Source: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY submitted to
A BIOENERGETIC APPROACH FOR CONTROL OF LISTERIA MONOCYTOGENES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0207508
Grant No.
2006-35201-17375
Project No.
NJ61903
Proposal No.
2006-01288
Multistate No.
(N/A)
Program Code
32.0
Project Start Date
Sep 1, 2006
Project End Date
Aug 31, 2010
Grant Year
2006
Project Director
Montville, T. J.
Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
CENTER FOR ADVANCED FOOD TECHNOLOGY
Non Technical Summary
Listeria monocytogenes kills over 500 people per year and is the leading bacterial cause of food recalls. It is especially dangerous to pregnant women, the immunocompromised. These bacteria grow or survive in harsh environments, making it difficult to control in ready-to-eat meats. This unique ability must be related to energy generation and conservation. However, little is known about how L. monocytogenes regulates its two major energy stores. This project examines how L. monocytogenes energy regulation (bioenergetics) controls its ability to grow under adverse conditions. We hypothesize that the ability of L. monocytogenes to balances its two energy currencies plays a critical role in response to adverse conditions. This is demonstrated by elucidating the physiological mechanisms by which L. monocytogenes regulates its energy interconverting enzyme: and genetically characterizing the role of this F0F1ATPase enzyme. This research identifies new targets for L. monocytogenes control and addresses a fundamental factor (i.e. bioenergetics) that influences colonization, multiplication, and the types of treatment/antimicrobials necessary to reduce listerial cell numbers. This foundational understanding of listerial biology provides a platform from which to develop new intervention strategies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7125010100030%
7125010102010%
7125010104030%
7125010108010%
7125010110020%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
1080 - Genetics; 1020 - Physiology; 1100 - Bacteriology; 1000 - Biochemistry and biophysics; 1040 - Molecular biology;
Goals / Objectives
Our research on PMF and ATP leads to the hypothesis that the ability to regulate F0F1ATPase activity, which balances energy reserves, plays a critical role in L. monocytogenes' response to adverse conditions. Objective 1 elucidates the physiological mechanisms by which L. monocytogenes regulates F0F1ATPase: a.) at the activity level: by demonstrating that membrane fluidity regulates F0F1ATPase activity, b.) through physiological adaptation: by confirming that the Acid Tolerance Response results in nisin resistance due to alterations in the abundance of F0F1ATPase c subunit, and c.) through mutation by demonstrating that F0F1ATPase abundance determines the acid sensitivity of nisin resistant mutants. Objective 2 genetically characterizes the role of the F0F1ATPase: a.) in two well-studied acid-sensitive/nisin resistant L. monocytogenes mutants, and b.) by creating specific F0F1ATPase mutants and correlating their phenotypes to preservative tolerance.
Project Methods
Objective 1a Membrane properties correlate with nisin resistance and antimicrobial sensitivity. ATPase activity is measured in proteoliposomes having different fluidities and fixed protein:lipid ratios. ATPase activity is also determined in proteoliposomes having different fluidities but fixed protein:lipid ratios. We also alter the protein (i.e. amount of ATPase):lipid ratios in proteoliposomes of fixed lipid composition and observe the effect of increased ATPase protein levels on ATPase activity and membrane fluidity. Objective 1b We confirm the identity of the 7.4 kDa down-regulated protein as the ATPase c subunit to verify that its downregulation causes nisin resistance during the Acid Tolerance Response. The proteins that Surface-Enhanced Laser Desorption Ionization, Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) characterizes as differentially-regulated are identified based on comparisons to the L. monocytogenes genome. Protein identities are confirmed using peptide sequencing of in-gel digestion fragments generated after polyacrylamide gel electrophoresis. Objective 1c Proteomic analysis of proteins from L. monocytogenes mutants. The relative abundance of the ATPase c subunit is determined using SELDI-TOF MS. . Objective 2a We hypothesize that the nisin-resistant strain NR30 has a mutation in the ATPase c subunit. To test this, strain NR30's nisin-resistance determinant(s) are cloned, expressed, and analyzed for the levels of ATP, PMF and ATPase activity. In addition, we create ATPase mutants and demonstrate the relationship between the gene and the acid tolerance/nisin resistance phenotype. Total DNA from L. monocytogenes nisin-resistant (NR30) cells is isolated and fragments are amplified using the pTV3 shuttle vector, in the E. coli MB2159 cells. The L. monocytogenes NR30 DNA library is used to transform the L. monocytogenes Lmdd cells with selection for the nisin resistance. The cloned L. monocytogenes NR30 DNA fragments containing determinant(s) for nisin resistance are sequenced and analyzed by comparing with the whole Listeria genome sequence. Objective 2b. To confirm that the c-subunit of the F0F1ATPase is responsible for acid-tolerance/nisin- resistance, degenerate PCR primers are used to generate four classes of mutants. The first class of mutants are designed using PCR primers based on conserved regions of ATPase subunits. The second set are generated using primers targeting the non-conserved regions. The third set is specifically designed using primers that target the residue(s) involved in proton transport. The fourth mutant class comprises deletions in genes coding for ATPase subunits. The constructed deletions are introduced into the L. monocytogenes chromosome. Mutants from each class are first tested for nisin-resistance by replicate plating, with positive clones subsequently screened for acid sensitivity and the ability to generate an Acid Tolerance Response. The mutations which give rise to acid-sensitive/nisin-resistant clones and those causing the ATR/nisin-resistant phenotype are verified by sequencing.

