Mechanisms of host-pathogen interaction between the bovine endometrium and the cholesterol dependent cytolysin of Trueperella pyogenes

Mechanisms of interaction between the bovine endometrium and the cytolysin of Trueperella pyogenes

Dairy cows commonly suffer bacterial disease of the uterus after parturition. We will find out how the most important bacteria damage the cells of the uterus and how the cells respond. The results will direct the development of new treatments.

IMPORTANCE
Dairy cows are important as they supply milk for human consumption. Production of milk depends on the fertility of the cow, because if there is no calf, there is no milk. Cows need to produce a calf each year to maintain milk production, so they must conceive about 3 months after calving. However, a common disease among dairy cows threatens this process. Dairy cows are particularly susceptible to infection of the uterus after calving. Every year almost half of all dairy cows develop a bacterial infection of the uterus, resulting in the discharge of copious amounts of pus from the genital tract, and causing the animal pain and suffering. The disease also reduces milk yields and causes infertility. Unfortunately, treatments have changed little in the last forty years and are not particularly effective, with many animals remaining infertile. Uterine disease costs the EU dairy industry 1.4 billion Euro each year for treatment, reduced milk yields and replacement of infertile animals. Farmers keep extra animals to replace infertile cows but these extra animals also have a damaging effect on the environment. Extra animals mean more strain on land and water resources, and increased production of greenhouse gases. So, there is an urgent need to improve the treatment of uterine disease.

CONTEXT
Several bacteria cause uterine disease, but only the presence of Trueperella pyogenes - literally "the pus producer" - is consistently associated with the severity of disease, tissue damage and infertility. There have been many clinical studies testing empirical treatments for uterine disease but with little success. Our idea is to start at the "other end" of the problem and find out the mechanisms linking Trueperella pyogenes with disease. All our strains of Trueperella pyogenes from the diseased uterus produce a toxin called pyolysin. Other disease-causing bacteria that infect different tissues in the human body produce similar toxins. These toxins are attracted to areas of cholesterol in the cell membrane where they form holes, which kills the cells. In preliminary work we have established that pyolysin also kills endometrial cells.

RESEARCH AIMS
Our research aims are to find out how the cells of the uterus respond to pyolysin; how sex hormones or metabolism affect the response to the toxin; and how cells detect pyolysin and defend themselves against it.

THE RESEARCH
First we will test how sensitive each of the cell types in the uterus is to pyolysin - what concentration of the toxin kills the cells and how the cells respond to lower concentrations. To aid in these experiments we have pure pyolysin, antibodies against pyolysin, and genetically manipulated on of our strains of T. pyogenes to remove the ability to produce pyolysin. We have many years of experience in collecting and using the cells from the uterus, and recently developed a new method of studying intact uterine tissue in the laboratory. To further understand the mechanisms of disease, we will work out how the hormones and metabolism that regulate endometrial function may also modify the interaction between pyolysin and cells. Finally, we will identify the mechanisms used by cells to detect or recognise pyolysin, and the ways in which they defend themselves against the toxin. We are encouraged that we can make a major step forward because, during preliminary work, we made an unexpected discovery of certain drugs that protect endometrial cells against pyolysin.

IMPACT OF THE RESEARCH
Our research will provide a platform for the rational design of new treatments to limit or prevent uterine disease in cattle.

Disease of the uterus after parturition is caused by bacteria that damage the endometrial lining of the uterus, affecting about 40% of dairy cows. The disease costs the EU dairy industry Euro1.4 billion annually for treatment, reduced milk production, and replacement of infertile animals. Trueperella pyogenes is the pathogen most correlated with endometrial damage, clinical signs and infertility. The major virulence factor of T. pyogenes is pyolysin and is produced by all uterine clinical isolates. Pyolysin is a cholesterol-dependent cytolysin, which creates pores in mammalian plasma membranes.

