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

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description This project aims to understand how Trueperella pyogenes and the bacterium's cholesterol dependent cytolysin (pyolysin, PLO) impact the bovine endometrium. We have made three advances during the first 9 months of the grant.

1. Endometrial stromal cells are remarkably sensitive to PLO
Purified populations of primary endometrial stromal cells are far more sensitive than endometrial epithelial cells or circulating white blood cells to cytolysis caused by native PLO from T. pyogenes (Fig 1). Furthermore, PLO is the main cytolytic agent of T. pyogenes because cells die when exposed to live T. pyogenes clinical isolates of bacteria but not to plo gene deletion mutant bacteria (T. pyogenes ?plo).
These two findings were included in a manuscript on Trueperella pyogenes, which is published in Biology of Reproduction.

2. Endometrial tissues mount inflammatory responses to T. pyogenes but not to PLO
Endometrial tissues mount inflammatory responses including the secetion of cytokines such as IL-6 when exposed to Trueperella pyogenes or T. pyogenes ?plo. The responses to bacteria likely depend on detection of bacterial lipoproteins by the innate immune system. Indeed, primary bovine endometrial cells and macrophages express the innate immune receptor (TLR2) necessary to detect bacterial lipoproteins (interim report for BB/I017240/1; manuscript under second review at Endocrinology). However, PLO does not simulate inflammatory responses by endometrial tissue, endometrial epithelial or stromal cells, or immune cells.
These findings are important for the present grant application to study the integration of energy metabolism with innate immunity, because we need to use factors that stimulate innate immunity. Clearly PLO is not relevant to the interaction of energy metabolism with innate immunity because is does not stimulate inflammation. However, the potential for metabolism impacting cytolysis is included in the existing grant BB/K006592/1. Therefore, we are not missing a scientific opportunity and we avoid any conflict of funding with our present application.

3. Pyolysin activates intracellular signalling pathways
In work to understand how cells respond to PLO, in the absence of an inflammatory response, we have discovered that endometrial stromal cells activate caspase-1 when treated with PLO, and there are changes in signalling pathways involving ERK1/2, p38a (MAPK14), JNK. Similarly, we are using Western blotting to examine the abundance of AKT, HIF-1a and Caspase-1 in our cells. We are currently studying which of these signalling pathways are involved in protection of endometrial cells against the PLO cytolysin.

4. The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 µM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5'-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-ß-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs. We have published a paper in FASEB Journal and a Review on this matter.

5. We have three angles of attack to enhancing cellular resilience to bacterial pore-forming toxins:

A. We are currently working on how manipulation of the mevalonate pathway modulates the resilience of cells against Pyolysin. Results are encouraging and we have a collaboration with the Department of Chemistry at Cardiff University to synthesise compounds to inhibit key enzymes. We have synthesised small molecules that inhibit a target in the cholesterol synthesis pathway. These novel molecules are not cytotoxic. We are now uncovering the biological data showing that these small molecules make cells more resilient or tolerant to cholesterol-dependent cytolysins. The work is at the discovery stage and we are building our underpinning data prior to protecting the IP. A manuscript is in preparation on the novel molecules.

B. We have identified components of the cholesterol pathway and their receptors that enhance the resilience of cells to cholesterol-dependent cytolysins. We are using a commercially available agonist for the target receptor, and we can inhibit toxin-induced cytolysis by > 80% using agonist concentrations < 1 nM. This is an exciting discovery, and whilst we do not hold the IP for the agonist, our suggested route of application and our target disease may have sufficient IP to warrant patent protection. We are finalising the underpinning research data for our IP consultants. A manuscript is also in preparation.

C. We have used inhibitors of enzymes in the mevalonate pathway to successfully enhance the resilience of bovine endometrial stromal cells and human cells against cholesterol-dependent cytolysins. We have two manuscripts in preparation covering the two parts of the cholesterol-synthesis pathway that confer protection against bacterial toxins.

6. Factors that influence endometrial cell physiology, also affect cell resilience against Pyolysin. Our latest work has been exploring how fundamental cellular pathways and nutrients might impact cellular resilience to cholesterol-dependent cytolysins. We have screened a range of nutrients, metabolites, and hormones for their effect on how cells respond to bacterial toxins. In particular, we have discovered that cellular resilience to pyolysin is dependent on carbon metabolism. Both glucose and glutamine deficits impact cellular resilience, and the effect can be rescued by anapleurotic supplementation of the Krebs cycle. A major manuscript is in preparation for submission in Q2 2017.
Exploitation Route We have published two research papers, and a review, on the role of dynamin and pyolysin.

