Final Report Summary - RECOIN (Studying the Mechanisms of Enhanced Pathogenesis<br/>in Polymicrobial Respiratory Co-Infection)
Summary description
Disease caused by microorganisms accounts for a significant financial and economic burden in the modern world. Taken together, viral, fungal and bacterial infections cost billions of pounds a year not only in human disease, leading to lost work hours, but also within an agricultural context where microorganisms can have a significant impact on domesticated animals such as beef and dairy cattle.
The initial event in the vast majority of this disease is the interaction of the pathogen with the host animal. Animals have many barrier systems in place including the skin, mucosal systems and sloughing epithelial cells lining outward-facing tissues. It is with these epithelial cells that pathogens must first interact in order to colonise the host. These initial interactions are complex and highly dependent upon specific facets of both the pathogen and the host, providing tropism for particular microorganisms. Influenza virus, for example, interacts with the respiratory epithelial cells, whereas Salmonella bacteria principally interact with the intestinal lining. Even after an initial contact, these host-pathogen interactions play a critical role in disease progression. Many fungal pathogens for example, upon entering the blood stream perform a second round of host interaction, with the cells of the blood-brain barrier.
Broadly, the main objectives of the project were:
(a) To develop a model system with which to study host-pathogen interactions
(b) To develop and use high temporal and spatial resolution 4-dimensional microscopy to study the mechanistic basis of host-pathogen interactions.
The tasks were as follows:
TASK 1: To source and establish cell models (RO1)
TASK 2: To develop or source bacterial strains (RO1)
TASK 3: To optimise imaging parameters (RO1)
TASK 4: To develop image analytical software (RO1-2)
TASK 5: Characterising bacterial adhesion (RO1)
TASK 6: Recapitulating co-infection (RO2)
TASK 7: Role of neuraminidase (RO2)
TASK 8: Receptor and Cellular Characterisation (RO2)
TASK 9: Grant application to expand the project into independent research area
Work performed and main results
TASK 1: To source and establish cell models (RO1):
Both primary cells (from human and ferret) and transformed cell lines were characterised for suitability for growth and imaging. Transformed cells were selected for reasons of cost and practicality.
TASK 2: To develop or source bacterial strains (RO1):
Streptococcus pneumoniae were transformed to express fluorescent proteins, however, under experimental conditions, the bacteria quickly degraded the fluorescent proteins as a stress response. Furthermore, in their natural state the bacteria form diplococci or chains making them significantly more difficult to automatically identify and track in software.
PROJECT REDIRECTION:
Having spent some months on Tasks 1 and 2, it was decided that in order to keep the project on track, the model system should be changed from a respiratory organ system to a model of the digestive system, whereupon more well established cell and bacterial systems could be utilised.
Tasks 1 and 2 were effectively repeated to validate the new direction. Cells representing intestinal epithelia were grown and characterised for their epithelial properties.
To compliment the cell system, a range of Salmonella strains were acquired. In line with the modified Task 2, these strains (including ones lacking certain adhesion factors) were manipulated to produce different fluorescent proteins.
TASK 3: To optimise imaging parameters (RO1):
Through collaboration with staff of the Birmingham Advanced Light Microscopy (BALM) facility and the hardware manufacturers, high resolution imaging was achieved with a suitable spatial resolution (equivalent to a volume of ~120µm x 120 µm x 30 µm) in quick enough succession (up to 50 frames per second) to acquire useful imaging data.
TASK 4: To develop image analytical software (RO1-2):
4-dimensional data sets were analysed using a plugin for the open-source analysis software ImageJ. The plugin is used to convert 4D imaging data into trajectory maps, that is to say, linked spatial co-ordinates in time, for each bacterium. A suite of custom-written scripts and macros were written to convert these data into metrics for analysis.
TASK 5: Characterising bacterial adhesion (RO1):
Initially, polarised Caco2 cell monolayers were exposed to GFP-labelled wild-type bacteria and imaged as the bacteria interacted with the host cells. The following parameters were selected for their biological significance: Instantaneous velocity, diffusion mode, dwell time and number of dwell events. Considering these factors, a number of novel observations were made that would not have been seen using conventional end-point adhesion assays that rely upon averaging of vast population-level data.
TASK 6: Recapitulating co-infection (RO2):
While preliminary investigations had begun into introducing a cognate viral pathogen such as norovirus or rotavirus into the system, there was not enough time to complete this task.
TASK 7: Role of neuraminidase (RO2):
Had the project successfully introduced a viral component into the system, and there were suitable reagents to block or study viral components, this Task would have detailed them. Given time restraints this task was not completed.