Our first objective is to determine the impact of T. pyogenes and pyolysin on primary endometrial stromal and epithelial cells, macrophages, and our intact-endometrium in vitro organ culture. We will measure cell survival, production of inflammatory mediators, and changes in intracellular signalling pathways, metabolites and cholesterol. The mechanisms underlying the differences in cellular sensitivity and responses will help explain the pathogenesis of uterine disease. Next, we will identify how the ability of host cells to respond and defend against pyolysin is modulated by factors that impact endometrial function - steroid hormones, energy balance, and oxygen tension. The mechanisms linking steroids or metabolism to host responses for any cytolysin are mostly unexplored, so our work will provide insights of wider relevance than just pyolysin. Finally, we will establish how host cells detect and defend against pyolysin by investigating ion fluxes, intracellular signalling pathways, innate immunity, and cellular cholesterol. To explore these cell defence mechanisms, we will use chemical inhibitors, active molecules and siRNA for signalling pathways, and we will manipulate cell cholesterol.

The results from our project will direct how the dairy, veterinary, pharmaceutical and animal health industries develop novel therapeutics against pyolysin to combat uterine disease.

Dairy cows are an important source of food for humans. We work on an endemic disease that compromises animal productivity, farm sustainability and food security world wide. Infection of the female genital tract with bacteria affects >90% of dairy cattle after parturition. Between 20% and 40% of postpartum animals develop acute clinical disease of the uterus with pain and the discharge of pus (metritis). Chronic clinical disease of the uterus with discharge of pus persists, beyond three weeks after calving in ~20% of animals (clinical endometritis). A further ~15% of animals have persistent inflammation of the endometrium without clinical signs (subclinical endometritis). Uterine disease incidence is rapidly increasing whilst cattle fertility has fallen to an all-time low. In the UK, >1,000,000 dairy cows have uterine disease each year, and the disease costs the EU dairy industry EURO1.4 billion/year for treatment, reduced milk production, and replacement of infertile animals.

Uterine disease is treated with antibiotics and/or hormones but the treatments have changed little over the last 40 years and are not particularly effective. The disease compromises food quality and safety, and antibiotic treatments risks transfer of antimicrobial resistance to humans consuming milk. Uterine disease compromises animal welfare, and limits the sustainability and profitability of dairy farming. Rearing extra animals to replace infertile cows also degrades the environment because these animals need more land and water, and emit more greenhouse gases. Reducing the incidence of uterine disease by a third would save the EU dairy industry Euro466 million/year. Restoring cattle fertility to levels attained in 1995 would reduce methane emissions by 10% or ~46 Mt CO2equivalent/year. Convergence of goals for industry, farmers and vets, society and government is an exciting prospect.

Postpartum infections damage the endometrium, which is the functional lining of the uterus necessary for normal reproductive cycles, conception and pregnancy. Although uterine disease is associated with several bacteria, Trueperella pyogenes is the most pathogenic. Only T. pyogenes infection is correlated with the severity of endometrial pathology, the severity of clinical disease and the extent of subsequent infertility. In our preliminary work, killing of endometrial cells depended on T. pyogenes secreting an exotoxin called pyolysin.

Pyolysin (PLO) is the major virulence factor of T. pyogenes and a member of the cholesterol-dependent cytolysin (CDC) family of exotoxins. The CDCs insert into the plasma membrane of mammalian cells to create pores that cause cytolysis. The highly conserved plo gene is expressed by all strains of T. pyogenes, and all our clinical isolates from the uterus produce functional PLO protein. However, almost nothing is known about the details of the interaction between PLO and endometrial cells, or the mechanisms of the cellular response to PLO. We have the tools and resources to address this gap in knowledge.

Defining the mechanisms involved in cell damage and defence induced by PLO is relevant to scientists studying host pathogen interactions. Identifying novel pathways to counter the action of PLO will inform scientists that work on CDCs and other pore-forming toxins. To guide disease control programmes we will communicate our understanding of how metabolism and hormones affect the interaction between PLO and the endometrium to veterinarians and farmers. Finally, understanding the mode of action of PLO in the endometrium will inform development of novel therapeutic approaches by animal health companies, and we already have long-term international R&D links with the two main players for uterine disease (Pfizer and Merck).

The proposed project will benefit dairy farmers and their animals, veterinarians, and the dairy, pharmaceutical and animal health industries.