Source: University of Michigan Medical School submitted to NRP
TARGETING BRANCHED CHAIN FATTY ACID SYNTHESIS TO CONTROL LISTERIA CONTAMINATION IN FOOD
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0225942
Grant No.
2011-67012-30682
Cumulative Award Amt.
$130,000.00
Proposal No.
2010-05362
Multistate No.
(N/A)
Project Start Date
Aug 15, 2011
Project End Date
Aug 14, 2014
Grant Year
2011
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
University of Michigan Medical School
1150 W. Medical Center Drive, 5751 Medical Sciences II
Ann Arbor,MI 48109
Performing Department
Microbiology and Immunology
Non Technical Summary
Listeria is a foodborne bacterial pathogen that causes listeriosis, a disease with a wide range of clinical manifestations including gastroenteritis, meningitis and spontaneous miscarriage. According to Centers for Disease Control and Prevention, a high mortality rate of 20% has been observed with infected individuals and an even higher mortality rate was reported for early neonatal infections. Moreover, Listeria is capable of propagating in diverse food products increasing the possibility of consumer exposure. As a result, Listeria contamination has been the cause of frequent food recalls costing government agencies and food industries a tremendous amount of time and money. For example, a listeriosis outbreak in 2008 killing at least four people was linked to Listeria contamination and was the cause of one of the biggest food recalls by Maple Leaf Foods Inc. with an estimated direct cost of 19 million dollars. Therefore, it is imperative to have a better understanding of the biological mechanism adapted by Listeria to persist in food processing and storage conditions normally inhospitable to microorganisms. Using bacteriocins, which are peptides with antimicrobial activity, as food preservatives or as components in surface treatments has been proposed as methods to prevent and control Listeria contamination. However, the possibility of bacteriocin usage in enriching resistant Listeria strains and increasing Listeria virulence potential needs to be addressed in order to design more effective applications of bacteriocins. This project will address both concerns and further identify novel inhibitory compounds that can simultaneously increase Listeria susceptibility to bacteriocins and decrease pathogenicity. This proposed project will define the role of branched chain fatty acids (BCFA), which are critical membrane components in Listeria, in bacteriocin resistance; investigate how exposure to bacteriocins affect Listeria pathogenesis; and discover antimicrobial agents that specifically target BCFA production to increase the susceptibility of Listeria to bacteriocins without activating virulence. When the project is complete, we will have a better basic understanding of the role of membrane fatty acids in Listeria bacteriocin resistance, establish how bacteriocins affect Listeria virulence, and identify novel anti-Listeria compounds that can enhance the activity of bacteriocins and control the prevalent Listeria contamination. The ultimate goal of this project is to provide a fundamental understanding of molecular determinants contributing to Listeria-related food safety issues and further introduce alternative approach to eliminate Listeria contamination more efficiently during food processing and storage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71234501100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3450 - Milk;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Listeria monocytogenes (Listeria) is a food-borne pathogen with the ability to survive after prolonged storage under refrigeration conditions, and thus is a notable cause of food contamination and recall of contaminated products. Listeria infection is generally associated with serious sequelae and a high mortality rate. As a result of the high economic cost associated with recall of contaminated food products and with medical treatments, it is imperative to define resistance mechanisms used by Listeria to survive food processing and storage protocols, and to design more efficient and cost-effective strategies for remediation. Antimicrobial agents such as bacteriocins, antimicrobial peptides produced by microorganisms, have been considered as candidates for in situ or ex situ treatments to control Listeria contamination. However, the emergence of resistant Listeria strains poses a significant challenge to general applications. Moreover, the effect of these antimicrobial peptides (AMP) on Listeria virulence is poorly understood. Therefore, this study is proposed to (1) define molecular determinants providing resistance against AMP in Listeria, (2) investigate how AMP exposure affects Listeria virulence, and (3) discover chemical compounds that increase the susceptibility of Listeria to AMP without increasing virulence. Using such compounds in combination with AMP could provide a synergistic approach to control Listeria contamination more efficiently than using AMP alone. This proposed study will demand the applicant's previous experiences in environmental microbiology and provide the applicant with new training in bacterial pathogenesis with the ultimate goal of discovering novel and practical solutions to Listeria contamination in food.
Project Methods
The proposed project will utilize basic microbiology and molecular biology techniques to define the role of branched chain fatty acids (BCFA) in Listeria monocytogenes resistance to antimicrobial peptides (AMP). This will be achieved by determining bacterial survival in different types of AMP using agar plates to quantify colony forming units or using flow cytometry to assess the integrity of bacterial cells. A L. monocytogenes mutant lacking the enzyme BKD necessary for BCFA synthesis has been created and used to address the contribution of BCFA by comparing survival phenotypes between the mutant and the wildtype bacteria. This project will also utilize biochemical techniques and tissue culture models of infection to determine the effect of AMP exposure on L. monocytogenes virulence. The production of virulence factor listeriolysin O (LLO) will be tested by hemolytic assay for LLO activity as well as immunoblotting for LLO abundance. The infectivity of L. monocytogenes will be tested by infecting tissue culture cell lines and determining the growth, phagosomal escape, and invasion of L. monocytogenes in response to AMP exposure. Finally, a high throughput small molecule screening will be performed to discover novel chemical inhibitors of the enzyme BKD as potential additives in preventing and controlling Listeria contamination. Findings from the proposed project will increase our current understanding of the mechanism of Listeria persistence in food storage and processing procedures. To promote knowledge learning and sharing, our findings will be regularly presented in national conferences to stimulate discussions and will be published in peer-reviewed journals for international access. The success of the project will be demonstrated by completion of all proposed experiments, recognition in scientific journals, and ultimately the discovery of novel anti-Listeria compound that will be beneficial for maintaining food safety for all Americans.

