Identification of an immunogenic Mannheimia haemolytica immunoglobulin-, fibronectin- and fibrinogen-binding protein differentially expressed in vitro

Bovine respiratory disease (BRD), or shipping fever , is the cause of large losses to the cattle industry annually and is primarily caused by Mannheimia haemolytica serotype (ST) 1. M. haemolytica, a normal flora respiratory tract bacterium, rapidly multiplies upon a stressful occurrence and colonizes the lower respiratory tract. This can lead to fibrinohemorrhagic pneumonia. Bacterial immunoglobulin-binding protein (IgBP) expression is known to play a role in the pathogenesis of a variety of organisms. Far-Western blot demonstrated the presence of an IgBP(s) in whole cell sonicates (WCSs) and a culture supernatant (CS) preparation from M. haemolytica ST 1. The IgBP(s) was isolated by affinity chromatography and used in a far-Western blot to show that the IgBP(s) also bound the extra-cellular matrix proteins (ECMPs) fibrinogen and fibronectin. ECMPs are known to play a role in colonization of some bacteria by acting as a bridge enabling binding between bacteria expressing extracellular matrix-binding proteins and epithelial cells. Flow cytometry showed the surface expression of an IgBP(s) on whole cell M. haemolytica. Immune sera from convalescent cattle showed an increased antibody response by Western blot to the 75.0 kDa IgBP when compared to sera from naive or acutely infected cattle. It was demonstrated that all 12 serotypes (1, 2, 5--9, 12--14, and 16) bound both bovine Fc IgG and sheep Fc IgG. Significant differences were not found between ST1, most frequently isolated in infections in cattle and ST2, isolated predominantly in sheep mannheimiosis. The effect of various M. haemolytica growth conditions on IgBP(s) expression was assessed by 2-D gel electrophoresis analysis. Membrane expression of M. haemolytica IgBP(s) was increased a minimum of two fold when M. haemolytica was grown in RPMI-1640 with 10%FBS, RPMI-1640 at 40°C and RPMI-1640 with 50 muM 2,2\u27-dipyridyl when compared to growth in RPMI-1640 at 37°C. The IgBP(s) constituted up to 19.9% of M. haemolytica membrane proteins when M. haemolytica was grown in RPMI at 40°C. This study shows a possible role in virulence for the M. haemolytica IgBP(s). It may enable M. haemolytica to evade the host immune response and may play a role in colonization through binding fibronectin or fibrinogen