Development and use of an in vitro air-liquid interface model of the bovine respiratory tract and multifaceted proteomic approaches to investigate host-pathogen interactions of Mannheimia haemolytica

In the present study, an air-liquid interface culture system was optimised for the use of bovine airway epithelial cells. This culture system has been adopted for other species, where a pseudostratified epithelium comprising multiple cell types and resembling the in vivo environment is produced. Several aspects of the epithelial culture process were optimised, including (i) the cell harvesting process and the expansion of cells in submerged culture, (ii) the substrate material used for air-liquid interface culture, the effects of collagen-coating of the substrate, and the substrate porosity, (iii) the oxygen tension (iv) growth factor (retinoic acid and epidermal growth factor) concentration used during culture and (v) the anatomical region of the respiratory tract from which the epithelial cells were harvested. The optimisation of the aforementioned conditions resulted in the production of an in vitro respiratory tract model that was more than 40% ciliated, pseudostratified with a layer of P63-expressing basal cells and capable of mucus and CC10 (Clara cell secretory protein) production. Thus, an in vitro model of the bovine respiratory tract was established to determine the host-pathogen interactions of M. haemolytica with the respiratory epithelium. A rigorous proteomic characterisation was carried out on the outer membrane of M. haemolytica. The Sarkosyl insoluble fractions of seven M. haemolytica and one M. glucosida isolates, representing a range of serotypes and host species, were prepared in triplicate. Tryptic digests of each sample were prepared using gel-based and gel-free proteomic digests before analysis by LC-MS/MS. Data was searched against the NCBI Uniprot database using MSCOT (Matrix Science). Assembly of the data revealed that a total of 83 unique outer membrane proteins were distributed amongst the eight Mannheimia spp. isolates. Fifty seven of the 83 OMPs were identified using both gel-free and gel-based methodologies with a further 18 and eight identified with a single methodology alone. Several proteins were identified in an isolate specific manner; two OMPs exhibited a strong pattern of serotype-specific identification. The YadA-like trimeric autotransporter was identified with full reproducibility in A2 serotype strains yet not in strains of different serotype. Conversely, the lipoprotein, plpD was identified in at least two replicates of all isolates except those of serotype A2 where it was not identified at all. The air-liquid interface culture system was used to study the adherence and colonisation of Mannheimia haemolytica to the bovine respiratory tract. In a pilot study, pathogenic bovine and ovine isolates M. haemolytica isolates were studied with bovine and ovine air-liquid interface airway epithelial cells. Bovine and ovine M. haemolytica isolates were found to adhere to the epithelial cells, six hours post-infection in the case of an ovine isolate and 24 hours in the case of a bovine isolate. Bovine isolates of M. haemolytica were predominantly found to invade and adhere to the sub-apical regions of the epithelium with relatively few bacteria found on the apical face. Adherence to the sub-apical epithelium represents the first description of M. haemoyltica causing invasive disease. Microscopic evidence of outer membrane vesicle production and exopolymeric material were also noted for one of the ovine isolates during infection. Following optimisation of the bovine air-liquid interface culture system, an experiment was conducted that examined the ability of M. haemolytica to adhere and colonise air-liquid interface cultures with epithelial cells derived from different anatomical regions of the respiratory tract. Cells derived from the nasopharynx, the mid-trachea and the secondary/tertiary bronchi were differentiated at ALI and infected with pathogenic and non-pathogenic bovine isolates of M. haemolytica. The non-pathogenic M. haemolytica isolate was found to adhere and colonise the nasopharyngeal derived cells better than either the bronchial or tracheal derived cells. Conversely, the pathogenic M. haemolytica isolate was found to have significantly greater adherence to the bronchial derived epithelial cultures and significantly higher adherence (P>0.0001) than the non-pathogenic isolate. The pathogenic M. haemolytica isolate was also found to colonise the nasopharyngeal-derived epithelial culture albeit with lower frequency than was observed for the bronchial-derived epithelial cells. The results presented herein demonstrate the development of a novel in vitro tool for studying the infection process of respiratory pathogens. The use of this tool to study the infection properties of M. haemolytica in combination with characterisation of the outer membrane proteome provides new insight into the process of colonisation and highlights the protein machinery that likely contributes to the process