LISTERIA MONOCYTOGENES EXOPOLYSACCHARIDE: STRUCTURE AND ROLES IN COLONIZATION AND PERSISTENCE ON PRODUCE SURFACES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0232378
• Project No.: WYO-491-13
• Project Start Date: Jan 1, 2013
• Project End Date: Sep 30, 2014

Project Director
Miller, KU.

Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000

Performing Department
Molecular Biology

Non Technical Summary
Listeria monocytogenes is a pervasive food-borne pathogen that causes hundreds of cases of the severe disease, listeriosis, in the USA every year. Although other food-borne bacteria such as Salmonella and E. coli cause more illnesses, L. monocytogenes is the deadliest of the common food-borne bacteria, having a mortality rate of approximately 20%. Infants, the elderly, immunocompromised individuals such as cancer patients, and pregnant women have the greatest risk of being infected. Traditionally, listeriosis cases have been associated with the consumption of contaminated meats, cheeses, smoked fish, and raw milk. However, over the last two decades the number of diseases caused by the consumption of contaminated fresh produce has significantly increased. Last year's multi-state outbreak of listeriosis caused by contamination of cantaloupe has been the deadliest (>30 deaths) outbreak caused by a food-borne pathogen in the USA in the last 80 years. L. monocytogenes is able to adhere to and grow on the surface of fresh produce such as cantaloupe in biofilms, i.e. multicellular communities embedded in extracellular substances secreted by bacteria. These substances facilitate bacterial attachment to surfaces and protect bacteria from environmental stressors. Among extracellular components of biofilms, exopolysaccharides are most commonly used by bacteria to attach to and grow on plant surfaces. The compositions of biofilms formed by L. monocytogenes on plant surfaces have not been characterized at the molecular level. An exopolysaccharide component of L. monocytogenes biofilms has recently been discovered by the PIs. The objectives of this study are to characterize the composition and structure of this novel exopolysaccharide, and to determine its role in L. monocytogenes attachment to produce and resistance to disinfectants and other control agents. This research will lead to a better understanding of the factors underlying the adherence and persistence of L. monocytogenes on produce and will guide efforts to prevent colonization and inhibit the growth of L. monocytogenes on produce.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1420	1000	25%
712	1420	1100	25%
712	1430	1000	12%
712	1430	1100	13%
712	1461	1000	12%
712	1461	1100	13%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables; 1461 - Peppers; 1420 - Melons;

Field Of Science
1100 - Bacteriology; 1000 - Biochemistry and biophysics;

Keywords

listeria monocytogenes

exopolysaccharide

produce contamination

Goals / Objectives
There are three Specific Aims to this project: 1. Composition and chemical structure of L. monocytogenes EPS. Many types of beneficial and pathogenic bacteria adhere to the surfaces of plants via exopolysaccharides (EPS). Currently the structure and function of EPS in L. monocytogenes is unknown. Our labs have identified L. monocytogenes genes involved in the synthesis of EPS and have developed mutant strains that either lack or over-express the polymer. Under this Aim we will isolate L. monocytogenes EPS from the over-expression strain, and determine its composition and structure. These studies will for the first time reveal the composition and structure of EPS synthesized by L. monocytogenes. 2. Function of EPS in the attachment and formation of L. monocytogenes biofilms on produce. Under this Aim, we will investigate how listerial EPS is involved in attachment of L. monocytogenes to cantaloupe and other produce, such as lettuce and red peppers, which have been identified as sources of L. monocytogenes contamination in previous listeriosis outbreaks. Experiments will be performed with EPS-negative, wild type, and EPS-over-producing strains, as well as with strains isolated from contaminated produce. EPS production will be quantified in these strains, and we will determine how attachment is correlated with EPS level. The studies will provide a thorough evaluation of the role of EPS in L. monocytogenes attachment to produce. 3. Function of L. monocytogenes EPS in resistance to environmental and food preservation stressors. In these experiments we will explore the function of EPS in the resistance of L. monocytogenes to environmental stress agents, including disinfectants and preservatives used in food decontamination and preservation. Experiments will be performed with produce-grown biofilms using our set of laboratory strains and selected environmental isolates that synthesize low or high levels of EPS. Several chemical and physical stressors will be tested in these experiments, including desiccation and bleach. These experiments will show for the first time the relationship between EPS production and resistance of the bacterium to control agents.

