Disentangling the stringent response to engineer novel anti-persister drugs

Project Information

DiStRes

Grant agreement ID: 864311

DOI

10.3030/864311

Project closed

EC signature date 6 March 2020

Start date 1 December 2020

End date 30 November 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

UNIVERSITE LIBRE DE BRUXELLES
Belgium

Periodic Reporting for period 3 - DiStRes (Disentangling the stringent response to engineer novel anti-persister drugs)

Reporting period: 2023-12-01 to 2025-05-31

Summary of the context and overall objectives of the project

Game-changing advances in understanding how bacteria modulates growth during stress
Researchers supported by the EU-funded DiStRes project have unraveled the major cues behind the regulation of the bacterial second messenger guanosine penta- and tetra-phosphate (pp)pGpp by bacterial stringent factors. This ubiquitous nucleotide “alarmones” coordinates growth and stress adaptation in bacteria.
As explained in the study published in the journals ‘Nature Chemical Biology’ and ‘Science Advances’, the research team obtained high resolution crystal structures of a complex of the enzyme Rel in different catalytic states (synthesis and hydrolysis of (pp)pGpp) and also activated off the ribosome by DarB, a bacterial regulatory protein and the structure and the first high resolution structure of SpoT, the full-length enzyme that destroys (pp)pGpp and re-establishes a fast-growing bacterial phenotype. These structural data, together with biophysical and microbiological measurements were combined to produce an overarching model that integrates all the allosteric checkpoints involved in the regulation of stringent factors.
While cryo-EM structures of the RSH synthetases have revealed the overall mechanism that controls how ppGpp is produced during stress, stabilising RSH enzymes in the ribosome, these structures did not have sufficient resolution at the active site level and for decades high resolution details on the catalytic states of RSH enzymes was lacking. Thus these proteins remained refringent to structural biology studies: purifying the enzymes proved to be very challenging, and the lack of information about its three-dimensional structure hinder developing molecules that would interfere with the (pp)pGpp metabolism. All this is now feasible given in the context of the structural biology breakthroughs of the DiStRes team.

Why stringent factors?
Determining how fast should one grows is one of the most important decisions for a pathogenic bacterium. If the bacterium grows too fast and overcommits, there will not be enough recourses for sustaining the cellular multiplication. If the bacterium grows too slow and does not exploit the nutritional recourses efficiently, it runs the risk of being too slow to establish a successful infection and will be eradicated by the immune system.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

A collaborative push to understand bacterial stress
The DiStRes team of Garcia-Pino at the Université libre de Bruxelles (ULB), joined forces with Lund University and the Université de Namur, to tackle the challenge of understanding the various regulatory layers of the different stringent factor. They focused on model organisms such as Thermus thermophilus, Escherichia coli, and Bacillus subtilis, and the important human pathogen Acinetobacter baumannii, a member of so-called ESKAPE group of bacterial pathogens of the greatest concern. The international team solved the three-dimensional structure of various stringent factors in different active catalytic states to cracked the molecular code that determines how fast these enzymes pulse. To slam on breaks on the growth rate, RSH enzymes assume a compact, mushroom-like shape. To let the bacterium grow at the maximum speed, they open up and ‘relaxed’ primed to bind the empty A-sites of ribosomes or engage other bacterial adaptor proteins that become active under particular stress signals.
Notably, one of the outcomes of these research identified a short ‘core’ domain located in between the N-terminal catalytic domains and C-terminal regulatory domains of stringent factors that is the linchpin of the active hydrolase conformation of the enzyme and key for the switch to active synthetase states. Interestingly, the propensity for the different active states correlated with the length and disorder of the core region in of the different families of stringent factors SpoT, Rel and RelA, which could mean that genetic expansion and contraction in the core is a defining evolutionary feature of the specialisation of RSH enzymes into (p)ppGpp synthetases or hydrolases. Since RSH enzymes can sense environmental stresses to modulate stress adaptation through coordinating (p)ppGpp synthesis and hydrolysis, further determining the control of the different states would be an exciting future research direction to enhance understanding of its role in stress signalling through (p)ppGpp hydrolysis.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

For the second part of the project, I anticipate that the development of a ppGpp biosensor to monitor alarmone levels in vivo constitute our major scientific challenge together with the discovery of novel molecules that regulate long stringent factors. Despite the obvious difficulties associated with the development of a novel biosensor, this is exacerbated by the fact that ppGpp is involved in the control of transcription, translation an dreplication. Having tool that allow us to monitor ppGpp levels in vivo, would again deliver another major breakthrough impinging beyond the field of the stringent response.
To address these challenges we plan to build on the mechanising framework we have developed and also incorporate to our research new technologies such as the use of Alphafold and modern artificial intelligence tools for structural biology that were not available when the project was launched.
In this regard we have constructed a large library of camelid nanobodies that bind RHS enzymes from E. coli, S. aureus and C. tepidum and we are going to screen this library to discover which nanobodies will inhibit the enzymes and induce a growth arrest phenotype. Based on the allosteric hot-spots we discover we will then design peptido-mimetics or screen for compounds that could bind these allosteric spots and modulate the enzymes.
We have also started two strategies to design the ppGpp biosensors. We will use an approach based on a riboswitch that responds to varying levels of ppGpp and a second approach based on the development of a chimera resulting from the fusion of a ppGpp binding protein and a fluorescent protein. Once a stable molecule that responds to ppGpp is obtained, it will be optimize by mutagenesis to generate a biosensor with a dynamic range that allows to study how the levels of ppGpp change during the cell cycle.
We are also in the process of establishing an approach that combines the use of Alphafold with traditional microbiology screening methods to discover novel RSH interacting molecules.

Cartoon how ppGpp regulates bacterial metabolism

Cartoon representing all the conformational states we have observed in long RSH enzymes

Structure of SpoT from A. baumannii in different states