Source: UNIV OF CONNECTICUT submitted to
CHARACTERIZATION OF NOVEL PATHWAYS INVOLVED IN MEDIATING PLANT-DERIVED MOLECULE INHIBITION OF STAPHYLOCOCCUS AUREUS INFECTION OF BOVINE MAMM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0227798
Grant No.
2012-67016-30210
Project No.
CONS-2011-02824
Proposal No.
2011-02824
Multistate No.
(N/A)
Program Code
A1221
Project Start Date
Jan 1, 2012
Project End Date
Dec 31, 2015
Grant Year
2012
Project Director
Govoni, K.
Recipient Organization
UNIV OF CONNECTICUT
(N/A)
STORRS,CT 06269
Performing Department
Animal Science
Non Technical Summary
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.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113499103010%
3113499104030%
3113499110010%
3114010103010%
3114010104030%
3114010110010%
Goals / Objectives
Mastitis is the most significant health problem on dairy farms and increased pathogen resistance to antibiotic treatments has demonstrated the necessity for a better understanding of the pathogenesis of this disease in dairy cattle and identification of innovative tools to control the disease. Reducing antibiotic usage in dairy cows using proven alternate approaches to maintain herd health for producing high quality and safe milk is important. Therefore, effective non-antibiotic based strategies for controlling mastitis need to be identified for the economic viability of dairies and improving public health. Based on our previous research, we have proposed two natural, GRAS-status plant molecules (trans-cinnamaldehyde and eugenol) that can potentially attenuate virulence in the mastitis pathogens, especially Staphylococcus aureus. We hypothesize that these plant molecules could potentially be used to modulate/attenuate bacterial virulence, and develop a new tool for controlling mastitis in cattle. These plant molecules being natural and food-grade will be better accepted by the producers and society without concerns for toxicity. In addition, a thorough understanding of the mechanism of pathogenesis and host-pathogen interactions is critical for the development of innovative approaches and tools to prevent mastitis in dairy cattle without the use of antibiotics. The objective of this proposal is to determine the molecular pathways by which two plant-derived molecules, namely trans-cinnamaldehyde and eugenol, reduce attachment and/or invasion of S. aureus at the pathogen and host cell level. The findings from the proposed studies will improve our understanding of the pathogen-host interaction, as well as identify novel mechanisms to develop strategies for controlling and/or preventing this costly disease. In addition, the findings from this research will be published in peer reviewed journals and presented at national meetings. This project will also provide training of graduate and undergraduate students in agricultural research.
Project Methods
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.

