Identification ofMannheimia haemolyticaAdhesins Involved in Binding to Bovine Bronchial Epithelial Cells

Abstract

Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by anti-M. haemolytica antibody. Western blot analysis of M. haemolytica proteins that bind to BBEC showed a dominant protein band with an apparent molecular mass of ϳ30 kDa. Peptide sequences for the 30-kDa BBEC-binding proteins, as determined by liquid chromatography-tandem mass spectrometry, matched two M. haemolytica surface proteins: heat-modifiable outer membrane protein A (OmpA) and lipoprotein 1 (Lpp1). Western blotting showed that the 30-kDa protein band is recognized by both anti-M. haemolytica OmpA and anti-Lpp1 antibodies. Furthermore, incubation with anti-OmpA and anti-Lpp1 antibodies significantly inhibited M. haemolytica binding to BBEC monolayers. In summary, these results suggest that OmpA and Lpp1 contribute to adherence of M. haemolytica to bovine respiratory epithelial cells.Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex (40, 54). The bacteria colonize the nasal cavity and tonsillar crypts in healthy cattle (18, 54). However, following stress or viral infection, M. haemolytica bacteria rapidly increase in number and descend into the lungs, leading to acute pneumonia (18,19). It is still not clear what mechanisms allow M. haemolytica to transform from a commensal organism to a pathogen that escapes clearance from the respiratory tract and invades the lung.The adherence of respiratory pathogens to the mucosal epithelium is a critical step in host colonization and infection (1). Previous studies demonstrated the ability of M. haemolytica to adhere to epithelial cells in vitro (7,20,52) and the mucosal surface of respiratory tissue explants (8,34). M. haemolytica produces several surface components that potentially can contribute to adherence. Fimbriae and glycocalyx were identified on M. haemolytica cells grown in culture and on bacteria associated with tracheal tissues isolated from experimentally infected cows (33, 34). However, the role of these structures in M. haemolytica adhesion has never been investigated. Similar to other gram-negative bacteria, M. haemolytica produces major outer membrane protein A (OmpA) (31, 55). This protein is also referred to as heat-modifiable outer membrane protein (OMP) or, in M. haemolytica, PomA (2, 31, 55). OmpA participates in specific binding to cell receptors and mediates adherence of several pathogenic bacteria to host cells (4,12,45,46,51). Based on sequence homology, it has been suggested that the OmpA of M. haemolytica might play a role in colonization of the respiratory tracts of cattle and sheep (13). M.haemoly...

Mannheimia haemolytica

“…94 Another outer membrane protein, OmpA, is a highly conserved protein that is involved in binding to specific host cell receptors in the upper respiratory tract, thereby playing a role in adherence and colonization and in generating host specificity. 37,63 Other studies have found that outer membrane proteins stimulate the release of nitric oxide, induce the expression of iNOS in interferon g-activated macrophages, increase actin polymerization, and modify oxidative burst in neutrophils. 57,84 Toxins and Extracellular Enzymes Leukotoxin M haemolytica LKT and LPS are well-characterized virulence factors with respect to their pathogenic role in the SF.…”

“…56,88 However, a locus encoding such an adhesin has not yet been identified. 53 Kisiela and Czuprynski 63 recently identified the heat-modifiable M haemolytica outer membrane protein OmpA and lipoprotein Lpp1, which are involved in adherence to the bovine bronchial epithelium.…”

Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15

“…E. coli OmpA has been shown to be an abundant protein and a predominant antigen, occurring at a copy number of approximately 100,000 copies per cell (27,45). With the demonstrated pathogenic functions of OmpA, such as epithelial adhesion and binding to fibronectin, and its immunogenicity, addition of rOmpA to M. haemolytica vaccines should be examined further (26,29,31).…”

Serotypic diversity and sequence variation of the ompA gene among Mannheimia haemofytica isolates from domestic ruminants

“…Based on sequence homology, Davies and Lee (2004) suggested that the OmpA of M haemolytica is involved in binding to bovine and ovine receptors, thus playing an important role in the colonization of the respiratory tracts of cattle and sheep. It has also been demonstrated that OmpA shows a specific interaction with the extracellular matrix (EMC) protein fibroncctin (Lo and Sorenscn 2007) and that it contributes to adher ence of M. haemolytica to bovine bronchial epithelial cells (BBEC) in vitro (Kisiela and Czuprynski 2009). Moreover, a recent study has shown that two different OmpA subclasses, OmpAl and OmpA2, could poten tially bind to different receptors in cattle and sheep (Ilounsomc et al 2011).…”