Progress 08/15/20 to 08/14/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training to two postdoctoral fellows, two PhD students, and one MS student, who all gained valuable experimental skills in microbial genetics, biochemistry, and plant-microbe interactions. The postdocs made significant discoveries that led to 2 patent applications, with them named as inventors. All participants had multiple opportunities to enhance their presentation skills through joint lab meetings with two other research groups, departmental seminars, and presentations at regional ASM (Rocky Mountain branch) meetings. In addition to technical training, the postdoctoral fellows and graduate students were involved in data analysis, drafting manuscripts as first authors, and contributing to manuscript revisions. These experiences provided them with comprehensive training in both scientific research and communication. How have the results been disseminated to communities of interest?In addition to 3published (and 1 submitted) peer-reviewed papers, the results of this project were presented as oral presentations at several national meetings, including the ASM Biofilms Conference (Chaarllotte, 2022) and the ASM Rocky Mountain Branch meetings (2022,2023), as well as at the 6th International Congress on Applied Microbiology (Amsterdam, 2024) and Wenner-Gren Symposium on Small molecule signaling across the tree of life (Stockholm, 2023); invited webinar in the ASM lectureship series; International Conference on Cyclic dinucleotide signaling (Wuhan, 2023). An article summarizing the project's findings for the general public was published in Reflections, the magazine of the College of Agriculture, Life Sciences, and Natural Resources. The discovery of the antibiofilm activity in maple wood extracts also contributed to the formation of a student-led startup, MayPall, which develops oral health products based on the antibiofilm properties of maple-derived polyphenols. ? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Listeria monocytogenes is a dangerous foodborne pathogen that frequently contaminates fresh produce, leading to severe outbreaks of listeriosis. The survival of Listeria on the surfaces of fresh produce has long been a mystery, particularly on inhospitable surfaces like whole cantaloupes, which were the source of the worst listeriosis outbreak in U.S. history in 2011. Unlike other resilient pathogens, Listeria monocytogenes does not form spores or other dormant cell types known for their toughness. Therefore, it was expected that these bacteria would not survive for extended periods in open air or withstand the acidic conditions they encounter in the stomach after consuming cantaloupe. Indeed, planktonically grown L. monocytogenes dies quickly when stored in air and is highly susceptible to acidic environments. Moreover, liquid-grown L. monocytogenes does not adhere well to the surfaces of fruits and vegetables. In this project, we discovered that this scenario changes dramatically when L. monocytogenes is able to produce the Pss exopolysaccharide (EPS), a key component of biofilms. We found that EPS plays a crucial role in the bacteria's ability to bind to the surfaces of fruits and vegetables, enabling them to form biofilms. Importantly, the EPS-biofilms are highly resistant to disinfectants, acids, and desiccation, allowing Listeria to survive on fresh produce far longer than previously expected. Listeria strains producing Pss EPS have up to 10,000 times a greater chance of contaminating fresh produce and surviving challenges like dehydration and stomach acid, compared to non-producing strains. In our search for compounds that could prevent the formation of these harmful biofilms, we found that maple (Acer) products--including maple sap, syrup, and maple wood extracts--are effective inhibitors of biofilm formation. We have identified several maple-derived polyphenols, such as nortrachelogenin glucoside, lariciresinol, isoscopoletin, and epicatechin gallate, that exhibit strong antibiofilm activity. Through further investigation, we deciphered the mechanism of action of these compounds, all of which inhibit the enzyme sortase A, responsible for anchoring surface proteins and exopolysaccharides on the surface of Listeria cells. This discovery underscores the potential of widely available, safe, and sustainable maple products as a novel tool to reduce the risk of listeriosis. The abundance and safety of maple products, along with the efficacy of the individual bioactive compounds, make them a promising natural solution to enhance the safety of fresh produce. Objective 1: Decipher the Regulatory Mechanisms Controlling Pss EPS Formation in Listeria monocytogenes To understand the regulatory mechanisms controlling Pss exopolysaccharide (EPS) formation, we pursued two directions. First, we focused on the role of the second messenger c-di-GMP, which is known to activate Pss synthesis. To understand the environmental stimuli promoting c-di-GMP synthesis, we invested in engineering a reported of c-di-GMP levels in Listeria monocytogenes. Two systems were engineered -- a transcriptional reporter based on the c-di-GMP-dependent transcription factor BldD from Streptomyces and a