Progress 08/15/11 to 08/14/14

Outputs
Target Audience: The award has enabled publications of two papers, which are viewd by the scientific community, three conference attendance, where research was presented to a wide range of scholars and scientists, and mentoring of undergraduate students, who have further advanced to PhD programs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has allowed the PI to attend three difference conferences for scientific discussions and networking. How have the results been disseminated to communities of interest? The results have been presented at conferences and publications. 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 have identified membrane branched chain fatty acids as critical determinants in providing resistance against AMP. Furthermore, we established that virulence regulation is closely in tune with modification of membrane fatty acid composition. We discovered that compounds such as butyrate, a common intestinal short chain fatty acid, is a potent chemical that dramatically modifies Listeria fatty acid composition and suppresses virulence.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Fatty acids regulate stress resistance and virulence factor production for Listeria monocytogenes. Sun Y1, Wilkinson BJ, Standiford TJ, Akinbi HT, O'Riordan MX. J Bacteriol. 2012 Oct;194(19):5274-84. doi: 10.1128/JB.00045-12. Epub 2012 Jul 27.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Regulation of bacterial pathogenesis by intestinal short-chain Fatty acids. Sun Y1, O'Riordan MX. Adv Appl Microbiol. 2013;85:93-118. doi: 10.1016/B978-0-12-407672-3.00003-4.


Progress 08/15/11 to 08/14/12

Outputs
OUTPUTS: During the reporting period, the PI has continuously been conducting and analyzing experiments as detailed in the proposal. During the process, the PI also mentored two students, an undergraduate student in University of Michigan and a student from the NIH-sponsored Post-baccalaureate Research Education Program (PREP), in designing, executing, and analyzing experiments. With the travel budget from the fellowship, the PI attended the general meeting of American Society for Microbiology in San Francisco, CA where she presented her findings in a poster format and disseminated her ideas to other conference participants, which include students, postdoctoral researchers, and faculties from around the country. At the conference, the PI also facilitated potential future collaborations for further advancing our current knowledge in controlling infections by foodborne pathogens. In addition to attending a national conference, the PI also gave an oral presentation to the University of Michigan Microbiome Research Initiative to further disseminate her findings and gather potential collaborations. PARTICIPANTS: Yvonne Sun is the principle investigator (PI) on this project and the sole person during the reporting period receiving salary support. All work performed for this project was conducted in University of Michigan Medical School. The PI receives mentorship from Dr. Mary O'Riordan in the department of Microbiology and Immunology, which also maintains shared facilities and equipment for departmental personnel. During the reporting period, the PI receives reagents provided from three separate collaborators: Dr. Brian Wilkinson from Illinois State University, Dr. Theodore Standiford from University of Michigan, and Dr. Henry Akinbi from University of Cincinnati. During the reporting period, this project provided significant professional training and development for the PI, a postdoctoral research fellow. In addition to daily scientific training in designing, executing, and analyzing experiments, the PI has opportunities to improve her oral presentation skills by showcasing her work at a national conference and an interdisciplinary organization in the University of Michigan. Moreover, her findings have led to a publication in which the preparation process provides training opportunities to improve the PI's writing and editing skills. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The PI has established the critical role of membrane fatty acid composition in a foodborne pathogen's ability to resist antimicrobial actions and modify its virulence expression. Results of her findings have been accepted for publication as listed in the next section. This finding expands our current understanding on how foodborne pathogens persist in harsh environment and will aid in designing better control strategies against contamination.

Publications

  • Sun, Y., Wilkinson, B.J., Standiford, T.J., Akinbi, H.T., and O'Riordan, M.X.D. 2012. Fatty acids regulate stress resistance and virulence factor production for Listeria monocytogenes. Journal of Bacteriology (in press).
  • Sun, Y., Esquilin-Lebron, K.J., and O'Riordan, M.X.D. 2012. Regulation of Listeria monocytogenes virulence by intestinal short chain fatty acids. American Society for Microbiology General Meeting, San Francisco, CA.
  • Esquilin-Lebron, K.J., Sun, Y., and O'Riordan, M.X.D. 2012. Intestinal envrionmental conditions tune Listeriolysin O protein production in Listeria monocytogenes. American Society for Microbiology General Meeting, San Francisco, CA.