Progress 01/01/12 to 12/31/15

Outputs
Target Audience:The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop and at the Joint Annual Meetings of the American Society of Animal Science and American Dairy Science Association in July 2014. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistics. Additionally, students have presented data at regional and national conferences allowing them to learn from experts in the field and improve written and oral communication skills. How have the results been disseminated to communities of interest?The results have been published in an abstract in the Journal of Animal Science, presented at regional scientific seminars, local stakeholder meetings and will be submitted for publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We determined that sub-inhibitory concentrations of two plant-derived antimicrobials, trans-cinnamaldehyde (TC; 0.006 %) and eugenol (EG; 0.03 %), reduce adhesion to and invasion of four strains (Thorn 17, DST 35, M9175 and Thorn 15) of Staphylococcus aureus (SA) to bovine primary mammary epithelial cells (MEC) using an in vitro culture system. To determine the potential mechanisms by which these plant molecules prevent adhesion and invasion, we explored changes in gene expression of virulence factors in SA and immune genes in MEC. As expected, when MEC are infected with SA, we observe increased expression of several virulence genes in SA and immune response genes in MEC. However, with treatment of TC and EG we observed altered expression of these immune and virulence factors, suggesting that these plant-derived antimicrobials may work to prevent SA infection of MEC through modulating gene expression. To further explore the effect of these plant molecules on SA gene expression, we sequences the entire transcriptome of SA in the absence and presence of TC or EG. Treatment of SA with TC resulted in 110 differentially expressed genes of which 3 were down-regulated greater than 2-fold and 10 were up-regulated greater than 2-fold. Treatment of SA with EG resulted in 222 differentially expressed genes of which 17 were down-regulated by greater than 2-fold and 13 were up-regulated greater than 2-fold. To gain a better understanding of the host-pathogen interaction during mastitis infection, we developed an in vitro model in which MEC were infected with SA and then RNA was isolated from both the MEC and SA for whole transcriptome analysis. RNA-Seq analysis determined that 706 genes in SA are differentially expressed when exposed to MEC and 1,260 genes were differentially expressed in MEC with infected with SA. These whole transcriptome analyses demonstrate that infection of MEC with SA causes several changes to both the host and pathogen, which contribute to infection. Additionally, TC and EG may be able to prevent infection by modulating the expression of these genes in SA. These studies demonstrate that novel methods to prevent or treat mastitis may be viable using TC or EG; however in vivo experiments are needed to confirm these findings. Lastly, there are several mechanisms at the host and pathogen level that contribute to mastitis infection and the host's immune response.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Jaganathan, D, Kollanoor-Johny, A, Venkitanarayanan, K, Kazmer, GW, Kuo, L, Wang, YB, and Govoni, KE. 2014. Plant-derived compounds, trans-cinnamaldehyde and eugenol, reduce adhesion and invasion of Staphylococcus aureus in bovine mammary epithelial cells in vitro. J Anim Sci Vol. 92, E-Suppl 2, p 182.


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:The findings from this research will be presented in peer-reviewed scientific journals and at national scientific meetings. Additionally the findings will be presented at local and regional seminars which target faculty and students. Changes/Problems:Several challenges were faced with optimizing RNA extraction from dual samples. We overcame these challenges and have excellent sequencing data from prokaryotic and eukaryotic RNA which originated from one sample. What opportunities for training and professional development has the project provided?This project has trained a Ph.D. student and several undergraduate students in microbiology and RNA-Seq. How have the results been disseminated to communities of interest?Results have been presented as an abstract at an international meeting and are being prepared for manuscripts in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?This project is coming to completion and the final results will be submitted for publication and used as preliminary data for future grant proposals.

Impacts
What was accomplished under these goals? To date we have determined that plant-derived antimicrobials, trans-cinnamaldehyde (TC) and eugenol (EG), reduced adhesion to and invasion of Staphylococcus aureus (S. aureus) to bovine mammary epithelial cells (MEC) in vitro. Specifically, using 4 strains of S. aureus and MEC from two different cows, we observed a 1 to 2-log CFU/mL reduction in adhesion when S. aureus and MEC were pre-treated with TC or EG (P < 0.05). Pre-treatment of S. aureus and MEC with TC or EG reduced invasion 2 to 4-log CFU/mL (P < 0.05). To determine potential mechanisms that contribute to TC and EG reduction of adhesion and invasion, we determined mRNA expression of prokaryotic and eukaryotic genes by real-time RT-PCR. In S. aureus, treatment with EG and TC reduced expression of virulence genes nuclease, intercellular adhesion, human leukocyte antigen, fibronectin binding protein-B, and fibronectin binding protein-A (P < 0.05) and increased expression of hlB (P < 0.05). Treatment of MEC with EG and TC increased expression of inflammatory markers complement, toll-like receptor-2, and Interleukin-8 (P < 0.05). To further elucidate the mechanisms by which the pathogen infects the host and the plant molecules prevent this infection, we performed RNA-Seq analysis. In the first experiment, prokaryotic mRNA was sequenced using Illumina MiSeq and 174 and 53 genes were differentially expressed with TC and EG treatment, respectively (P < 0.05). The second experiment used dual RNA-Seq to determine the host-pathogen interaction by determining prokaryotic and eukaryotic mRNA expression using Illumina NextSeq. These data are currently being aligned and analyzed.

