CHARACTERIZATION OF NOVEL PATHWAYS INVOLVED IN MEDIATING PLANT-DERIVED MOLECULE INHIBITION OF STAPHYLOCOCCUS AUREUS INFECTION OF BOVINE MAMM

Mastitis is the most economically significant disease in dairy cows. Antibiotic treatments against mastitis are not always successful for multiple reasons, including emergence of drug resistant pathogens. Moreover, antibiotics are common harmful drug residues detected in milk, raising a public health hazard. Thus, there is a need for developing alternate tools to antibiotics for controlling mastitis, for which a thorough understanding of mastitis pathogenesis and host-pathogen interactions is critical. We demonstrate that two food-grade, plant molecules, trans-cinnamaldehyde and eugenol decreased the invasion of S. aureus in bovine mammary epithelial cells. Moreover, trans-cinnamaldehyde substantially down-regulated several virulence genes in Staphylococcus aureus, suggesting that trans-cinnamaldehyde and eugenol may be used to attenuate virulence of pathogens and control mastitis. This study will characterize the molecular pathways involved in the interaction between S. aureus and bovine mammary epithelial cells using high-throughput transcriptome analysis, and determine the mechanisms by which trans-cinnamaldehyde and eugenol reduce pathogen attachment and/or invasion at bacterial and host cell levels. This research will identify novel pathways involved in host-pathogen interactions, generating critical information for developing new approaches to control mastitis. This could permit curtailed usage of antibiotics in cattle, leading to decreased emergence of antibiotic-resistant pathogens and drug residues in milk, thereby improving economic opportunities for dairy farmers, animal health and public health in general.

To test our hypothesis, we will utilize the novel method of whole transcriptome sequencing to identify key factors involved in mediating the effects of plant-derived molecules on pathogen attachment and/or invasion of the host. Specifically, mammary epithelial cells will be challenged with Staphylococcus aureus and treated with sub-inhibitory doses of trans-cinnamaldehyde and eugenol. We will determine the effect of trans-cinnamaldehyde and eugenol on the attachment and invasion of mammary epithelial cells by the pathogens. In addition, we will perform whole transcriptome analysis on both prokaryotic and eukaryotic RNA from cells treated with and without trans-cinnamaldehyde and eugenol and challenged with and without pathogen. Lastly, we will use real-time PCR to validate key genes identified in the transcriptome analysis. Transcriptome analysis will be completed using statistical methods including QuEST, F-seq, GisGenome, and Markov models.