TASK 8: Receptor and Cellular Characterisation (RO2):
Due to time restraints, this task was not completed.
TASK 9: Grant application to expand the project into independent research area:
Despite the novelty of the technique, the amount of time that was required to develop the technique precluded forming enough data to form the basis for a novel research program. It is our hope that the work will form part of a broader publication in the future.
Final results and impact
The change of model organ system after completion of Tasks 1 & 2, set back the project by a considerable number of months. Added to this the amount of time required for the development of imaging protocols and the validation of data analysis techniques, the project ran out of time before reaching Tasks 6-8. Tasks 1-5, however were highly successful and produced some novel and interesting results, briefly covered below.
After acquisition and quantitation of a large amount of data, we found that many parameters could be gleaned from high resolution 4D imaging experiments. The choice of parameters depended largely on the model system. For example, when looking at Salmonella interactions with host epithelial cells, the most useful (and scientifically interesting) parameters extracted from our analysis were the dwell times and the number of dwell events. By comparing wild-type Salmonella with identical bacteria lacking known adhesins (for example, those encoded by the Salmonella Pathogenicity Island 1), we saw a reduction in the average dwell time of Salmonella, but not the number of interactions. This supports published data in the field but adds a potential mechanistic understanding; that SPI1 affects the strength of adhesion for bacteria already bound to cells, but does not affect the initial interactions, which are likely mediated by another class of adhesins.
These and similar findings are important because they are hard or impossible to see using conventional techniques. Furthermore, through collaboration with other researchers at Birmingham, the technique has been applied to other systems including those of Cryptococcus neoformans in brain endothelia. Finally, although it does not require the same resolution, the principal of the technique has also been used to track and analyse the migration of fibroblasts in two spatial dimensions over time, with the intention of moving the system to true 4D in the future to better understand the migration of cells within a 3D matrix.
Having overcome the major technical hurdles of the project, the system can be easily applied to this and other projects for wide-reaching effect. For example:
• introducing viral agents such as norovirus and rotavirus to study the effects of co-infection as per Annex 1
• elucidating the mechanism of blocking by small adhesion inhibitors
• studying the mechanism of action of antibacterials/peptides that alter the host cells or microorganism cell surface
Importantly, this also provides the project with sustainable applications for the future.
Training and Transfer of Knowledge
TRAINING ACTIVITIES: There have been several opportunities for training activities, including taking on supervision for graduate students and a 10-week project student who provided assistance with the data analysis aspects of the project. Further training has been provided through involvement in Journal Club meetings to critically assess published articles, grant-writing workshops and figure and manuscript preparation.
KNOWLEDGE TRANSFER: As per Annex 1, B2.1 Dr. Mason has been involved with groups at Birmingham University including collaborations with Prof. Tim Mitchell (Gram positive pathogens), Prof. Laura Piddock (bacterial host-pathogen interactions), Prof. Adam Cunningham (Salmonella biology), Prof. Ian Henderson (Salmonella adhesins) and Prof. Robin May (pathogenic yeast biology) to facilitate the imaging and analysis of experimental data. Furthermore, he has taken on several Masters-level teaching assignments including helping to plan and run a Midlands Integrative Biological Training Partnership (MIBTP) workshop on Imaging an Post Acquisition Analysis. This has given Dr. Mason the chance to inspire and teach future generations of Research Scientists at Birmingham. In addition to this Dr. Mason's extensive knowledge has been shared with his working groups for unrelated project such as the analysis of Fluorescent Recovery After Photobleaching (FRAP) experiments and the quantitative analysis of Western Blots.
During his time at Birmingham Dr. Mason has also, through collaboration and integration, expanded his own skill-set. This includes, learning to handle and manipulate the gram positive pathogen S. pneumoniae, learning how to culture and purify pseudoparticle viruses as well as being exposed to and leveraging advanced imaging hardware.
INTEGRATION ACTIVITES: Throughout the project, Dr. Mason has been fully integrated into the University of Birmingham, through attendance at several different group lab meetings, seminars, Journal Club meetings and involvement in the University Mentorship scheme. Furthermore, he has attended seminars and meetings of both the Centre for Human Virology (CHV) and The Institute for Microbiology and Infection (IMI), which together constitute the majority of virology, bacteriology, mycology and parasitology conducted at the University of Birmingham.
In summary, the goals of the project in terms of Knowledge Transfer, Integration and Teaching have been achieved in a highly successful manner. The techniques developed in this project have far reaching applicability and have strengthened collaboration between individuals and groups, both at the University of Birmingham and further afield.
