Source: UNIV OF WISCONSIN submitted to
(P)PPGPP-MEDIATED ANTIBIOTIC TOLERANCE IN THE GRAM-POSITIVE BACTERIUM BACILLUS SUBTILIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1006577
Grant No.
(N/A)
Project No.
WIS01875
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2015
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Project Director
Wang, JA, .
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
Non Technical Summary
Sub-therapeutic and therapeutic doses of antibiotics are commonly applied to livestock in agriculture to enhance growth and treat disease. An increasing concern is the emergence and spread of antibiotic resistant bacteria, including drug-resistant human pathogens. To prevent microbial pathogens from reoccurrence during food production, it is critical to understand how microbes survive antibiotic stress.We discovered a mechanism by which the stress-inducible nucleotide (p)ppGpp promotes antibiotic tolerance in Gram positive bacteria, and identified targets of (p)ppGpp that are conserved in Gram-positive pathogens such as B. anthracis, B. cereus, E. faecalis and S. aureus. We will explore how to increase the efficacy of antibiotic treatment of Gram-positive pathogens by disrupting this pathway, thus promote agriculture productivity. We will develop effective strategies to prevent persistence. Because persistence is often the first step in development of antibiotic resistance in pathogenic bacteria, some of which infect humans and animals alike, we can effectively reduce the presence of antimicrobial-resistant human pathogens in meat and thus promote Food Safety.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71238401100100%
Goals / Objectives
The major goal of this project is to understand how Gram-positive bacteria tolerate antibiotic treatments. Understanding this basic mechanism will provide new directions in antagonizing pathogenic Gram-positive bacteria by reducing their tolerance.
Project Methods
We will use genetic, cell biological and biochemical approaches to conduct the research. For example, we engineer mutants of bacteria that are more sensitive to antibiotic stress, and then trying to treat wild type cells in a way that would mimic the mutations. By doing so, we develop effective strategies that potentiate antibiotic efficacy.Evaluation: We hope to publish major data papers and reviews on peer reviewed journals.

Progress 10/01/15 to 09/30/18

Outputs
Target Audience:The target audience is the agricultural and health service communities. My effort is acquiring knowledge of how bacteria survive antibiotic treatment. This knowledge will be delivered to the scientific community to assist the development of strategies to potentiate antibiotic efficacy, thus promoting agricultural development in Wisconsin. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project trains two graduate students: Jessy Tse-Barra, who has graduated with a PhD last year, and based on her training in my lab, was hired as a Scientist at a microbiology company, Rapid Micro Biosystems. Currently, the project is training Jin Yang, who will be graduating in one or two years. How have the results been disseminated to communities of interest?Through publications (please see publication list). In addition, I have given many invited seminars both on campus, throughout USA and abroad. My audience includes students, postdocs, professors from universities, as well as from industry and government agency. 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 characterized diverse pathways of persister formation in Gram-positive bacteria: develops stochastically, induced by starvation or antibiotic treatment, and identified depletion of GTP as a common mechanism. We showed, for the first time, single cell entrance to persistence as GTP levels drop beneath a threshold, and demonstrated GTP depletion enable most cells to survive lethal antibiotics. GTP depletion is mediated by the nucleotide (p)ppGpp, whose spontaneous accumulation triggers switch-like persister dynamics instead of gradual entrance to dormancy. (p)ppGpp is also inducible by starvation and lethal/sublethal concentrations of antibiotics, which allows adaptive tolerance. The multiplepathways entering persistence require different (p)ppGpp synthetases, suggesting that persistence via the (p)ppGpp-GTP dichotomy is an evolved trait which can be exploited to potentiate antibiotic efficacy.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Danny K. Fung, Jessica L. Tse, Jeremy W. Schroeder, David Ying, Jue D. Wang. Spontaneous and antibiotic-induced persister formation by (p)ppGpp-GTP dichotomy.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Tippapha Pisithkul, Jeremy W. Schroeder, Edna A. Trujillo, Polkrit Yeesin, Tai Chaiamarit, Joshua J. Coon, Jue D. Wang, and Daniel Amador-Noguez. Metabolic remodeling during biofilm development of Bacillus subtilis


