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In-depth characterization of the metabolic effect of the bacterial alamorne ppGpp

Award Number: K99GM135536
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP
PERIOD OF PERFORMANCE START DATE: 02/01/2020
PERIOD OF PERFORMANCE END DATE: 09/01/2021

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In-depth characterization of the metabolic effect of the bacterial alamorne ppGpp - Project Summary Bacterial cells are extremely efficient in adapting to environmental stresses. As a prime example, they synthesize a second messenger called ppGpp in response to starvation. Accumulation of ppGpp in bacteria arrests growth and reprograms cellular physiology to promote survival. These effects of ppGpp is required for antibiotic persistence that allows bacteria to survive the treatment of antibiotics to which they do not encode genetic resistance. At molecular levels, ppGpp reprograms gene expression, and targets many other proteins involved in translation and small-molecule metabolism. As a preliminary study, I synthesized a photo-crosslinking probe of ppGpp and used this probe to capture and identify about 30 new ppGpp-binding proteins in E. coli. Recently, a ppGpp riboswitch has been discovered in Gram-positive organisms. Intriguingly, E. coli transcriptome has a rich secondary-structure landscape, but the capability of these secondary structures in binding to small-molecule metabolite has not been explored. Therefore, in Aim 1, I will use a photo-crosslinking approach to capture binding partners of ppGpp from E. coli transcriptome, and identify these transcripts using RNA-seq. Hit interactions will be validated biophysically, and their putative effects on translation efficiency or the transcript stability will be examined in vivo. Additionally, in my preliminary study, I also compared metabolite profiles in E. coli before and shortly after ppGpp induction, and found strong perturbation of dozens of essential metabolites. This perturbation of cellular metabolism goes much beyond known direct effects of ppGpp. It is unclear how ppGpp induction drives the inhibition of various metabolic pathways to effectively arrest bacterial growth and promote persistence. In Aim 2, I hypothesize that “ppGpp-sensitive” metabolites, i. e., those whose levels perturbed by ppGpp induction, serve as a proxy of ppGpp to regulate cellular metabolism. I will seek discovering protein-metabolite interactions responsible for this indirect effect of ppGpp. To this end, I will use time-resolved metabolomics to identify candidate metabolites and enzymes whose levels/activities perturbed contemporaneously with ppGpp accumulation. I will then screen for protein-metabolite interactions among these candidates using an ultrafiltration-based assay. Briefly, I will purify each protein of interest (POI) to high homogeneity. Then, I will subject a mixture containing a library of all candidate metabolites and a single POI to ultrafiltration. Analyzing the filtrate using MS should reveal a decrease of the POI’s cognate ligands. I will validate any novel interactions identified using biochemical, structural, and genetic approaches. Together, the proposed research will explore two new aspects of ppGpp signaling, namely RNA targeting and indirect effects on cellular metabolism via ppGpp-sensitive metabolites. This study may lead to the discovery of key regulatory interactions required for bacterial persistence, and these interactions may serve as targets for anti-persistence drug design.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2022 ( Subtotal = -$49,713 )
2022	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	4/18/2022	NON-COMPETING CONTINUATION	-$49,713
2022	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	2	12/14/2021	NON-COMPETING CONTINUATION	$0
														Subtotal = -$49,713

Issue Date FY: 2021 ( Subtotal = $100,000 )
2021	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	2	1/28/2021	NON-COMPETING CONTINUATION	$100,000
2021	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	8/24/2021	NON-COMPETING CONTINUATION	$0
														Subtotal = $100,000

Issue Date FY: 2020 ( Subtotal = $100,000 )
2020	2020	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	1	1/20/2020	NEW	$100,000
														Subtotal = $100,000

Grand Total All Awards = $150,287

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In-depth characterization of the metabolic effect of the bacterial alamorne ppGpp

Award Number: K99GM135536

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP

PERIOD OF PERFORMANCE START DATE: 02/01/2020

PERIOD OF PERFORMANCE END DATE: 09/01/2021

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