Three papers are in preparation on how manipulating the cholesterol-synthesis pathway enhances cellular resilience to pore-forming toxins.

We have a collaboration with the Department of Chemistry at Cardiff University to synthesise compounds to inhibit key enzymes that affect cell resilience against pyolysin.

A major research paper is in preparation to describe the role of cellular metabolism and the sensitivity to cholesterol-dependent cytolysins. We think we have a substantive step forward here. However, we are likely to only have the time and resource to publish what we set out in the grant application, which are the descriptive findings. To determine the underlying mechanisms we will need further substantial work, extending into new areas of discovery; as a consequence, I am writing further funding applications.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Industry is exploring the use of inhibitors of cholesterol synthesis pathways for protecting dairy cows against uterine disease. We are collaborating with 3 pharmaceutical companies on potential routes to develop a preventative agent for postpartum uterine disease in cattle. One of our partners is planning a study to take our work forward in vivo. We have also used the knowledge from this project to generate an in vivo model of uterine disease in cattle for collaborators at the University of Florida, USA.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description PAR-13-204:Research in Biomedicine and Agriculture
Amount $1,861,408 (USD)
Funding ID 1R01HD084316-01A1 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 10/2016 
End 05/2021
 
Title A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes 
Description Piersanti RL, Zimpel R, Molinari PCC, Dickson MJ, Ma Z, Jeong KCC, Santos JEP, Sheldon IM, Bromfield JJ 2019 A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Journal of Dairy Science, 10.3168/jds.2018-15595 Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade =1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact This model is now in use in the USA to study the impact of uterine disease on ovarian function for animal and human health. 
 
Title Recombinant Pyolysin 
Description We have generated recombinant cholesterol-dependent cytolysin, pyolysin 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact We have developed collaborations and subsequent publications sharing our resource. 
 
Description MSD 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution We are exploring how to help Merck Inc (MSD) to prevent uterine disease in dairy cattle.
Collaborator Contribution Merck are seeking novel methods to increase the robustness of dairy cattle against uterine disease.
Impact Series of teleconferences and sharing of data and standard operating procedures.
Start Year 2018
 
Description Mevalonate pathway chemistry 
Organisation Cardiff University
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Developing the chemistry of nitrogen-containing bisphosphonates to modulate the mevalonate pathway. We are leading the biological evaluation of the compounds.
Collaborator Contribution Prof Ruedi Allemann, Head of Chemistry, is leading the development of the chemistry of the compounds.
Impact Studentship funded by the Welsh Government Ser Cymru programme
Start Year 2015
 
Description Supplying Trueperella pyogenes isolates 
Organisation French National Institute of Agricultural Research
Department INRA Rennes Centre
Country France 
Sector Public 
PI Contribution We supply fully sequenced isolates of our clinical isolates of Trueperella pyogenes for research in vitro and in vivo
Collaborator Contribution We are conducting collaborative projects, and seeking further funding in some cases.
Impact In vivo animal model (Confidential IP)
Start Year 2015
 
Description Supplying Trueperella pyogenes isolates 
Organisation University of Florida
Department Department of Animal Science
Country United States 
Sector Academic/University 
PI Contribution We supply fully sequenced isolates of our clinical isolates of Trueperella pyogenes for research in vitro and in vivo
Collaborator Contribution We are conducting collaborative projects, and seeking further funding in some cases.
Impact In vivo animal model (Confidential IP)
Start Year 2015
 
Description Supplying Trueperella pyogenes isolates 
Organisation Zoetis
Country United States 
Sector Private 
PI Contribution We supply fully sequenced isolates of our clinical isolates of Trueperella pyogenes for research in vitro and in vivo
Collaborator Contribution We are conducting collaborative projects, and seeking further funding in some cases.
Impact In vivo animal model (Confidential IP)
Start Year 2015
 
Description Supplying recombinant cholesterol-dependent cytolysin pyolysin and mutants 
Organisation Vilnius University
Country Lithuania 
Sector Academic/University 
PI Contribution We are generating and sharing our recombinant cholesterol-dependent cytolysin pyolysin and mutant forms of the toxin to academic partners.
Collaborator Contribution We collaborated on a project that examined the activity of pyolysin in model memebranes and their biophysical properties
Impact The project that yielded a publication in BBA Biomembranes
Start Year 2016
 
Description Biennual innovation meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Developed IP
Developed product operating plans
Innovated for research

Patent application submitted
Year(s) Of Engagement Activity 2010,2011,2012,2013
 
Description Bishopstons Mens Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk to men on infertility in women and animals

Local interactions with people interested in science
Year(s) Of Engagement Activity 2015