Progress 09/01/06 to 08/31/10

Outputs
OUTPUTS: This project developed new knowledge on acid resistance and nisin resistance of Listeria monocytogenes, an important food borne pathogen. This is significant because acid is a major barrier to listerial growth. Similarly, nisin is a new method for controlling this pathogen and understanding bacterial resistance to it can lead to new control methods. This information was shared through publication and through exchange of students with other universities. The most significant output was the recognition of this research when Dr. Montville was awarded the Institute of Food Technologists' Bernard Oser Award for Food Ingredient Safety in 2008. PARTICIPANTS: Thomas Montville, PI. Primary responsible for the microbial physiology aspects of the project. Michael Chikindas, Co. PI. Primary responsible for genetic aspects of project. Mohammed Badoui Najjar, Doctoral candidate who worked on physiological aspects. Received Ph.D. now working at FDA. Ruth Wirwan, Post Doctoral Associate, now working in industry. Danielle Voss, undergraduate research assistant, now pursunig doctoral studies. Collaboration with Martin Weidman, Cornell University TARGET AUDIENCES: The target audience is other researchers, regulators who need new methods of listeria control, and food manufacturers who need to understand how this organisms resists acids and preservative. PROJECT MODIFICATIONS: None

Impacts
This research produced a fundamental shift in our understanding of acid tolerance in Listeria monocytogenes. We correctly identified the importance of an acid pumping enzyme. However, it's regulation was not by any established mechanism. Unfortunately, this novel finding could not be pursued as USDA no longer funds this area. This project afforded doctoral training to a student now employed at FDA and to a post-doctoral researcher now working in industry. Several undergraduate students were afforded the opportunity to work in a research laboratory. As a result, one of these is now pursuing a doctoral degree.

Publications

  • Badaoui Najjar, M, Chikindas, M. and Montville, T.J. 2007. Changes in Listeria monocytogenes membrane fluidity in response to temperature stress. Appl. Environ. Microbiol.73:6429-6435.
  • Badaoui Najjar, M.Z., Chikindas, M.L., and Montville, T.J. 2009. The acid tolerance response alters membrane fluidity and induces nisin resistance in Listeria monocytogenes. Probiotics Antimicrob. Prot. 1:130-135. DOI 10.1007/s12602-009-9025-8 in.
  • Badaoui Najjar, M.Z. 2009, Listeria monocytogenes adjusts its membrane fluidity, ATPase activity and atpE transcription levels in response to cold and acid stress. Ph.D. dissertation, the Graduate School-New Brunswick, Rutgers the State University.
  • Montville, T.J. 2009. Membrane fluidity regulates Listeria monocytogenes ATPase. Tokyo Institute of Technology. Tokyo, Japan.
  • Montville, T.J. 2009. The role of membrane fluidity and transcriptional regulation on the F0F1ATPase of Listeria monocytogenes. Second International Symposium on Probiotics and Antimicrobial Peptides. St-Malo, France.
  • Bonnet, M., Ravi, M., Chikindas, M.L. and Montville, T.J. 2006. A bioenergetic mechanism for nisin resistance induced by the acid tolerance response of Listeria monocytogenes. Appl. Environ. Microbiol.72:2556-2563.


Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: This period's research has produced a new understanding of how previous exposure to acid "hardens" Listeria monocytogenes to subscequent acid exposure. Thus, the use of acid washes on (for example) cucumbers that would subsequently be fermented to pickles would have a negative effect of food safety. Furthermore, we examined the effect of this acid hardening on the effectiveness of the bacteriocin, nisin. (Nisin is a natural antimicrobial protein made by some bacteria that ferment dairy products.) We found that acid hardened cells were cross-resistant to nisin. These data suggest that the use of acid to decrease listerial load may be detrimental under some circumstances and must be use selectively. These findings were reported in one peer reviewed paper and one international conference (see below). The project resulted in the graduation of one doctoral student who is now employed at the FDA/University of Maryland Joint Center for Food Safety (JIFSAN). PARTICIPANTS: Drs. Thomas Montville and Michael Chikindas were the PIs on this project and were assisted by the Postdoctoral Research Associate Ruth Wirawan and the Graduate Assistant Mohamed Badaoui Najjar. Dr. Richard Ludescher provided assistance with fluorospectrophotometry, and there was an informal collaboration with Dr. Brian Wilson. As external member of Badaoui Najjar' dissertation committee, Dr. Bassam Annous of the USDA provide additional guidance to the project. TARGET AUDIENCES: The immediate audience for this information is researchers in the area of microbial food safety and the federal regulators responsible for the food processing practices that assure it. Eventually this information will diffuse to food processors to the benefit of the eating public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research has provided a better understanding of when and where acid washes can be used. While they were previously used indiscriminately as a blanket intervention to improve food safety, our results indicate that in some cases, they can have the opposite effect. The doctoral level scientist trained in this project is expected to have a continuing positive influence on food safety through his association with JIFSAN.

Publications

  • Baadaout Najjar, M.Z., Chikindas, M.L., amd Montville, T.J. 2009. The acid tolerance response alters membrane fluidity and induces nisin resistance in Listeria monocytogenes. Probiotics and Antimcrobial. Proteins. (published ahead of print, DOI 10.1007/s12602-009-9025-8 in)
  • Montville, T.J. 2009. The role of membrane fluidity and transcriptional regulation on the F0F1ATPase of Listeria monocytogenes. Second International Symposium on Probiotics and Antimicrobial Peptides. St-Malo, France. (Abstract)


Progress 09/01/07 to 08/31/08

Outputs
OUTPUTS: The first objective of the project is to elucidate the mechanism(s) which regulate FOF1ATPase activity. Part of this has been accomplished by demonstrating that the membrane fluidity is regulated in wild-type cells but not mutant strains which are incapable of making branched chain fatty acids (BFAs); the ATPase activity was correlated with membrane fluidity. However, when the BFA precursors were added to the mutant, normal fluidity was restored without the corresponding change in ATPase activity. This led to an analysis using realtime PCR, which demonstrated that the regulation occurs at the transcriptional level. These results have been disseminated through publication and discussions at national meetings which have led to collaborations with scientists at Memorial Sloan Kettering Hospital and Cornell University. PARTICIPANTS: Dr. Thomas Montville, PD Dr. Michael Tchikindas, Co-PD Dr. Ruth Wirwan, responsible for genetic studies Mr. Mohamed Badaoui Najjar, responsible for physiological studies Ms. Shanta Addeeb, USDA National Need Fellow Dr. Martin Weidman, Cornell University, informal collaboration on membrane fluidity of Listeria mutants Dr. Michael Glickman, Memorial Sloan Kettering Cancer Center, informal collaboration on membrane fluidity of Mycobacteria TARGET AUDIENCES: Targets include, members of the food industry who wish to control listeria in foods, scientists who study bioenergetics as an underlying theme to microbial physiology, and researchers interested in the dynamics of the microbial membrane and its use as a model system. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
These findings have resulted in several changes in knowledge. 1. In contrast to the generally accepted dogma, we have demonstrated that the membrane composition is not the sole determinant of its fluidity. 2. Although the BFA mutant and wild-type had the expected differences in fluidity and ATPase activity, complimenting the mutation reversed the former, but not the later. 3. The F0F1 appears to be regulated at the transcriptional level.

Publications

  • Badaoui Najjar, M. and T. J. Montville. 2007. Changes in Listeria monocytogenes membrane fluidity and behavior in response to temperature. New York Institute of Food Technologists. (Poster)
  • Badaoui Najjar, M. and T. J. Montville. 2007. Changes in Listeria monocytogenes membrane fluidity in response to temperature. American Society of Microbiology. (Poster).
  • Badaoui Najjar, M, Chikindas, M. and Montville, T.J. 2007. Changes in Listeria monocytogenes membrane fluidity in response to temperature stress. Appl. Environ. Microbiol.73:6429-6435.
  • Badaoui Najjar, M, Chikindas, M. and Montville, T.J. 2008.Impact of Changes in Membrane Fluidity on ATPase Activiy of Listeria monocytogenes. Abs Ann. Meeting American Society for Microbiology.
  • Badaoui Najjar, M. and T. J. Montville. 2008. Acid Tolerance Response Induces Nisin Resistance and Membrane Fluidity Alterations in Listeria monocytogenes. Abs. Ann. Meeting Institute of Food Technologists.


Progress 09/01/06 to 09/01/07

Outputs
In the two months since the initiation of this project, team members have been selected, oriented to laboratory protocols, and are beginning studies on membrane fluidity.

Impacts
We expect this research to provide mechanisms which will reduce the growth of listeria in foods and thereby reduce listeriosis and its associated mortality

Publications

  • No publications reported this period