02. A Lateral view in rainbow ribbon diagram. B Top view of the rainbow ribbon diagram. C Alignment between the close structure OmpC of E. coli, which is represented by purple lines, and MhMP by the rainbow ribbon diagram. D Prediction of ligand binding site; the consensus binding residues of Omp 38 of Delftia acidovorans are represented by blue sticks and the ligand malate by green spheres. MhMP in gray color diagram (before as rainbow ribbon).

Figure 8

Molecular docking between MhHM model (green ribbon diagram) and bovine lactoferrin (blue ribbon diagram). The residues in sticks representation show the possible amino acids involved in the docking between the two proteins [47].

Figure 9

Molecular docking between MhMP model (green ribbon diagram) and bovine lactoferrin (blue ribbon diagram). The residues in sticks representation show the possible amino acids involved in the docking between the two proteins [47].