Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

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 Rel Msm in the presence of designed inhibitors in a dosedependent 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.

Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments

“…Interestingly, the activity of inhibitory compounds analogous to Rel proteins [synthetic (p)ppGpp] has been described for Mycobacterium spp. (419). The impacts of these molecules on long-term persistence, biofilm disruption, and downregulation of (p)ppGpp have been analyzed in vivo in M. smegmatis (419).…”

“…(419). The impacts of these molecules on long-term persistence, biofilm disruption, and downregulation of (p)ppGpp have been analyzed in vivo in M. smegmatis (419). Another compound, pyrazinoic acid (POA), has been described for M. tuberculosis and showed significant inhibition of persister cells (420).…”

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle

“…The stringent response and persistence in B. burgdorferi 3855 vaccines and antimicrobials (Wexselblatt et al, 2013;Syal et al, 2017).…”

Crystallographic and solution structure of the N‐terminal domain of the Rel protein from Mycobacterium tuberculosis

“…Because of the central role of Mt Rel in the pathogenic bacterium Mtb , knowledge about its individual structural domains, their mechanism(s) and regulation for maintaining their functions are essential and may provide a platform to design agents against its enzymatic activity, as demonstrated by the recently identified inhibitors targeting the synthetic activity of Mt Rel . Here, an enzymatically active Mt Rel NTD was generated, enabling the determination of its first crystallographic structure and its solution shape, revealing a dimeric arrangement in solution.…”