c-di-GMP-dependent riboswitch-based system from Clostridia. However, the dynamic range of these reporters was limited (~2-3-fold), which made them unsuitable for large-scale screening of factors affecting c-di-GMP levels. A recently engineered fluorescent reporter may offer a more robust alternative for this analysis. In the second direction, we investigated transcriptional control of the pss operon. We identified PssR as a key transcriptional activator of the pss operon. PssR is a MerR-family transcription factor that is activated by peptide antibiotics such as thiostreptone. This finding suggests that L. monocytogenes activates EPS synthesis as a protective response against antibiotics produced by soil bacteria like Streptomyces. This discovery provides new insight into the role of EPS in the environmental survival of L. monocytogenes. Objective 2: Identify Antibiofilm Compounds in Tree Extracts and Characterize the Mechanism of EPS-Biofilm Inhibition This objective has proved to be the most productive and impactful in the project. Given the known antibiofilm activity of aqueous maple (genus Acer) wood extracts against L. monocytogenes, we screened various compounds derived from these extracts. We identified several key compounds with antibiofilm activity, including nortrachelogenin glucoside, lariciresinol, isoscopoletin, and epicatechin gallate. We further investigated the mechanism of action of these compounds and found that all of them they inhibit sortase A, a critical enzyme that anchors surface proteins to the cell wall peptidoglycan. Inhibition of sortase significantly reduces L. monocytogenes' ability to attach to plant surfaces, diminishing attachment by almost one order of magnitude. Additionally, these compounds impair the ability of L. monocytogenes to anchor the Pss EPS to the cell surface, thus making it more vulnerable to environmental insults such as desiccation and disinfectants. These findings suggest that affordable, widely available, and safe maple wood extracts--or individual chemicals such as epicatechin gallate (a major polyphenol found also in green and black tea)--can be used to prevent biofilm formation by L. monocytogenes on fresh produce. We anticipate that these discoveries (resulted in 2 patent applications) will be incorporated into food safety practices in fresh produce processing facilities. Objective 3: Uncover the Origin of EPS-Independent Appendages and Characterize Their Role in Listerial Plant Biofilms We extensively characterized the EPS-independent appendages observed in electron micrographs of L. monocytogenes biofilms formed on wood and cantaloupe rind surfaces. Initially, we hypothesized that these appendages were protein-based. However, our analysis revealed that these appendages are a mixture of extracellular DNA and nanotubes, not proteins as previously thought. Importantly, our characterization of the role of these appendages in L. monocytogenes colonization of fresh produce surfaces indicated that their contribution is minimal. This finding supports our understanding that Pss EPS plays the dominant role in plant surface colonization by L. monocytogenes, reinforcing the importance of EPS in the environmental survival of the pathogen. These objectives collectively deepen our understanding of Listeria monocytogenes' biofilm formation on fresh produce and offer viable strategies for combating foodborne listeriosis through natural, safe, and sustainable approaches.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
The Listeria monocytogenes exopolysaccharide significantly enhances colonization and survival on fresh produce.
Fulano AM, Elbakush AM, Chen LH, Gomelsky M.
Front Microbiol. 2023 Apr 27;14:1126940.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lignan-containing maple products inhibit Listeria monocytogenes biofilms on fresh produce.
Elbakush AM, Fulano AM, Gomelsky M.
Front Microbiol. 2023 Oct 19;14:1258394
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Maple compounds prevent biofilm formation in Listeria monocytogenes via sortase inhibition.
Elbakush AM, Trunschke O, Shafeeq S, R�mling U, Gomelsky M.
Front Microbiol. 2024 Sep 16;15:1436476
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Progress 08/15/22 to 08/14/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two postdoctoral fellows and a PhD student were the main drivers of the reported progress. All have had several opportunities to practice their presentation skills at joint lab meetings with two other gorups. They presented their findings at a regional ASM meeting (Rocky Mountain branch) and ASM Biofilms meeting. The postdoctoral fellows drafted twomanuscripts. How have the results been disseminated to communities of interest?Resutls of this study were presented at the Rocky Mountain BraanchASM meeting andnational ASM Biofilms meeting (Charlotte, NC). What do you plan to do during the next reporting period to accomplish the goals?Objectives 1. Characterize the regulatory mechanisms controlling pss gene expression by the newly identified regulator PssR. 2. Furthercharacterize the mechanism of EPS-biofilm inhibition by the maple-derived polyphenols, and publish manuscripts describing these indings. 3. This objective has been completed.
