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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have successfully developed and optimized a three-dimensional differentiated airway epithelial cell model at air-liquid interface (ALI) of the ovine respiratory tract for use in infectious disease research. We have optimized a range of parameters including pore density of the substrate membrane, media type, ambient oxygen tension, and the concentration of key hormones and growth factors including retinoic acid, epidermal growth factor and triiodothyronine. We have assessed the effect of these parameters on cellular differentiation, composition and thickness of the epithelial cell layer, tight junction integrity, degree of ciliation, cilia-beating, presence of goblet cells and production of mucus. These findings have been published (O'Boyle et al. [2018] PLoS ONE, 13(3), e0193998). We have also performed detailed time-course experiments in which we have monitored proliferation and differentiation of the epithelial cell layer from days 0 to 42. We have established that the epithelial cell layer is fully differentiated by day 21 and remains healthy until day 42. These findings are important because they indicate that the model is "fit-for-purpose" for an extended period of time and this will increase it's usefulness and likely uptake by other researchers. This work has also been published (O'Boyle et al. [2017] PLoS ONE 12(7), e0181583). We have carried out novel infection time-course studies of differentiated ovine airway epithelial cells using a panel of eight M. haemolytica isolates representing pathogenic and commensal strains. In these studies, we have followed the course of infection over 5 days and have demonstrated that pathogenic and commensal strains of M. haemolytica differ in their ability to invade and infect ovine airway epithelial cells; pathogenic strains invade and rapidly multiply within the epithelial layer whereas commensal strains are unable to do so. Importantly, we have demonstrated for the first time that M. haemolytica invades airway epithelial cells. These findings have important implications for our understanding of ovine pneumonic pasteurelllosis and for the development of improved prevention and treatment measures. This study is complete and a manuscript is being prepared for publication. Overall, our data demonstrate that the model provides a highly effective tool for studying host-pathogen interactions within the ovine respiratory tract; it is capable of differentiating between virulent and avirulent strains of M. haemolytica and can be used for long-term infection studies (up to 5 days). Crucially, we have identified features of infection that have not previously been described and have developed an in vitro model that has significant potential for replacing/reducing the use of sheep in infectious disease research. In addition, we have also demonstrated that the model can be used as a tool to study the effects of antibiotics in a system that more closely mimics the in vivo environment than does standard minimum inhibitory concentration (MIC) assays. This work was conducted in collaboration with our industrial partner, MSD Animal Health. Furthermore, by scaling-up the size and number of cultures infected, we have recovered sufficiently-large numbers of bacteria to allow outer membrane proteins (OMPs) to be recovered. We have demonstrated that OMP profiles (in SDS-polyacrylamide gels) of bacterial cells recovered from airway epithelial cell cultures are different to those obtained from bacteria grown under normal laboratory conditions. We are currently assessing these differences using proteomic approaches with the intention of identifying OMPs that are uniquely up-regulated during the infection process. Finally, we have also compared three different approaches for constructing gene deletions (knock-outs) in M. haemolytica. Diverse approaches were used in order to attempt to develop new tools and maximise the potential for successful generation of a mutant. The lktA leukotoxin-encoding gene was selected as a deletion target for all three approaches as it has a critical involvement in pathogenesis and deletions can be assessed visually for a lack of haemolysis on blood agar. The allelic exchange system developed by the industrial partner (MSD Animal Health) performed best and we subsequently constructed deletion mutants of M. haemolytica lacking selected OMPs (autotransporter proteins) that are either known or predicted to be involved in adherence and colonisation. Preliminary infection experiments were performed with these deletion mutants. In conclusion, this project has been highly successful. We have established and optimized a three-dimensional differentiated airway epithelial cell model (at ALI) of the ovine respiratory tract. We have used the model as an in vitro tool to investigate infection by M. haemolytica and to investigate antibiotic transport and efficacy. Finally, we have demonstrated using proteomic approaches that bacteria from infected epithelial cells express a unique repertoire of OMPs that are currently under investigation. Therefore, the model has significant potential for furthering our understanding of the pathogenesis of respiratory tract infections and for the development of new and improved therapeutics and vaccines.
Exploitation Route The three-dimensional airway epithelial cell model of the ovine respiratory tract that we have developed and optimized may be used by others studying bacterial and viral infections of the ovine respiratory tract. Our findings are also relevant to researchers studying respiratory tract infections of other animal species. The use of our model to study antibiotic activity is novel and has significant potential for studying antimicrobial activity leading up to animal experimentation and clinical trials. We have also demonstrated that bacteria recovered from infected cells have a different repertoire of outer membrane proteins than do bacteria grown under standard laboratory conditions. We are currently attempting to identify these proteins using proteomic approaches. Therefore, the model could be used to identify previously unrecognized proteins that have key roles in infection in vivo. Such proteins could represent key targets for improved vaccines and/or antimicrobials. We have demonstrated that the model could be used in experiments instead of using animals; therefore, the model has significant potential 3Rs-related impact.
Sectors Agriculture

