Abstract
Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters’ temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis via pyrophosphate transfer from an ATP molecule to the 3’ position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on RelSeq (from S. equisimilis), still the only bifunctional Rel protein for which a GDP-bound “synthetase-ON” structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of RelSeq by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from S. aureus. Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.
Highlights
Persistence is a very elegant bet hedging strategy adopted by bacteria to survive hostile conditions, such as nutrient starvation and antibiotic treatment (Lewis, 2010; Maisonneuve and Gerdes, 2014)
The position of the corresponding catalytic residues in the RelTt post-catalytic structure is similar to RelP pre-catalytic state, indicating that a conformation change seems to be necessary for the activation of RelSeq
The glutamic acid residue of all three proteins is located in a rigid secondary structure element (E174 in RelP-β5, E323 in RelSeq-β7 and E345 in RelTt-β7, respectively) and its interaction with the Mg2+ ion is mediated in RelP by two water molecules
Summary
Persistence is a very elegant bet hedging strategy adopted by bacteria to survive hostile conditions, such as nutrient starvation and antibiotic treatment (Lewis, 2010; Maisonneuve and Gerdes, 2014). This temporary antibiotic-tolerant phenotype plays a starring role in the difficult treatment and eradication of chronic and recurrent infections (Conlon et al, 2015) and possibly in the insurgence of actual antimicrobial resistance (Cohen et al, 2013; Barrett et al, 2019). Intrigued by the role played by the stringent response, a stress-induced signalling cascade in bacterial survival (Irving et al, 2020), we decided to focus our attention on the triggering event of the cascade, i.e. the accumulation within the cell of guanosine tetra- or pentaphosphate, collectively called (p) ppGpp and often referred to as alarmone (Kushwaha et al, 2019). (p)ppGpp is an histrionic second messenger that plays pleiotropic effects in bacterial cells and has been directly linked to the insurgence of persisters and antimicrobial resistance (Wu et al, 2010;Kanjee et al, 2012;Liu et al, 2015;Kamarthapu et al, 2016;Dutta et al, 2019;Diez et al, 2020)
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