Survival and dissemination of enteric pathogens through activation and subsequent inhibition of programmed cell death pathways

After being ingested on infected food pathogenic bacteria are taken up by cells in the intestine, entering the cells and attempting to grow. An infected cell recognizing the danger undergoes a tightly controlled form of cell suicide known as apoptosis. Thus the cell removes itself and the danger whilst simultaneously sending out warning signals to the immune system. E. coli and Salmonella are two of the most common food poisoning associated pathogens in the U.K. Based on our knowledge of the body's response to infection we would expect cells to undergo apoptosis when they come into contact with E. coli and Salmonella. In the case of these pathogens however the infected cells survive for some time following infection allowing the bacteria to grow within the infected cell. This extra time for growth is crucial, allowing these pathogens time to multiply and cause a more serious prolonged infection. Through this proposal we aim to understand how these pathogens are prolonging the life of infected cells. This work will have relevance for numerous bacterial pathogens, many of which we now know also attempt to interfere with this process of apoptosis during infection.

Our previous work has shed new light on the complex interactions occurring in Salmonella infected cells. During infection the bacteria deliberately target proteins in the cell that respond to the infection by inducing cell suicide. In particular one extremely potent host cell enzyme called caspase-3, or the 'executioner caspase' is targeted. This is the key enzyme in inducing cell suicide, killing the cell quickly but in a controlled fashion. Due to the destructive power of caspase-3 its activation is tightly regulated, making its targeting by bacterial pathogens all the more surprising.

However instead of trying to prevent caspase-3 from working these bacteria initially try to harness its activity bypassing the stringent controls put in place to ensure caspase-3 activity is kept in check. We now know Salmonella infection is activating caspase-3 by a unique mechanism and understanding and exploiting this is a key objective for this proposal.

Activation of an enzyme as destructive as caspase-3 is a risky strategy for a pathogen that only has a limited time to try to grow within an infected cell. Salmonella and E. coli however have developed a mechanism to control the destructive power of the enzyme. Host cells have a natural recycling system called the proteasome that is used to take old or damaged proteins and break them down and use the building blocks to form new proteins. These bacterial pathogens tap into this recycling system, using their own proteins to mimic proteins from the cell that sort host proteins for recycling. Using this tactic the bacteria divert caspase-3 from its normal role, causing the host cell to inadvertently send it for recycling. This delays cell death meaning the bacteria can now multiply within the cell.

This tactic we believe is replicated by numerous other bacterial pathogens during infection, meaning this proposal will have repercussions for the study of numerous microbial infections.

Bacterial pathogens are now known to interact intimately with apoptotic pathways, promoting their persistence through selective activation and inhibition of these pathways. Here we will characterize these pathogen-apoptosis interactions building on novel findings from my laboratory.

Initial studies will characterize the novel mechanism by which the enzyme is activated by a translocated effector protein in circulating immune cells. Although we have identified the effector responsible, its interaction and that of other effectors with the many pathways regulating apoptosis remain unresolved. We will elucidate the apoptotic response during early stages of infection, generating a comprehensive picture of bacterial interactions with this crucial process.

Induction of apoptosis is a high-risk strategy for a bacterial pathogen but we have identified an innovative bacterial approach to delaying cell death, use of the cell's own ubiquitination machinery. Ubiquitin ligase mimics are well characterized in a number of pathogens and these bacterial proteins are crucial to the unique phenomenon of bacterial induced turnover of caspase-3 that we see during infection. This tagging of caspase-3 with ubiquitin to induce its proteasomal degradation aids in the maintenance of the intracellular replication niche for an increased length of time, enabling bacterial growth and promoting persistence.

Understanding bacterial interactions with these pathways can identify new bacterial protein targets enabling therapeutic intervention or the generation of vaccine candidates to alleviate both acute and persistence infection.

Many, if not all bacterial pathogens, interact with apoptotic pathways. Our recent discovery of direct activation of caspase-3 by S. Typhimurium in order to promote infection has already had an important impact on the field of infectious disease work. Building on this discovery by elucidating the mechanisms employed by S. Typhimurium to not only activate but also control caspase-3 is the priority in this proposal. Dissemination of the findings will benefit others in the field researching other acute and persistent microbial infections. Activating or preventing cell death is a key part of many types of infection and identifying the mechanisms and bacterial proteins involved will allow for further research into their potential therapeutic targeting. In this regard this proposal will have implications for research into the survival of pathogens within their livestock and human hosts, shedding light on a novel pathogenic mechanism that has far reaching relevance in the field.

The overarching goal of this research is to gain an understanding of bacterial pathogens' ability to undermine the apoptotic process. The potential for exploitation of this finding for use in therapy is great as this project studies two novel discoveries in caspase-3 control, direct activation and its inhibition through ubiquitination. Elucidating how bacteria undermine caspase-3 through the use of bacterial effectors will potentially allow for vaccine mediated intervention. This will result in potential vaccine candidates that can be tested, an area that the mentor for this project Professor Mark Roberts has many years experience and success in.

So while the field of infectious disease research has much to gain from this proposal in the longer term this research will also I believe prove invaluable to understanding the basic cellular biology of apoptosis. Bacteria in this proposal are being used by us to highlight potential weak points in the regulation and control of apoptosis, weak points which without the use of bacteria would remain undiscovered. Elucidating the mechanisms employed by Salmonella to undermine caspase-3, specifically in regard to SipA which directly activates the enzyme, could lead to the development of therapies for directly stimulating apoptosis in cells where it is defective. In this regard collaborations have been put in place with to share our findings with researchers at the Beatson Institute for Cancer Research in Glasgow with a view to testing selected bacterial proteins in appropriate animal models in the future. Understanding the mechanism behind direct activation of caspase-3 is of great significance and potentially has a great many therapeutic uses but this activation may be very complex and must be understood completely.

Understanding apoptosis and in particular defective apoptosis has become an important part of my research in the past number of years. Presently I collaborate with the Medical Genetics Unit at Yorkhill Children's Hospital in Glasgow where I am analyzing samples from patients that are heterozygous for caspase-3 expression in an attempt to better understand caspase-3 regulation. The work undertaken in my laboratory has positively benefitted these medical professionals and patients and aided in the diagnosis of a highly unusual genetic condition. In addition we have recently instigated collaborations with researchers studying veterinary pathogenesis at the University of Glasgow and at the Moredun Research Institute. We hope that through these collaborations we can apply our work to reduce the carriage of pathogenic organisms in livestock.