Disease caused by microorganisms accounts for a significant financial and economic burden in the modern world. Taken together, viral, fungal and bacterial infections cost billions of pounds a year not only in human disease, leading to lost work hours, but also within an agricultural context where microorganisms can have a significant impact on domesticated animals such as beef and dairy cattle.
The initial event in the vast majority of this disease is the interaction of the pathogen with the host animal. Animals have many barrier systems in place including the skin, mucosal systems and sloughing epithelial cells lining outward-facing tissues. It is with these epithelial cells that pathogens must first interact in order to colonise the host. These initial interactions are complex and highly dependent upon specific facets of both the pathogen and the host, providing tropism for particular microorganisms. Influenza virus, for example, interacts with the respiratory epithelial cells, whereas Salmonella bacteria principally interact with the intestinal lining. Even after an initial contact, these host-pathogen interactions play a critical role in disease progression. Many fungal pathogens for example, upon entering the blood stream perform a second round of host interaction, with the cells of the blood-brain barrier.
Broadly, the main objectives of the project were:
(a) To develop a model system with which to study host-pathogen interactions
(b) To develop and use high temporal and spatial resolution 4-dimensional microscopy to study the mechanistic basis of host-pathogen interactions.
The tasks were as follows:
TASK 1: To source and establish cell models (RO1)
TASK 2: To develop or source bacterial strains (RO1)
TASK 3: To optimise imaging parameters (RO1)
TASK 4: To develop image analytical software (RO1-2)
TASK 5: Characterising bacterial adhesion (RO1)
TASK 6: Recapitulating co-infection (RO2)
TASK 7: Role of neuraminidase (RO2)
TASK 8: Receptor and Cellular Characterisation (RO2)
TASK 9: Grant application to expand the project into independent research area
Work performed and main results
TASK 1: To source and establish cell models (RO1):
Both primary cells (from human and ferret) and transformed cell lines were characterised for suitability for growth and imaging. Transformed cells were selected for reasons of cost and practicality.
TASK 2: To develop or source bacterial strains (RO1):
Streptococcus pneumoniae were transformed to express fluorescent proteins, however, under experimental conditions, the bacteria quickly degraded the fluorescent proteins as a stress response. Furthermore, in their natural state the bacteria form diplococci or chains making them significantly more difficult to automatically identify and track in software.
PROJECT REDIRECTION:
Having spent some months on Tasks 1 and 2, it was decided that in order to keep the project on track, the model system should be changed from a respiratory organ system to a model of the digestive system, whereupon more well established cell and bacterial systems could be utilised.
Tasks 1 and 2 were effectively repeated to validate the new direction. Cells representing intestinal epithelia were grown and characterised for their epithelial properties.
To compliment the cell system, a range of Salmonella strains were acquired. In line with the modified Task 2, these strains (including ones lacking certain adhesion factors) were manipulated to produce different fluorescent proteins.
TASK 3: To optimise imaging parameters (RO1):
Through collaboration with staff of the Birmingham Advanced Light Microscopy (BALM) facility and the hardware manufacturers, high resolution imaging was achieved with a suitable spatial resolution (equivalent to a volume of ~120µm x 120 µm x 30 µm) in quick enough succession (up to 50 frames per second) to acquire useful imaging data.
TASK 4: To develop image analytical software (RO1-2):
4-dimensional data sets were analysed using a plugin for the open-source analysis software ImageJ. The plugin is used to convert 4D imaging data into trajectory maps, that is to say, linked spatial co-ordinates in time, for each bacterium. A suite of custom-written scripts and macros were written to convert these data into metrics for analysis.
TASK 5: Characterising bacterial adhesion (RO1):
Initially, polarised Caco2 cell monolayers were exposed to GFP-labelled wild-type bacteria and imaged as the bacteria interacted with the host cells. The following parameters were selected for their biological significance: Instantaneous velocity, diffusion mode, dwell time and number of dwell events. Considering these factors, a number of novel observations were made that would not have been seen using conventional end-point adhesion assays that rely upon averaging of vast population-level data.
TASK 6: Recapitulating co-infection (RO2):
While preliminary investigations had begun into introducing a cognate viral pathogen such as norovirus or rotavirus into the system, there was not enough time to complete this task.
TASK 7: Role of neuraminidase (RO2):
Had the project successfully introduced a viral component into the system, and there were suitable reagents to block or study viral components, this Task would have detailed them. Given time restraints this task was not completed.