Impacts
What was accomplished under these goals?
The following advances have been achieved in Objectives 1-3. Objective 1: Decipher the Regulatory Mechanisms Controlling Pss EPS Formation inListeria monocytogenes We investigated transcriptional control of the pss operon. We identified PssR, a protein encoded in the gene upstream of the pss operon, as a key transcriptional activator of the pss operon. We have identified the potential binding sites ofPssR, an shown that its critical role as an activator of pss operon expression. PssRis a MerR-family transcription factor that is activated by peptide antibiotics such as thiostreptone. This finding suggests thatL. monocytogenesactivates EPS synthesis as a protective response against antibiotics produced by soil bacteria likeStreptomyces. This discovery provides new insight into the role of EPS in the environmental survival ofL. monocytogenes. Objective 2: Identify Antibiofilm Compounds in Tree Extracts and Characterize the Mechanism of EPS-Biofilm Inhibition The Pss exopolysaccharide (EPS) enhances the ability of the foodborne pathogenListeria monocytogenesto colonize and persist on surfaces of fresh fruits and vegetables. Eradicating listeria within EPS-rich biofilms is challenging due to their increased tolerance to disinfectants, desiccation, and other stressors. Recently, we discovered that extracts of maple wood, including maple sap, are a potent source of antibiofilm agents. Maple lignans, such as nortrachelogenin-8'-O-β-D-glucopyranoside and lariciresinol, were found to inhibit the formation of, and promote the dispersion of pre-formedL. monocytogenesEPS biofilms. However, the mechanism remained unknown. Here, we report that these lignans do not affect Pss EPS synthesis or degradation. Instead, they promote EPS detachment, likely by interfering with an unidentified lectin that keeps EPS attached to the cell surfaces. Furthermore, the maple lignans inhibit the activity ofL. monocytogenessortase A (SrtA) in vitro. SrtA is a transpeptidase that covalently anchors surface proteins, including the Pss-specific lectin, to the cell wall peptidoglycan. Consistent with this, deletion of thesrtAgene results in Pss EPS detachment from listerial cells. We also identified several additional maple compounds, including epicatechin gallate, isoscopoletin, scopoletin, and abscisic acid, which inhibitL. monocytogenesSrtA activityin vitroand prevent biofilm formation. Molecular modeling indicates that, despite their structural diversity, these compounds preferentially bind to the SrtA active site. Since maple products are abundant and safe for consumption, our finding that they prevent biofilm formation inL. monocytogenesoffers a viable source for protecting fresh produce from this foodborne pathogen. Objective 3: Uncover the Origin of EPS-Independent Appendages and Characterize Their Role in Listerial Plant Biofilms We extensively characterized the EPS-independent appendages observed in electron micrographs ofL. monocytogenesbiofilms formed on wood and cantaloupe rind surfaces. Initially, we hypothesized that these appendages were protein-based. However, our analysis revealed that these appendages are a mixture of extracellular DNA and nanotubes, not proteins as previously thought. Importantly, our characterization of the role of these appendages inL. monocytogenescolonization of fresh produce surfaces indicated that their contribution is minimal. This finding supports our understanding that Pss EPS plays the dominant role in plant surface colonization byL. monocytogenes, reinforcing the importance of EPS in the environmental survival of the pathogen.
Publications
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Progress 08/15/21 to 08/14/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major challenge is to identify factors that stimulate EPS synthesis. This line of research requires c-di-GMP reporter. In Objective 1, we have spent significant efforts in making a c-di-GMP reporter, but have not yet succeeded atengineering a reporter with a sufficient dynamic range. What opportunities for training and professional development has the project provided?A postdoctoral fellow and a PhD student were the main drivers of the reported progress. Both have had several opportunities to practice their presentation skills at joint lab meetings with two other gorups.They presented their findings at a regional ASM meeting (Rocky Mountain branch).The postdoctoral fellow drafted the first manuscript. How have the results been disseminated to communities of interest?An article for the general public has been drafted for the College of Agriculture magazine Reflections. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We are well on our way to engineer a c-di-GMP reporter to test a variery of compounds for activation of c-di-GMP synthesis, the limiting factor in EPS synthesis. We have also started and will continue to use a so-called Congo Red assay to monitor EPS production directly. Objective 2: After identifying the active anti-EPS component, NCGG, we're focusing on mechanisms ofits action. In this research, we developed new tools to gain mechanistic insights (e.g. a pss-lacZ transcriptional fusion). Objective 3: We have completed analysis of the EPS-independent appendages and determined that they represent extracellular DNA and nanotubes. These components have been characterized by other groups, yet our analysis shows that theyplay noimportant role in fresh produce colonization, we willnot be pursuing this line of research much further.
