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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.
Highlights
Mannheimia haemolytica is an opportunistic Gramnegative bacterium that belongs to the Pasteurellaceae family
Bovine hololactoferrin was not used as a sole iron source by M. haemolytica, and bovine apolactoferrin showed a bactericidal effect against this bacterium In iron-chelated BHI medium (Additional file 1), both strains of M. haemolytica required 80 μM iron for growing, under the conditions tested in this work; this bacterial species was not able to grow when bovine hololactoferrin (BholoLf) was added as a sole iron source to the iron-chelated BHI broth, even with 80 μM iron derived from BholoLf (Figure 1)
Bovine lactoferrin mainly binds to two M. haemolytica outer membrane proteins To determine whether M. haemolytica possesses bovine lactoferrin (BLf) binding proteins, overlays were performed with extracted OMP and binding to horseradish peroxidase (HRP)-coupled BholoLf and Bovine apoLf (BapoLf) was explored
Summary
Mannheimia haemolytica is an opportunistic Gramnegative bacterium that belongs to the Pasteurellaceae family. This bacterium is part of the bovine respiratory disease (BRD), which causes important economic losses in livestock. The influence of the environment, stressing factors, and infections by viruses and bacteria, causes BRD; these factors seem to alter the bovine upper respiratory-tract epithelium allowing M. haemolytica to colonize it, escape clearance and move from the nasopharynx to the lungs, leading to pneumonia [1]. M. haemolytica does not produce siderophores; receptors for siderophores have been found in its genome. M. haemolytica genes coding HmbR1 and HmbR2 proteins to capture hemoglobin and the use of this ferrous protein as an iron source have been demonstrated [19]
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