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience is the agricultural community of Wisconsin. We acquired knowledge of how soil bacteria and pathogens survive antibiotic treatment. This knowledge will be delivered to the scientific community to assist the development of strategies to potentiate antibiotic efficacy and prevent antibiotic resistance. Together, our efforts will promote agricultural development and public health in Wisconsin. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In December 2017, the graduate student successfully defended her Ph.D thesis and is now interviewing for scientist positions in Microbiology industry. How have the results been disseminated to communities of interest? We submitted several publications. What do you plan to do during the next reporting period to accomplish the goals?My graduate student Jin Yang will be working to identify and characterize direct targets of(p)ppGpp in the Gram positive pathogen Bacillus anthracis. Preliminary results indicate that there are 1% of the proteome is regulated by (p)ppGpp. Jin will verify these results and examine the functions of these interactions in protecting B. anthracis during starvation and antibiotic stress.

Impacts
What was accomplished under these goals? We have concluded from our study that there is a highly conserved mechanism that is responsible for the failure of antibiotics in killing Gram positive bacteria. The knowledge we gained provides the keys to reducing Gram-positive bacteria's ability to survive antibiotics. A major culprit of antimicrobial treatment failure is dormant, antibiotic-tolerant persisters, yet how persisters are generated remain poorly un­­derstood. Using a single cell reporter for Gram-positive persisters, we show that persisters can form via diverse pathways: induced by starvation, switched on stochastically in unstressed cells, and in response to antibiotic treatment. All three pathways commence from intracellular accumulation of the bacterial alarmone (p)ppGpp, mediated through its antagonizing effect on GTP, resulting in phenotypic switch from rapid growth to dormancy. (p)ppGpp spontaneously accumulates via dual-action of two (p)ppGpp synthetases RelA and SAS1. (p)ppGpp is induced by sublethal concentrations of antibiotic treatment­ via SAS2 to increase adaptive antibiotic tolerance. The strikingly conserved role of (p)ppGpp in persistence and the diverse pathways leading to (p)ppGpp accumulation, implicates that persistence is a highly regulated cellular process shaped by diverse evolutionary pressure. In addition, we made the following observations: It was proposed in 2016 that ATP depletion, not ppGpp, is associated with persistence in Gram positive bacteria S. aureus. (Conlon BP, Rowe SE, Gandt AB, Nuxoll AS, Donegan NP, Zalis EA, Clair G, Adkins JN, Cheung AL, Lewis K. Nat Microbiol. 2016 1:16051. doi: 10.1038/nmicrobiol.2016.51). We have tested this model extensively in S. aureus and B. subtilis. We show that ATP depletion leads to (p)ppGpp accumulation in both organisms, and loss of (p)ppGpp synthetases compromised antibiotic tolerance in ATP-depleted cells. This indicates that (p)ppGpp is responsible for persistence in these organisms during starvation. We have performed a Tn-seq experiment to identify genetic factors other than ppGpp that are involved in persistence. We identified two pathways, the purine/pyrimidine de novo biosynthesis pathway, and the manganese homoestasis pathway. Our characterization of these two pathways suggest that they may interact with the (p)ppGpp pathway to regulate persistence.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Vadia S, Tse JL, Lucena R, Yang Z, Kellogg DR, Wang JD, Levin PA. Curr Biol. 2017 Jun 19;27(12):1757-1767.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Fung DK , Anderson BW, Tse JL, Wang JD. Nucleotide second messengers: (p)ppGpp and cyclic dinucleotides. Bacillus, Cellular and Molecular Biology, Second Edition.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Berti et al."Evolution of Antibiotic Tolerance During Oxacillin, Daptomycin and Dalbavancin Therapy Results in Breakthrough Staphylococcus aureus Bacteremias". Open Forum Infect Dis. 2017 Fall; 4(Suppl 1): S14. PMC5631970