Impacts
What was accomplished under these goals?
Listerial survival on the surfaces of fresh produce has remained somewhat of a mystery--especially on inhospitable surfaces such aswhole cantaloupes, which werethe source of the worst listeriosis outbreak in US history in 2011. Listeria monocytogenes does not form spores or other dormant cell types known for resilience. Further,these bacteria are not expected to survive inopen air for long periods of time. Neither are they expected to withstand the exposure to strong acid, which they face in the stomach following cantaloupe consumption. Indeed, planktonically grown L. monocytogenes die quickly upon storage in air and do not withstand exposure to hydrochloric acid particularly well. Furthermore, liquid-grown L. monocytogenes does not attach well to the surfaces of fruits and vegetables. This picture changes drastically when it can synthesize an exopolysaccharide (EPS) and forms EPS-basedbiofilms. In the reported period, we made significant progress in two areas. Weuseda new biofilm model, where pieces of wood or fresh produce are incubated in a minimalliquid medium inoculated with L. monocytogenes, and colonization is assessed after 1-2 days.We quantifiedthe impact of listerial EPS-biofilms formed onthe surfaces of fresh produce in this model. We found that the EPS-synthesizing listerial strains colonize surfaces of diverse kinds of fresh produce that caused recent listeriosis outbreaks(includingcantaloupe rinds, cantaloupe pulp, cut celery, and lettuce)by 2- to 12-fold better thanthe EPS-negative strains. Next, wemeasured tolerance of the EPS-biofilms formed on fresh produce to two major stressors affecting infectivity of the contaminated fresh produce, i.e. desiccation and acid stress. Desiccation is the condition experienced by bacteria during fresh produce storage and transportation.Acid stress is experienced by bacteria passing through stomach acid.L. monocytogenes in the EPS-biofilms on fresh producewere found to be 6-16-fold more tolerant to desiccation, andsurvived exposure to strong acid approximately 12-116-fold better than the EPS-negative strains. We surmise thatL. monocytogenes in the EPS-biofilms has 100-to-10,000-fold higher chanceof causing foodborne illness, compared to the EPS-negative strains.We suggest thatlisterial EPS-biofilms explainthe mysterious resilience of Listeria on the surfaces of fresh produce. The second major advance in the reportingperiod is our identification ofthe chemical present in maple wood extracts that strongly inhibits listerial EPS formation. We tested25 majorcomponents ofthe methanol maple extract reported in the literature fortheir anti-EPS activity. The lignan, nortrachelogenin-8'-O-b-D-glucopyranoside (NCGG), has emerged as the active anti-EPS ingredient. At 120 microM, NCGG inhibits EPS formation on the surfaces of cantaloupes, celery and lettuce by 100-to1000-fold. Because NCGG is present inmaple syrup, a sweetener derived from sugar maple and broadly consumed in North America, it likely belongs to the generally regarded as safe (GRAS) category. The GRAS status of NCGG opens thepossibility ofdeveloping NCGG-containing products from maple wood that can coat fresh produce (such as cantaloupes) and drastically decrease listerial colonization. Further, other wood sources containing NCGG may be used, e.g., star jasmine (Trachelospermum jasminoides), pecan or hickory. Objective 1. Decipher the regulatory mechanisms controlling Pss EPS formation in listeria. The levels of intracellular second messenger c-di-GMP determine the level of the exopolysaccharide (EPS) production. To monitor c-di-GMP levels in L. monocytogenes, a transcriptional lacZ reporter involving the c-di-GMP-dependent transcription factor Streptomyces BldD, and BlldD-dependent reporter gene was constructed. Unfortunately, the dynamic range of this reporter proved to be low, ~ 2-fold, when high c-di-GMP (delta pdeB/pdeC/pdeD) and low c-di-GMP (EGD-e) strains were compared. Such low range is not conducive for screening of the factors affecting c-di-GMP level changes. Using a different strategy, we have constructed ac-di-GMP-dependent riboswitch placed upstream of the reporter GFP gene. Unfortunately, this new reporter has also proved to have a limited dynamic range, unsuitable for c-di-GMP measurements inL. monocytogenes high- and low c-di-GMP strains.Currently, we are pursuing a third approach based on a recently engineered by us tightly controlled gene expressionsystem inL. monocytogenes. Objective 2. Identify an antibiofilm component in tree extracts and characterize the mechanism of EPS-biofilm inhibition. The key finding in this objective is our identification of the active anti-EPS compound, NCGG, present in various kids of maple trees(Acer saccharum, A. rubrum, A. palmatum, A. platanoides, A. saccharum) as well as in maple syrup. Currently, we are investigating the mechanisms through which NCGG inhibits EPS formation. So far, we have excluded c-di-GMP synthesis, EPS degradation and EPS pss operon gene expression. By exclusion, it appears that NCGG inhibits biosynthesis of the Pss EPS. Objective 3. Uncover the origin of EPS-independent appendages and characterize their role in listerial plant biofilms. We spentsignificant effort in characterizing the EPS-independent appendages observed in the electron micrographs of the listerial biofilms formed on pieces of wood and cantaloupe rinds. Our analysis suggests that these are non-protein appendages, representing a mixture of extracellular DNA and nanotubes. Importantly, our characterization of the roles of these appendages in listerial colonization of the surfaces of fresh produce suggests that these roles are minimal. These findings indirectly confirm the major role that EPS plays in plant surface colonization by L. monocytogenes.