Publications


    Progress 01/01/14 to 12/31/14

    Outputs
    Target Audience: The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop and at the Joint Annual Meetings of the American Society of Animal Science and American Dairy Science Association in July 2014. Changes/Problems: We found the process of RNA-Seq challenging since we could not separate the eukaryotic and prokaryotic RNA before sequencing. So we spend a lot of time optimzing the dual-RNA-Seq procedures. We have promising preliminary results and will use this method for RNA-Seq analysis. What opportunities for training and professional development has the project provided? The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistic. How have the results been disseminated to communities of interest? The results have been shared with scientists at the University of Connecticut, during the PD workshop and at national meetings. What do you plan to do during the next reporting period to accomplish the goals? We plan to repeat the experiments in a third animal, extract RNA from all three animals, complete RNA-Seq analysis, and publish the findings in a peer-reviewed journal.

    Impacts
    What was accomplished under these goals? To date we have determined that sub-inhibitory concentrations of two plant-derived antimicrobials, trans-cinnamaldehyde (TC; 0.006 %) and eugenol (EG; 0.03 %), reduce adhesion to and invasion of four strains of Staphylococcus aureus (SA) in bovine primary mammary epithelial cells (MEC). Specifically, for strain Thorn 17, pre-exposure of SA (5 h) and MEC (12 h) to EG reduced SA adhesion to and invasion of MEC by ~1.6 ± 0.01 log10 CFU/mL (control = 6.67 ± 0.01 log10 CFU/mL; P < 0.0001) and 2.8 ± 0.11 log10 CFU/mL (control = 3.9 ± 0.02 log10 CFU/mL; P < 0.0001), respectively. Pre-exposure of SA and MEC to TC reduced SA adhesion to and invasion of MEC by ~2.2 ± 0.02 log10 CFU/mL (control = 6.7 ± 0.02 log10 CFU/mL; P < 0.0001) and 2.85 ± 0.08 log10 CFU/mL (control = 3.94 ± 0.01 log10 CFU/mL P<0.0001), respectively. Similar results were observed with strains DST 35, M9175 and Thorn 15. Real- time quantitative PCR data revealed that PDAs down-regulated critical virulent genes of SA (intercellular adhesion and nuclease) and modified immune genes of MEC (complement, toll-like receptor2, interleukin1-beta and interleukin6; P ≤ 0.05). Preliminary analysis of SA and MEC RNA from these experiments has been completed using the Dual RNA-Seq procedure using Hi-Seq 2000 from Illumina.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 54. Jaganathan, D, Kollanoor-Johny, A, Venkitanarayanan, K, Kazmer, GW, Kuo, L, Wang, YB, and Govoni, KE. 2014. Plant-derived compounds, trans-cinnamaldehyde and eugenol, reduce adhesion and invasion of Staphylococcus aureus in bovine mammary epithelial cells in vitro. J Anim Sci Vol. 92, E-Suppl 2, p 182.


    Progress 01/01/13 to 12/31/13

    Outputs
    Target Audience: The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop in October 2013. Changes/Problems: We have faced several challenges with isolating RNA for RNA-Seq analysis. In particular, obtaining enough prokaryotic RNA from samples in which S. aureus invasion has been reduced or prevented has posed a challenge. We are working with more samples to increase our yield. We also anticipate finding a challenge with mapping our RNA-Seq data to the bovine genome. However, more publications are available now with sequence data that we will use to map to our findings. What opportunities for training and professional development has the project provided? The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistic. How have the results been disseminated to communities of interest? The results to date have been shared with students and faculty at the University of Connecticut and with other scientists at the annual PD workshop in October 2013. What do you plan to do during the next reporting period to accomplish the goals? We plan to repeat the expeirments using different strains of S. aureus. In addition, we will complete isolationf of RNA and whole transcriptome analysis using RNA-Seq. The current results are being presented at the ASAS meeting in July 2014 and a manuscript is in preparation.

