Periodic Reporting for period 4 - PP-MAGIC ((Photo-)Control of Persisters: Targeting the Magic Spot)
Período documentado: 2024-09-01 hasta 2025-08-31
The COVID-19 pandemic has underscored how vulnerable modern societies are to infectious diseases. While viral infections have dominated attention, bacterial infections are also rising and becoming harder to treat as many species evolve into multidrug-resistant strains. Beyond acquired resistance, bacterial persistence poses a major challenge: some bacteria drastically slow or halt their metabolism, surviving antibiotics that target active cells.
We aim to develop new chemical tools and compounds to better understand and ultimately overcome bacterial persistence, improving metabolism-dependent antibiotic therapies. Persistence is regulated by small signalling molecules called magic spot nucleotides (MSN), closely linked to e.g. inorganic polyphosphate metabolism. This project seeks to elucidate how MSN control bacterial metabolism and identify ways to interfere with this regulation, potentially enhancing antibiotic efficacy. We also investigate how bacteria modulate interactions with hosts -humans or plants- via MSN and related messengers such as inositol pyrophosphates.
This work tackles a pressing societal challenge: infections by persistent bacteria that threaten human health and agriculture. Using a chemical biology and medicinal chemistry approach, we integrate synthetic and analytical chemistry, biochemistry, and microbiology to understand and manipulate MSN function.
Conclusions of the Action
We developed efficient synthetic routes to a wide range of MSN, including previously inaccessible structures such as isotope-labelled, light-activated (caged), and cell-permeable analogues - yielding general insights into small-molecule permeability. Using these tools, we showed for the first time that light-triggered MSN release can modulate growth of bacteria. We also designed pull-down probes to identify nucleotide-binding proteins in pathogenic and non-pathogenic bacteria, revealing key players in the stringent response and potential crosstalk with other alarmones like dinucleoside polyphosphates. Our molecules are now used by other laboratories, extending the project’s impact.
Beyond synthesis and biochemistry, we established the most sensitive analytical platform for quantifying MSN in bacteria and infected plants. Capillary electrophoresis–mass spectrometry (CE-MS) with heavy-isotope standards showed that plants produce MSN upon infection. We also examined changes in polyphosphate and inositol pyrophosphate levels, and developed luminescence-based sensors to distinguish polyphosphates, nucleotides, and MSN, enabling high-throughput inhibitor screening. Finally, we initiated the design of bisubstrate analogue inhibitors.
In summary, this work delivers new synthetic, analytical, and biochemical tools to probe bacterial stress signalling, deepens understanding of persistence, and offers promising strategies for next-generation antibiotics.
We aim to develop new chemical tools and compounds to better understand and ultimately overcome bacterial persistence, improving metabolism-dependent antibiotic therapies. Persistence is regulated by small signalling molecules called magic spot nucleotides (MSN), closely linked to e.g. inorganic polyphosphate metabolism. This project seeks to elucidate how MSN control bacterial metabolism and identify ways to interfere with this regulation, potentially enhancing antibiotic efficacy. We also investigate how bacteria modulate interactions with hosts -humans or plants- via MSN and related messengers such as inositol pyrophosphates.
This work tackles a pressing societal challenge: infections by persistent bacteria that threaten human health and agriculture. Using a chemical biology and medicinal chemistry approach, we integrate synthetic and analytical chemistry, biochemistry, and microbiology to understand and manipulate MSN function.
Conclusions of the Action
We developed efficient synthetic routes to a wide range of MSN, including previously inaccessible structures such as isotope-labelled, light-activated (caged), and cell-permeable analogues - yielding general insights into small-molecule permeability. Using these tools, we showed for the first time that light-triggered MSN release can modulate growth of bacteria. We also designed pull-down probes to identify nucleotide-binding proteins in pathogenic and non-pathogenic bacteria, revealing key players in the stringent response and potential crosstalk with other alarmones like dinucleoside polyphosphates. Our molecules are now used by other laboratories, extending the project’s impact.
Beyond synthesis and biochemistry, we established the most sensitive analytical platform for quantifying MSN in bacteria and infected plants. Capillary electrophoresis–mass spectrometry (CE-MS) with heavy-isotope standards showed that plants produce MSN upon infection. We also examined changes in polyphosphate and inositol pyrophosphate levels, and developed luminescence-based sensors to distinguish polyphosphates, nucleotides, and MSN, enabling high-throughput inhibitor screening. Finally, we initiated the design of bisubstrate analogue inhibitors.
In summary, this work delivers new synthetic, analytical, and biochemical tools to probe bacterial stress signalling, deepens understanding of persistence, and offers promising strategies for next-generation antibiotics.