TASK 8: Receptor and Cellular Characterisation (RO2):
Due to time restraints, this task was not completed.
TASK 9: Grant application to expand the project into independent research area:
Despite the novelty of the technique, the amount of time that was required to develop the technique precluded forming enough data to form the basis for a novel research program. It is our hope that the work will form part of a broader publication in the future.
Final results and impact
The change of model organ system after completion of Tasks 1 & 2, set back the project by a considerable number of months. Added to this the amount of time required for the development of imaging protocols and the validation of data analysis techniques, the project ran out of time before reaching Tasks 6-8. Tasks 1-5, however were highly successful and produced some novel and interesting results, briefly covered below.
After acquisition and quantitation of a large amount of data, we found that many parameters could be gleaned from high resolution 4D imaging experiments. The choice of parameters depended largely on the model system. For example, when looking at Salmonella interactions with host epithelial cells, the most useful (and scientifically interesting) parameters extracted from our analysis were the dwell times and the number of dwell events. By comparing wild-type Salmonella with identical bacteria lacking known adhesins (for example, those encoded by the Salmonella Pathogenicity Island 1), we saw a reduction in the average dwell time of Salmonella, but not the number of interactions. This supports published data in the field but adds a potential mechanistic understanding; that SPI1 affects the strength of adhesion for bacteria already bound to cells, but does not affect the initial interactions, which are likely mediated by another class of adhesins.
These and similar findings are important because they are hard or impossible to see using conventional techniques. Furthermore, through collaboration with other researchers at Birmingham, the technique has been applied to other systems including those of Cryptococcus neoformans in brain endothelia. Finally, although it does not require the same resolution, the principal of the technique has also been used to track and analyse the migration of fibroblasts in two spatial dimensions over time, with the intention of moving the system to true 4D in the future to better understand the migration of cells within a 3D matrix.
Having overcome the major technical hurdles of the project, the system can be easily applied to this and other projects for wide-reaching effect. For example:
• introducing viral agents such as norovirus and rotavirus to study the effects of co-infection as per Annex 1
• elucidating the mechanism of blocking by small adhesion inhibitors
• studying the mechanism of action of antibacterials/peptides that alter the host cells or microorganism cell surface
Importantly, this also provides the project with sustainable applications for the future.
Training and Transfer of Knowledge
TRAINING ACTIVITIES: There have been several opportunities for training activities, including taking on supervision for graduate students and a 10-week project student who provided assistance with the data analysis aspects of the project. Further training has been provided through involvement in Journal Club meetings to critically assess published articles, grant-writing workshops and figure and manuscript preparation.
KNOWLEDGE TRANSFER: As per Annex 1, B2.1 Dr. Mason has been involved with groups at Birmingham University including collaborations with Prof. Tim Mitchell (Gram positive pathogens), Prof. Laura Piddock (bacterial host-pathogen interactions), Prof. Adam Cunningham (Salmonella biology), Prof. Ian Henderson (Salmonella adhesins) and Prof. Robin May (pathogenic yeast biology) to facilitate the imaging and analysis of experimental data. Furthermore, he has taken on several Masters-level teaching assignments including helping to plan and run a Midlands Integrative Biological Training Partnership (MIBTP) workshop on Imaging an Post Acquisition Analysis. This has given Dr. Mason the chance to inspire and teach future generations of Research Scientists at Birmingham. In addition to this Dr. Mason's extensive knowledge has been shared with his working groups for unrelated project such as the analysis of Fluorescent Recovery After Photobleaching (FRAP) experiments and the quantitative analysis of Western Blots.
During his time at Birmingham Dr. Mason has also, through collaboration and integration, expanded his own skill-set. This includes, learning to handle and manipulate the gram positive pathogen S. pneumoniae, learning how to culture and purify pseudoparticle viruses as well as being exposed to and leveraging advanced imaging hardware.
INTEGRATION ACTIVITES: Throughout the project, Dr. Mason has been fully integrated into the University of Birmingham, through attendance at several different group lab meetings, seminars, Journal Club meetings and involvement in the University Mentorship scheme. Furthermore, he has attended seminars and meetings of both the Centre for Human Virology (CHV) and The Institute for Microbiology and Infection (IMI), which together constitute the majority of virology, bacteriology, mycology and parasitology conducted at the University of Birmingham.
In summary, the goals of the project in terms of Knowledge Transfer, Integration and Teaching have been achieved in a highly successful manner. The techniques developed in this project have far reaching applicability and have strengthened collaboration between individuals and groups, both at the University of Birmingham and further afield.