    Impacts
    What was accomplished under these goals? To date we have confirmed that our primary mammary gland cell population is primarily mammary epithelial cells using immuno-histochemistry staining for multi-cytokeratin. In addition, we have determined the sub-inhibitory concentrations of TC as 0.006% and 0.0125% and EG as 0.03%. These concentrations do not inhibit mammary epithelial cell growth in culture. Consistent with our preliminary findings in MAC-T cells, TC inhibits attachment of S. aureus (6.1 ± 0.2, 5.4 ± 0.3, and 4.6 ± 0.5 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) and invasion (3.4 ± 0.05, 2.4 ± 0.09, and 0.8 ± 0.2 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) when incubated with S. aureus alone. When both S. aureus and mammary epithelial cell are incubated with TC, attachment (5.1 ± 0.2, 4.2 ± 0.2, and 2.5 ± 0.3 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) and invasion (3.0 ± 0.02, 1.1 ± 0.16, and 0 log CFU/mL; control, 0.006%, and 0.0125% TC) are further reduced. Pre-treatment of S. aureus with EG reduced attachment (5.6 ± 0.1 vs. 4.3 ± 0.3 log CFU/mL; control vs. EG) and invasion (3.0 ± 0.3 vs. 1.4 ± 0.3 log CFU/mL; control vs. EG) of mammary epithelial cells. When both S. aureus and mammary epithelial cell were incubated with EG attachment (5.7 ± 0.04 vs. 3.8 ± 0.07 log CFU/mL; control vs. EG) and invasion (3.2 ± 0.03 vs. 0 log CFU/mL; control vs. EG) were further reduced. Statistical analysis is in progress. Data presented are the average of three separate experiments.

    Publications


      Progress 01/01/12 to 12/31/12

      Outputs
      OUTPUTS: This project has provided collaboration between the Department of Animal Science and the Statistics Department at UConn. In addition, it has contributed to the training of a Ph.D. candidate in Animal Science and two Ph.D. candidates in Statistics. An undergraduate student in Animal Science has also received laboratory skills training through this project. PARTICIPANTS: Kristen Govoni, Kumar Venkitanarayan, Gary Kazmer, Devi Jaganathan, Cameron Smart; University of Connecticut, Department of Animal Science. Lynn Kuo, Patrick Harrington, Yu-Bo Wang; University of Connecticut, Statistics Department. TARGET AUDIENCES: This work targets scientists, faculty, graduate students and undergraduates. This work will be presented at national meetings, in peer-reviewed scientific journals and local and national seminars. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      This project is still in the preliminary stages of development. To date, we have established the primary mammary epithelial cell line from five different cows. These cells have been expanded and stored in cryo tanks for proposed experiments. In addition, we are in the process of optimizing RNA extraction from both prokaryotic and eukaryotic RNA from the same cell culture well. We have also repeated the attachment and invasion assays completed in our preliminary data (MAC-T cells) using primary mammary epithelial cells. We have confirmed successful attachment and invasion of S. Aureus using one strain. After several trial experiments with different MOI (eukaryotic to bacterial cell) of 1:10,1:20,1:30,1:100, we found that 1:100 i.e. 10^5 : 10^7 (eukaryotic to bacterial cell) showed adequate invasion of Staphylococcus aureus into the bovine mammary epithelial cells in cell culture. The invasion with 1:100 MOI was determined to be around 4 log CFU / ml. The invasion rate with other MOI was found to be < 3 log CFU/ml. We have decided to use an MOI of 1:100 using staphylococcus strain DST 35. Further this MOI will be checked with other strains of mastitis causing Staphylococcus aureus (DST 38 and DST 4) for its invasion ability into the bovine mammary epithelial cells.

      Publications

      • No publications reported this period