Project Methods
Specific Aim 1. Composition and chemical structure of L. monocytogenes EPS. EPS will be obtained for chemical composition and structure analysis from our L. monocytogenes EGD-e mutant strain that overproduces EPS. To obtain EPS, cells will be grown in liquid medium and EPS removed from the cell surface by stirring. After pelleting cells by centrifugation, residual proteins will be removed by trichloroacetic acid precipitation, EPS will be precipitated from the culture medium using ethanol, precipitated EPS will be taken up in water and dialyzed against water to remove small molecules, and dialyzed EPS will be freeze-dried to obtain a crude EPS fraction. The next steps of EPS structural analysis will be to determine the monomer composition of the polymer, identify the types of linkages between monomer units, and determine its 3D structure by NMR spectroscopy. Specific Aim 2. Function of EPS in the attachment and formation of L. monocytogenes biofilms on produce. The analysis of the role of EPS in produce attachment and biofilm formation will be performed using a wild type lab strain (EGD-e), and EPS-negative and EPS-over-producing derivatives of EGD-e constructed in the lab. Experiments also will be performed with strains associated with outbreaks of produce origin. All strains will be assayed to determine the level of EPS they synthesize. The produce items that will be studied include cantaloupes, lettuce, and peppers. Data will be evaluated to determine if the number of cells recovered from the produce items is correlated with the EPS synthesis levels of the strains. For selected strains, we will examine the structure of the biofilms formed on produce surfaces by scanning electron microscopy. Specific Aim 3. Function of L. monocytogenes EPS in resistance to environmental and food preservation stressors. In these experiments we will expose biofilm cells grown on plastic microtiter plates and cantaloupe rinds to stress agents, and then determine survival of cells in the biofilm. Experiments will be conducted with our three lab strains that produce high, intermediate, and no EPS, and selected strains isolated from produce-associated listeriosis outbreaks. As stressors, we will evaluate nine agents and conditions that are known to impact L. monocytogenes viability. For disinfectants/sanitizers we will evaluate benzalkonium chloride and hypochlorous acid (bleach), which are used for control of L. monocytogenes growth in food processing plants. The food preservation agents that will be evaluated include heat, cold, pH variation, high salt concentration, sodium lactate, and the bacteriocin nisin. We also will evaluate the resistance of the strains to desiccation. Strains will be compared to one another to determine if the level of EPS in the biofilm affects cell survival.

Progress 01/01/13 to 09/30/14

Outputs
Target Audience: The target audiences that were reached in this reporting period were 1) scientists working in the area of food safety and microbiology that read our PLoS Pathogens journal article on Listeria exopolysaccharide (EPS), and 2) food producers who have read our UW Ag Experiment Station Field Days Bulletin report on preliminary characterization of the composition of listerial EPS and identification of genes required for its synthesis. Both of these publications are listed in the "Products" section of the report. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training. This research award has contributed to the training of a PhD-level graduate student at the University of Wyoming (Volkan Koseoglu, Dept. of Molecular Biology, 1.0 FTE). Training has consisted of one-on-one mentoring in the research lab. The PI, Co-PI, and the project collaborator, Dr. Teresa E. Lehmann of the Chemistry department have all contributed to the training of Volkan. Professional Development. The research award has supported the professional development of the PI, Co-PI, and one graduate student. Regional Meetings. The student, Volkan Koseoglu gave a presentation covering part of the research project at the Rocky Mountain Branch of the ASM in Laramie, WY in April 2014. Univ. of Wyoming Meetings. The student V.K. presented his findings on the structure and properties of L. monocytogenes EPS at the annual symposium for the Molecular Cellular Life Sciences graduate program at the Univ. of Wyoming. Through meeting presentations, the student has had the opportunity to further develop his presentation skills. Volkan also wrote the manuscript that is under review currently at the journal Molecular Microbiology, which has strengthened his writing skills. How have the results been disseminated to communities of interest? Some of the outcomes of the research conducted in the 2014 reporting period were disseminated to the non-scientist community via publication of our report in the UW Ag Experiment Station Field Days Bulletin. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this reporting period, the complete chemical structure of L. monocytogenes EPS was determined by NMR spectroscopy. The PI's graduate student, Volkan Koseoglu, purified EPS from the cell surface of our EPS-over-producer strain that maintains high cyclic dimeric GMP (c-di-GMP) levels. The EPS was sent to the Complex Carbohydrate Research Center at the Univ. of Georgia, where two NMR spectroscopists, Dr. Christian Heiss and Dr. Parastoo Azadi developed methods to solubilize the EPS for NMR experiments. From work performed in the preceding year, we knew that this EPS consisted of N-acetylmannosamine (ManNAc) and galactose (Gal) in a two to one ratio. Using a combination of 1D- and 2D-NMR methods, the repeat unit of the EPS was determined to be {4)-β-ManpNAc-(1→4)-[α-Galp-(1→6)]-β-ManpNAc-(1→}. This is the first time an EPS has been characterized from this bacterium. Volkan Koseoglu also carried out a more complete analysis of genes required for ManNAc-Gal EPS synthesis. It was determined that synthesis requires all five genes in the polysaccharide synthesis (pss) operon (genes lmo0527-0529). These genes encode respectively a protein of unknown function (PssA), a likely deacetylase that may modify some of the ManNAc residues in the polymer (PssB), the EPS synthase (PssC), a synthase scaffolding protein (PssD), and the c-di-GMP receptor (PssE). In addition we determined that the PssZ gene (lmo1913) located elsewhere on the chromosome encodes a dedicated glycosylhydrolase that degrades ManNAc-Gal EPS. These findings are significant in that they may lead to better approaches to remove L. monocytogenes from food processing plants, etc. The results of these studies have been incorporated in a manuscript under review by Molecular Microbiology, and may be published this year.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen, L.-H., K�seoglu, V. K., G�vener, Z. T., Myers-Morales, T., Reed, J. M., D'Orazio, S. E. F., Miller, K. W., and Gomelsky, M. 2014. PLoS Pathog. 10, e1004301. "Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes."
• Type: Other Status: Published Year Published: 2014 Citation: Koseoglu, V. K., Gomelsky, M., and Miller, K.W. 2013. University of Wyoming Agricultural Experiment Station Field Days Bulletin. "Listeria monocytogenes exopolysaccharide: structure and roles in colonization and persistence on produce surfaces."
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Koseoglu, V. K., Miller, K. W., and Gomelsky, M. 2014. Rocky Mountain Branch of the American Society for Microbiology Spring Meeting, April 19. "Composition, Biosynthesis, and Degradation of C-di-GMP Regulated Listeria monocytogenes Exopolysaccharide."
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: K�seoglu, V. K., Heiss, C., Azadi, P., Topchiy, E., G�vener, Z. T., Lehmann, T. E., Miller, K. W., and Gomelsky, M. 2015. Revision submitted to Mol. Microbiol. "Listeria monocytogenes exopolysaccharide: Origin, composition, biosynthetic machinery, and C-di-GMP-dependent regulation."

