Metritis is the result of dysbiosis of uterine microbiota caused by an over-growth of F. necrophorum, B. pyogenes, and P. levii. These bacteria are opportunistic pathogens commonly found in the uterus of healthy cows. It is still unclear how/why they remain commensal or what factors trigger their pathogenic transformation within the uterus. The major goal of the project is to develop solutions for maintaining uterine homeostasis through an improved understanding of the mechanisms by which these opportunistic pathogens regulate or activate host inflammatory responses. To achieve the goal of the project, we propose the following aims.Aim 1 will identify the interactions of F. necrophorum, B. pyogenes, and P. levii for enhancing growth, colonization, and pathogenicity. The objectives of Aim 1 are to: 1) determine bacterial growth rates in culture media, separately and together; 2) determine the capability of these bacteria to form biofilms, separately and together; 3) determine the in vitro cytotoxicity of these uterine pathogens against bovine endometrial epithelial cells (BEND).Aim 2 will characterize the in vitro metabolic profiles of F. necrophorum, B. pyogenes, and P. levii to determine what metabolic pathways are involved in their interactions and what bacterial metabolites are associated with endometrial infection. The objectives of Aim 2 are to: 1) determine carbon sources utilized by F. necrophorum, B. pyogenes, and P. levii; 2) identify bacterial products that are toxic to BEND; 3) characterize extracellular and intracellular metabolites produced by these 3 bacterial species during infection of BEND.Aim 3 will investigate the host responses to F. necrophorum, B. pyogenes, and P. levii using a 3-D culture model of the endometrium. The objectives of Aim 3 are to: 1) develop a 3-D tissue culture model of the endometrium; 2) identify transcriptomes for both bacteria and BEND using dual RNA-Seq; 3) determine the cytokine/chemokine response by BEND.Our approach for an in vitro infection model of F. necrophorum, B. pyogenes, and P. levii using a 3-D culture of bovine endometrial epithelial cells will expand our knowledge regarding molecular mechanisms of bacterial interaction and their effects on uterine homeostasis and the development of metritis. The identification of metabolites that modulate bacterial interaction and host response may lead to new intervention strategies for the prevention and treatment of metritis and improve the overall health and reproductive performance of dairy herds.