Food and Drink

Pharmaceuticals and Medical Biotechnology

 
Description We presented the model at Glasgow Explorathon 2016 (part of European Researchers' Night), held at the Glasgow Science Center, in September 2016 (over 350 visitors attended our display). We designed and presented an interactive display that focused on our development of a three-dimensional model of the respiratory tract for studying infectious disease and replacing/reducing the use of animals in scientific research. There were opportunities for the general public to observe animal tissue and our 3-D tissue culture samples by microscopy and have face-to-face discussions with the researchers about the project, the principles of the 3Rs and other alternatives to animal research. In this way, we demonstrated how lung tissue from slaughtered animals (derived from a local abattoir) can be used to generate a living epithelial layer in the laboratory (with beating cilia and mucus-producing goblet cells). We highlighted how this model of the respiratory tract can subsequently be used in scientific research to study the interactions of pathogens with the respiratory tract. In this way, the model can be used to better understand respiratory disease processes and ultimately help in the design of improved vaccines to prevent infection. We emphasized how the model can be used in this way without performing experiments on living animals; thus, the development of our model will reduce the use of animals in scientific research and will therefore have a beneficial impact on the 3Rs. The successful development of our three-dimensional model of the ovine respiratory tract for infectious disease research will lead to improved understanding of economically-important respiratory tract infections of sheep and ultimately to improved methods of prevention and treatment. For example, in collaboration with our industrial sponsor MSD Animal Health, we have used the model to compare the action of selected antibiotics on infection with M. haemolytica. Therefore, our findings have potential economic and societal impacts and could be used in the "agriculture, food and drink" and "pharmaceutical and "medical biotechnology" sectors.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Title Development of a three-dimensional differentiated airway epithelial cell of the ovine respiratory tract 
Description We have developed a three-dimensional differentiated airway epithelial cell model of the ovine respiratory tract for use in infectious disease research. Epithelial cells are derived from the trachea of freshly killed sheep (from a local abattoir), expanded in tissue culture flasks and seeded onto semi-permeable membranes. At confluency, an air-liquid interface is introduced and the growth conditions manipulated such that the cells differentiate to form a pseudostratified layer that mimics the natural in vivo epithelium. We have used the model to study infection by Mannheimia haemolytica - an important respiratory tract pathogen of sheep. We have demonstrated that the model represents an excellent in vitro tool for studying respiratory tract pathogens and has significant potential for studying the pathogenesis of a wide range of pathogens. The development of this model is likely to impact on the 3Rs by replacing and reducing the number of sheep used in infectious disease research. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2016 
Provided To Others? Yes  
Impact We have also used the model to study antibiotic diffusion across the respiratory and demonstrated that the model has potential in pharmaceutical research for testing drug efficacy in an in vivo-like environment. MSD Animal Health have expressed an interest in the use of this model for testing anti-microbial drugs. 
 
Description Provison of antibodies by Zoetis 
Organisation Zoetis
Country United States 
Sector Private 
PI Contribution Preliminary discussions have taken place with Zoetis, one of the world's largest animal health companies, regarding the provision of reagents (antibodies against Mannheimia haemolytica and bovine respiratory disease virus) for use in our research. We propose to use these antibodies to explore the use of our model as an in vitro tool for screening potential vaccine candidates. Further discussions are planned for 13th March 2019 about collaboration in this area.
Collaborator Contribution Our partner (Zoetis) have agreed in principle to supply reagents (antibodies) for use in our research.
Impact None at present. This is a very recent collaboration.
Start Year 2017
 
Description Use of differentiated bovine airway epithelial cells for drug and vaccine testing 
Organisation Zoetis
Country United States 
Sector Private 
PI Contribution Development of the AEC model as an in vitro tool for drug and vaccine testing.
Collaborator Contribution Development of the AEC model as an in vitro tool for drug and vaccine testing.
Impact None yet
Start Year 2020
 
Description Public engagement event at European Researchers' Night (Explorathon) held at Glasgow Science Center 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact My team designed and presented an interactive display for Glasgow Explorathon 2016, part of the European Researchers Night, held at the Glasgow Science Center in September 2016. This was entitled "Life and death: alternatives to animal research" and was funded by a NC3Rs public engagement award. The event was attended by members of the general public and school parties, as well as by staff and students representing various Scottish institutions; we had over 350 visitors. The event was also attended by a representative from the NC3Rs.

Our interactive display highlighted the overall objectives and principles of the NC3Rs and focused on our development of a three-dimensional model of the bovine respiratory tract for studying infectious disease and replacing/reducing the use of animals in scientific research. The activity involved the use of a "hands-on" model of the respiratory tract, animal tissue and the 3-D tissue-culture model itself; these were supported by posters explaining the processes involved in the creation of our airway epithelial cell model (reusable components of the display will also be used for future engagement events). There were also opportunities for the public to observe animal tissue and our 3-D tissue culture samples by microscopy and have face-to-face discussions with the researchers about the project, the principles of the 3Rs and other alternatives to animal research. In this way, we demonstrated how lung tissue from slaughtered cattle (derived from a local abattoir) can be used to generate a living epithelial layer in the laboratory (with beating cilia and mucus-producing goblet cells). We highlighted how this model of the respiratory tract can subsequently be used in scientific research to study the interactions of pathogens with the respiratory tract. In this way, the model can be used to better understand respiratory disease processes and ultimately help in the design of improved vaccines to prevent infection. We emphasized how the model can be used in this way without performing experiments on living animals; thus, the development of our model will reduce the use of animals in scientific research and will therefore have a beneficial impact on the 3Rs.

On the night we had roughly 350 interactions with the general public, mostly families with young children and young adults. We were able to have discussions on both the importance of the 3Rs and how researchers are reducing the use of animals in research, as well as the work performed by our research group and the impacts our investigation will have on researching economically important animal pathogens. This sparked a lot of interest from the general public where we were able to highlight the activities of the NC3Rs. We were also able to engage with the public to increase interest in microbiology and host-pathogen interactions, particularly in younger children. Audience opinion was evaluated during the event, and we will use this feedback to inform future public engagement events.
Year(s) Of Engagement Activity 2016