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audiences that were reached in this reporting period were 1) scientists working in the area of food safety that read our American Society for Microbiology abstract on Listeria exopolysaccharide (EPS), and 2) food producers who have read our UW Ag Experiment Station Field Days Bulletin report on the role of the EPS in resistance to disinfectants. Both of these publications are listed in the "Products" section of the report. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training. This research award has contributed to the training of two PhD-level graduate students at the University of Wyoming (Volkan Koseoglu, Dept. of Molecular Biology, 1.0 FTE; Elena Topchiy, Dept. of Chemistry, 0.1 FTE). For both students, training has consisted of one-on-one mentoring in the research lab. The PI and Co-PI have contributed to the training of Volkan Koseoglu. The project collaborator, Dr. Teresa E. Lehmann of the Chemistry department, has contributed to the training of Elena Topchiy. Professional Development. The research award has supported the professional development of the PI, Co-PI, and two graduate students. National Meetings. The PI, Co-PI, and the student Volkan Koseoglu attended the annual meeting of the American Society for Microbiology in Denver, CO, in May 2013, where V.K. presented a poster on the properties of the L. monocytogenes EPS. The three participants learned about new research in related fields of study and established contacts in the international research community. Regional Meetings. The two students, V.K. and E.T. gave presentations of selected aspects of the research at the Rocky Mountain branch of the ASM in Fort Collins, CO in April 2013. Univ. of Wyoming Meetings. The student V.K. presented his findings on the structure and properties of L. monocytogenes EPS at the annual symposium for the Molecular Cellular Life Sciences graduate program at the Univ. of Wyoming in September 2013. Through meeting presentations, the two students have had the opportunity to develop their presentation skills. How have the results been disseminated to communities of interest? The outcomes of the research in the 2013 reporting period were disseminated to the non-scientist community via publication of our report in the UW Ag Experiment Station Field Days Bulletin. (See “Products” section). What do you plan to do during the next reporting period to accomplish the goals? In the coming reporting period we plan to refine the composition analysis of listerial EPS by using a purer preparation of the polysaccharide. This will also allow for the accurate determination of the linkages between the monosaccharide residues in the polymer. We also plan to complete the writing of a manuscript on the composition of the EPS and submit it for publication.