During the final period, the project achieved several milestones that advanced our understanding of how small signaling molecules called magic spot nucleotides (MSN) function in living systems. We identified and validated proteins that interact with and regulate ppGpp, a central MSN, and showed how these partners influence cellular processes such as membrane depolarization. In parallel, we completed the development of photoactivatable MSN - light-sensitive molecular versions that can be modified, for example by introducing stable isotopes, sugars, or polycations to improve cellular uptake. These molecules were successfully tested in bacteria and other models, demonstrating broad biological applicability. A major breakthrough was the first delivery of MSN into living bacteria and their release on demand by irradiation. This enabled real-time tracking of MSN metabolism via capillary electrophoresis–mass spectrometry (CE–MS), which we refined into the most sensitive platform currently available for such studies. We also developed selective chemical sensors that detect MSN or related inorganic polyphosphates, enabling measurements in biological samples and high-throughput screening.
Detailed analyses of MSN levels in Escherichia coli during growth and nutrient stress revealed an unexpected coupling with another class of molecules, dinucleoside polyphosphates, suggesting stress messengers are more tightly linked than previously thought. We also began establishing a method to monitor MSN turnover using ¹⁸O-labeled water, useful for following metabolic fluxes in living cells.
Our work on chemical inhibitors of the bacterial enzyme RelA progressed substantially; we synthesized a diverse library of compounds to help clarify how MSN production is regulated in different bacterial species.
Throughout the project, we created an extensive toolbox of MSN derivatives, including authentic standards, isotope-labeled references, photoactivatable compounds, and affinity probes. We developed efficient new synthetic routes, such as chemoenzymatic approaches that now enable access to structures previously extremely difficult to obtain. Using CE–MS, we discovered that bacterial infection triggers MSN production in plants and addressed their molecular origin. We also observed oscillations in MSN levels correlating with other stress signals, revealing new directions for studying bacterial adaptation. The results were widely disseminated through 11 peer-reviewed publications, with three more in preparation. Team members presented findings at international conferences and workshops, earning poster prizes and winning science slams for the public. Importantly, the new analytical tools, datasets, and molecular probes are freely available to the scientific community, ensuring others can build upon this research.
Detailed analyses of MSN levels in Escherichia coli during growth and nutrient stress revealed an unexpected coupling with another class of molecules, dinucleoside polyphosphates, suggesting stress messengers are more tightly linked than previously thought. We also began establishing a method to monitor MSN turnover using ¹⁸O-labeled water, useful for following metabolic fluxes in living cells.
Our work on chemical inhibitors of the bacterial enzyme RelA progressed substantially; we synthesized a diverse library of compounds to help clarify how MSN production is regulated in different bacterial species.
Throughout the project, we created an extensive toolbox of MSN derivatives, including authentic standards, isotope-labeled references, photoactivatable compounds, and affinity probes. We developed efficient new synthetic routes, such as chemoenzymatic approaches that now enable access to structures previously extremely difficult to obtain. Using CE–MS, we discovered that bacterial infection triggers MSN production in plants and addressed their molecular origin. We also observed oscillations in MSN levels correlating with other stress signals, revealing new directions for studying bacterial adaptation. The results were widely disseminated through 11 peer-reviewed publications, with three more in preparation. Team members presented findings at international conferences and workshops, earning poster prizes and winning science slams for the public. Importantly, the new analytical tools, datasets, and molecular probes are freely available to the scientific community, ensuring others can build upon this research.
We explored how magic spot nucleotides (MSN) help bacteria and plants respond to stress. We first developed chemical methods to synthesize these complex molecules quickly and diversely and now have MSN that can be tracked inside cells or activated by light, giving scientists powerful tools to study their functions. Our methods are already used by other groups and have opened new possibilities for studying cellular communication.
A key achievement was the development of analytical techniques that detect MSN in living organisms at extremely low concentrations. Using these, we showed for the first time that bacteria and plants both use MSN during infection, and that the molecules’ origin can be assigned. This discovery sheds light on how pathogens and hosts interact at the molecular level.
We also delivered MSN directly into living cells and controlled their release with light. This breakthrough allows observation of cellular reactions to MSN level changes and enables real-time study of stress signaling. Such precision had never been achieved for this class of molecules.
Finally, light-sensitive MSN helped identify proteins interacting with these messengers, revealing new pathways and links between stress-related signaling systems.
Together, these advances establish a research platform combining chemistry, biology, and analytics. The results will help scientists understand how cells adapt to stress, providing knowledge that may guide new strategies in biotechnology, agriculture, and medicine.
A key achievement was the development of analytical techniques that detect MSN in living organisms at extremely low concentrations. Using these, we showed for the first time that bacteria and plants both use MSN during infection, and that the molecules’ origin can be assigned. This discovery sheds light on how pathogens and hosts interact at the molecular level.
We also delivered MSN directly into living cells and controlled their release with light. This breakthrough allows observation of cellular reactions to MSN level changes and enables real-time study of stress signaling. Such precision had never been achieved for this class of molecules.
Finally, light-sensitive MSN helped identify proteins interacting with these messengers, revealing new pathways and links between stress-related signaling systems.
Together, these advances establish a research platform combining chemistry, biology, and analytics. The results will help scientists understand how cells adapt to stress, providing knowledge that may guide new strategies in biotechnology, agriculture, and medicine.