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Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria
Kirtimaan Syal1, Kelly Flentie2, Neerupma Bhardwaj1
1Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore, India.
Abstract:
Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
Insights
Researchers designed novel inhibitors targeting (p)ppGpp synthesis, a key bacterial survival mechanism. These compounds effectively reduced bacterial stress responses and biofilm formation in mycobacteria, showing promise as new antimicrobial agents with low toxicity.
Area of Science:
- Microbiology and Molecular Biology
- Drug Discovery and Development
Background:
- Bacteria possess stringent response pathways, regulated by (p)ppGpp, enabling survival under stress conditions like starvation.
- The Rel enzyme's (p)ppGpp synthetase activity is crucial for mycobacterial stress response and host persistence.
- Targeting (p)ppGpp synthesis presents a novel strategy for inhibiting bacterial survival.
Purpose of the Study:
- To design and synthesize novel inhibitors of (p)ppGpp synthesis.
- To evaluate the efficiency of these inhibitors using enzyme kinetics.
- To assess the phenotypic effects of the inhibitors on mycobacteria, including stress response, persistence, and biofilm formation.
Main Methods:
- Synthesis and characterization of new (p)ppGpp synthesis inhibitors using mass spectrometry and NMR spectroscopy.
- Enzyme kinetics and isothermal titration calorimetry to study Rel enzyme inhibition and binding kinetics.
- Phenotypic assays in *Mycobacterium smegmatis* and *Mycobacterium tuberculosis* for long-term persistence, biofilm formation/disruption, and *in vivo* (p)ppGpp levels.
- Toxicity assessment using MTT and hemolysis assays; cell permeability confirmed by mass spectrometry.
Main Results:
- Designed inhibitors significantly and dose-dependently inhibited (p)ppGpp synthesis by RelMsm.
- Compounds demonstrated inhibition of long-term persistence, biofilm formation, and biofilm disruption in *M. smegmatis*.
- Inhibitors reduced *in vivo* (p)ppGpp levels in *M. smegmatis* and inhibited biofilm formation in *M. tuberculosis*.
- Tested compounds showed no toxicity to H460 cells or human red blood cells and exhibited cell membrane permeability.
Conclusions:
- Novel inhibitors targeting (p)ppGpp synthesis are effective against mycobacterial stress response and biofilm formation.
- These compounds demonstrate potential as non-toxic antimicrobial agents against pathogenic mycobacteria.
- Further development of these inhibitors could lead to new therapeutic strategies for treating mycobacterial infections.