Impacts
What was accomplished under these goals? During this reporting period, a graduate student in the PI's lab, Volkan Koseoglu, under the direction of the PI and Co-PI, purified and determined the chemical composition of a cell surface exopolysaccharide (EPS) produced by the important foodborne pathogen Listeria monocytogenes. He also showed that this EPS contributes to the survival of listerial cells exposed to disinfectants such as bleach. L. monocytogenes is a pervasive foodborne pathogen that causes hundreds of cases of the severe disease, listeriosis, in the USA every year. Although other foodborne bacteria such as Salmonella and E. coli cause more illnesses, L. monocytogenes is the deadliest of the common foodborne bacteria, having a mortality rate of approximately 20%. Infants, the elderly, immunocompromised individuals such as cancer patients, and pregnant women have the greatest risk of being infected. The results obtained through NIFA support of this project will ultimately be useful for limiting the growth of L. monocytogenes in food production facilities and retail stores. Aim 1 Progress. A significant amount of effort was devoted to this aim--"Composition and chemical structure of L. monocytogenes exopolysaccharide (EPS)"--during the reporting period. Note that the results discussed here have not yet been reported in the publications listed under the "Products" section of this report. However, a manuscript containing these results will be submitted in 2014. Under this aim Volkan Koseoglu has succeeded in purifying a major listerial cell surface EPS that is under c-di-GMP control. The composition of this EPS has been determined by the Complex Carbohydrate Research Center at the University of Georgia, Athens, GA. The EPS consists of two monosaccharides, galactose and N-acetylmannosamine, in a ratio of two to one, with perhaps trace amounts of glucose. To our knowledge, this is the first EPS of L. monocytogenes whose composition has been determined. Under the "Aim 3 Progress" section below, the role of this EPS in protection of cells against disinfectants and desiccation is described. Aim 2 Progress. There is one result to report at this time for this aim of the project--"Function of EPS in the attachment and formation of L. monocytogenes biofilms on produce."We have analyzed wild type and EPS overexpression strain cells grown in liquid culture by scanning electron microscopy (SEM) to determine if EPS fibers can be visualized in our strains. SEM images showed fibers connecting cells in the EPS overproduction strain, but not in the wild type strain. Therefore we presume that these fibers may be the galactose/N-acetylmannosamine polymer that we have purified from the listerial cell surface under Aim 1. Aim 3 Progress. Advances were made during the reporting period on this aim--"Function of L. monocytogenes EPS in resistance to environmental and food preservation stressors." In these experiments, Volkan Koseoglu compared the susceptibility of wild type L. monocytogenes and an EPS-overproducing strain to hydrogen peroxide (H2O2), sodium hypochlorite (bleach), and benzalkonium chloride (BC). Cells in aqueous suspension were challenged with 200 mM H2O2, 1600 ppm sodium hypochlorite, or 100 ppm BC for 10 minutes, then plated for enumeration of surviving cells. The EPS-overproducing strain displayed a 2-log protection to both H2O2 and BC treatment, and a 5-log protection to sodium hypochlorite compared to the wild type strain. In addition, the susceptibility of the two strains to desiccation was compared by incubating dried samples of cells for up to seven weeks and then determining the number of surviving cells remaining in samples. The EPS-overproduction strain proved to be 5-fold less susceptible to desiccation than the wild type strain. These experiments indicate that listerial EPS helps protect cells against environmental challenges, and further suggest that this polysaccharide may contribute to survival of the bacterium in food production and retail facilities. The results summarized here were reported at the 2013 ASM meeting and are included in the PLoS Pathogens manuscript that is currently under review. (See "Products" section).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Koseoglu, V. K., Chen, L.-H., Miller, K. W., and Gomelsky, M. 2013. Abstr. Amer. Soc. Microbiol. "A c-di-GMP-dependent cellulosic exopolysaccharide produced by Listeria monocytogenes that enhances resistance to disinfectants and desiccation."
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Chen, L.-H., Koseoglu, V. K., Guvener, Z. T., Myers-Morales, T., Reed, J. M., D'Orazio, S. E. F., Miller, K. W., and Gomelsky, M. 2014. PLoS Pathogens, under review. "Cyclic di-GMP-dependent signaling pathways in the pathogenic firmicute Listeria monocytogenes."
• Type: Other Status: Published Year Published: 2013 Citation: Koseoglu, V. K., Gomelsky, M., and Miller, K.W. 2013. University of Wyoming Agricultural Experiment Station Field Days Bulletin, pp. 153-154. "Listeria monocytogenes exopolysaccharide: structure and roles in colonization and persistence on produce surfaces."