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience will be the agricultural community of Wisconsin. My effort is acquiring knowledge of how bacteria survive antibiotic treatment. This knowledge will be delivered to the scientific community to assist the development of strategies to potentiate antibiotic effacacy, thus promoting agricultural development in Wisconsin. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training Activities The Hatch project has provided opportunity for my graduate student Jessica Tse to conduct research into mechanisms of (p)ppGpp-mediated antibiotic tolerance in Gram positive bacteria. During the last year, Jessica has made great strides in becoming more knowledgeable about her project. She has obtained the majority of data and will be able to start writing her PhD thesis in half a year. She has fulfilled her one semester teaching practum and her professional career education program that is required for obtaining a PhD degree at University of Wisconsin. She is planning to graduate December 2017 and will be working in microbiology-related industry after graduation. Professional development Jessy has attended multiple meetings with topics covering bacterial stress response and antibiotic tolerance, including the Perlman Symposium on antibiotic discovery and development and the International Bacteria and Phage Conference, and presented with talks or posters about her work. Most notibly, Jessy has given an invited talk in the annual meeting of the American Society for Microbiology entitled "(p)ppGpp controls intracellular GTP levels to promote antibiotic tolerance in Bacillus subtilis.", and received very positive responses from world leading microbiologists. How have the results been disseminated to communities of interest?I have given more than ten talks in the last year about (p)ppGpp's effect in Gram positive bacteria, to a variety of Wisconsin, national and international audiences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Antibiotics are used to treat bacteria infections to restore human health. Antibiotics are also commonly applied to livestock in agriculture to enhance growth and treat disease. However, we are running out of effective antimicrobial treatment methods due to the world-wide emergence and spread of antibiotic resistant bacteria, especially drug-resistant human pathogens. Bacteria's ability to develop antibiotic resistance depends on a poorly understood phenomenon called antibiotic tolerance. Our major goal is to understand the mechanism of bacterial antibiotic tolerance. Our research has a strong fundamental impact on agriculture and food safety. It will provide knowledge that will allow the scientific and industrial communities to develop methods to minimizing the occurrance of antibiotic resistance, reducing this major threat to human and agriculture. We discovered that the stress-inducible molecule (p)ppGpp is critical for antibiotic tolerance of Gram-positive bacteria, and that (p)ppGpp's ability to reduce intracellular GTP levels is important for antibiotic tolerance. We plan to verify this hypothesis and further dissect the downstream targets of (p)ppGpp/GTP that are important for antibiotic tolerance. Our three goals are: 1. Test the hypothesis that (p)ppGpp/GTP mediate antibiotic tolerance by regulating transcription We have verified that (p)ppGpp/GTP indeed regulate transcription globally, however, we have yet to find evidence that any candidates with altered transcription are important for antibiotic tolerance. 2. Test the hypothesis that (p)ppGpp/GTP mediate antibiotic tolerance by regulating translation We have directly measured in vivo translation using radioactive incooperation assay, and obtained evidence suggesting that translation is unlikely the most critical factor affecting antibiotic tolerance. 3. Test the hypothesis that (p)ppGpp/GTP mediate antibiotic tolerance by preventing replication-transcription conflicts We have obtained evidence supporting the idea that for certain antibiotics, for example, flouroquinoline based drugs such as ciprofloxacin, (p)ppGpp increases survival of bacteria by preventing replication-transcription conflicts. In addition to the three objectives above, we have expanded our approaches to obtain new knowledge beyound our original hypotheses. 4. We performed fluorescent labeling of cell wall synthesis, and found that (p)ppGpp/GTP mediate tolerance to the antibiotics that target cell wall biosynthesis, such as vancomycin, by arresting cell wall biosynthesis. 5. In collaboration with Petra Levin in Washington University, we have shown that (p)ppGpp levels are also affected by fatty acid biosynthesis pathways, allowing cells to survive antibiotics such as ceruleum. 6. In collaboration with Warren Rose in UW-Madison, we have shown that elevated (p)ppGpp is also responsible for antibiotic tolerance of the human pathogen S. aureus.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Vadia S, Tse JL, Wang JD, Levin PA. Flux through lipid synthesis dictates cell size.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Fung DK , Anderson BW, Tse JL, Wang JD. Nucleotide second messengers: (p)ppGpp and cyclic dinucleotides. Bacillus, Cellular and Molecular Biology, Second Edition. (in press).