Molecular interactions of Mannheimia haemolytica with the bovine and ovine respiratory tracts using three-dimensional tissue engineering approaches

Background. Respiratory disease complex is a multifactorial condition of cattle and sheep that involves interactions between different bacterial and viral pathogens and causes significant economic losses to livestock industries worldwide. Although various bacterial species are associated with bovine and ovine respiratory disease, Mannheimia haemolytica is considered to be the principal bacterial pathogen involved. There is an urgent need to develop more effective vaccines and antibiotics against M. haemolytica but progress towards improved antimicrobials is hampered because the pathogenesis of M. haemolytica is poorly understood and protective antigens are ill-defined. A major reason for this is that current in vitro methods available to investigate the molecular interactions of this pathogen with the host respiratory tract (RT) are poorly developed. Consequently, there is a compelling need to develop in vitro methods that can be used to study the interactions of pathogens with the bovine and ovine RTs. A relatively new and hugely promising approach involves the three-dimensional (3-D) culture of differentiated primary airway epithelial cells grown at an air-liquid interface (ALI). In this method, a polarized and fully differentiated pseudostratified epithelium containing both ciliated and non-ciliated cells is produced and this provides an excellent physiologically-relevant in vitro mimic of the RT for the study of both short- and long-term host-pathogen interactions. There are approximately 100 proteins in the outer membrane of M. haemolytica but very little is known about the roles of these outer membrane proteins (OMPs) in host-pathogen interactions. Bacterial protein expression is very different under in vitro and in vivo growth conditions and the key proteins in bacterial infection are those that are specifically expressed in vivo. Quantitative proteomic analysis of bacterial cells growing in contact with the 3-D epithelial cell models at an ALI represents a powerful and novel approach that will allow the identification of key target proteins involved in interactions with the host RT. The use of the 3-D airway models also represents an innovative approach for studying antimicrobial efficacy in vitro.

Aims and objectives. The project aims to use novel 3-D tissue engineering approaches to investigate molecular interactions of M. haemolytica with the bovine and ovine RTs. Bioinformatics and proteomic approaches will be used to identify key proteins, with particular emphasis on OMPs, that are up- and down-regulated during interactions of selected strains with airway epithelial cells. The roles of OMPs identified in this way, in processes associated with adherence and colonization, will be confirmed by assessment of knockout mutants in the airway epithelial cell models. Consequently, we will gain a much improved understanding of the molecular basis of host-specificity and virulence of M. haemolytica and identify potential protein targets for more effective disease management. The models will also be used in a proof-of-concept approach to assess the antimicrobial effects of antibiotics on M. haemolytica under conditions that mimic those encountered in vivo. The 3-D models will support the future selection of new in vitro compounds prior to animal clinical trials.

Applications and benefits. The proposed project is an industrial collaboration with MSD Animal Health and will result in the commercialisation and exploitation of the scientific data generated. The project will lead to the identification of bacterial proteins which could represent targets for new drug candidates with innovative modes-of-action. In addition, the 3-D models will allow the future selection of new in vitro-active compounds that may be tested in subsequent animal trials. Therefore, the proposal is likely to have major economic and societal benefits.

Objectives. The overall aim of the project is to use novel 3-D tissue engineered airway models to investigate molecular interactions of M. haemolytica with the bovine and ovine RTs. Bacterial proteins that are regulated during these interactions will be identified and, with emphasis on outer membrane proteins (OMPs), proteins involved in adherence and colonization confirmed. In this way, we will gain an improved understanding of the molecular basis of host-specificity and virulence of M. haemolytica and identify potential protein targets for more effective disease management. The models will also be used to assess the antimicrobial effects of selected antibiotics on M. haemolytica under conditions that mimic those encountered in vivo.

Methods. Bovine and ovine airway epithelial cells will be obtained from respiratory tract material derived from freshly slaughtered animals at a local abattoir. Airway cells will be grown to confluence on porous membrane inserts as submerged cultures and differentiation triggered by creating an air-liquid interface. The integrity, structure and functioning of the epithelium will be monitored by biochemical and morphological characterization to determine the optimal "window" for infection studies. The adherence and colonization characteristics of six M. haemolytica isolates will be monitored both quantitatively and using microscopic approaches for up to 4 days. Adherent bacteria will be separated from epithelial cells at different stages of infection and quantitative proteomic approaches used to assess the regulation of proteins, with emphasis on OMPs, during the colonization process. The potential roles of proteins in adherence and colonization identified in this way will be confirmed by the creation of targeted mutants and by assessment of their subsequent interactions with airway cells. The antimicrobial activities of six antibiotics against three bovine isolates of M. haemolytica will also be assessed using the bovine model.

This joint research proposal in collaboration with MSD Animal Health involves the development and use of new and innovative 3-D tissue engineering methodologies and cross-disciplinary approaches to investigate molecular interactions of M. haemolytica with the bovine and ovine respiratory tracts (RTs). At the end of the project, we will have an improved understanding of the molecular mechanisms involved in the early stages of pneumonic pasteurellosis and will have identified new protein targets for the management of disease. The novel methods and approaches employed in the project will be of value to national and international researchers studying a wide range of RT pathogens infecting cattle, sheep and other species. The cross-disciplinary nature of the proposed project will be relevant to a wide range of academic disciplines. Therefore, there are a large number of potential academic beneficiaries and the proposal is likely to have significant academic impact. The development of novel approaches for studying host-pathogen interactions of the RT, together with the identification of potential vaccine antigens and drug targets, will make a significant contribution to combating an important infectious disease of farmed animals and will have a major impact on improving animal health, a BBSRC priority area. The development of new antimicrobials will enhance the efficiency and performance of the livestock industries and will contribute to wealth creation and economic prosperity both within the UK and globally. Therefore, the proposal is likely to make a major economic and societal impact. The successful development and validation of the 3-D models for studying bacterial RT infections of cattle and sheep will lead to the replacement and reduction of these animals in scientific research. In addition, the models are likely to become more widely used to study pathogens of other animal species. Therefore, the proposal will have a significant impact on the 3Rs initiative, also a BBSRC priority area. The development and use of the 3-D models to replace and reduce the use of animals in scientific research, in the current societal climate, will have wider interest to the general public and within schools and higher education.

The impact of the proposal will be enhanced by engagement with specific beneficiaries and stakeholders, both nationally and internationally, to allow the timely development of the 3-D models in other areas. This will be achieved as follows:

(1) Industrial beneficiaries. Industrial collaboration with MSD Animal Health will result in the commercialisation and exploitation of scientific knowledge. The project will lead to the identification of bacterial proteins which could represent targets for new drug candidates with innovative modes-of-action. In addition, the 3-D models will allow the future selection of new in vitro-active compounds that may be tested in subsequent animal trials. The appointed PDRA will receive training with the company at Schabenheim, Germany.

(2) Specific academic beneficiaries. Prof. Anthony Confer is one of the world's leading authorities on bovine respiratory disease and related vaccine research. Prof. Confer has agreed that the appointed PDRA may visit his laboratory for the purpose of technology transfer and to receive training in techniques specific to his laboratory. Dr. Geraldine Taylor is Head of Vaccinology at the Pirbright Institute and has agreed to collaborate on the future development of the 3-D models to perform co-infection studies involving bacterial and viral pathogens. Dr. Jayne Hope is a research scientist at the Roslin Institute, Edinburgh and has agreed to collaborate on the future development of the 3-D models to investigate the innate immune response of airway epithelial cells to bacterial pathogens involved in respiratory disease; this will provide a clearer understanding of early immunological events likely to influence the outcome of host-pathogen interactions.