Publications
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Progress 08/15/20 to 08/14/21
Outputs
Target Audience:Scientific community. Changes/Problems:The major challenges havebeen due to COVID-19, and involved limited access to the laboratory in 2020-21, inability to hire international postdoc and graduate student (until summer-2021), and consequences of the severe COVID-19 illness of the graduate student involved in the project that forced her toquit graduate school. What opportunities for training and professional development has the project provided?One Ph.D. student was trained in this project. Unfortunately, severe COVID-19 illness shortened her involvement in the project and forced her to exit Graduate School. How have the results been disseminated to communities of interest?A paper was presentedat the International Association for Food Protection, IAFP-2021. What do you plan to do during the next reporting period to accomplish the goals?A postdoctoral fellow and new graduate studenthave been recruited to work on the project. The postdoc is expected topursue objectives 1 & 3, and graduate student will pursue objective 2.
Impacts
What was accomplished under these goals?
Because of COVID-19 restrictions regarding the use of the laboratories during 2020-21 as well asrestrictions on international travel that precluded hiring international postdoctoral fellows and graduate students,progress in year 1 was limited. AseriousCOVID-19 illness of the graduate studentinvolved in this project from the beginning, has ultimately resulted in her exiting Graduate school. Thisunfortunate circumstance further limited ourprogress in year 1. Objective 1. Decipher the regulatory mechanisms controlling Pss EPS formation in listeria. The levels of intracellular second messenger c-di-GMP determine the level of the exopolysaccharide (EPS) production. To monitor c-di-GMP levels in L. monocytogenes, atranscriptional lacZ reporter involving the c-di-GMP-dependent transcription factor Streptomyces BldD, and BlldD-dependent reporter gene was constructed. Unfortunately, the dynamic range of this reporter prroved to be low, ~ 2-fold, when highc-di-GMP (delta pdeB/pdeC/pdeD) andlow c-di-GMP(EGD-e) strains were compared.Such low range is not conductive for screening of the factors affecting c-di-GMP level changes. Our attempts to improve the dynamic range by adjusting expression levels of the components (BldD, lacZ) did not succeed. Therefore, a new strategy, involving a c-di-GMP-dependent riboswitch will be attempted next. Objective 2.Identify an antibiofilm component in tree extracts and characterize the mechanism of EPS-biofilm inhibition. An improtant finding in this objective involved our characterization of various parts of maple trees, and realization that the anti-EPS activity is present in all components (wood, leaves, bark). Further, we found that all maple species that we tested (Acersaccharum, A. rubrum, A.palmatum, A.platanoides)contain the active compound(s) that is capable of breaking down listerial EPS and/or preventing its formation. Further, we found that commercial maplesyrup contains this compund, which provides us a stable and abundant source of the compound, and opens the pathfor its purification and characterization. (A patent application has been filed by University of Wyoming.) Objective 3.Uncover the origin of EPS-independent appendages and characterize their role in listerial plant biofilms. Nothing done thus far.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
AG Tajani, J Carr, A Elbakush, B Bisha, M Gomelsky - IAFP 2021
Evaluation of an enzymatic treatment to control listerial biofilm on produce.
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