Periodic Reporting for period 4 - NascenTomiX (Ribosome inhibition by nascent or antimicrobial peptides)
Reporting period: 2021-12-01 to 2023-05-31
During translation by the ribosome, nascent proteins pass through a long exit tunnel before being released into the cell. The NascenTomiX project focuses on nascent proteins called arrest peptides, which halt their own translation by interacting with the ribosomal interior. In some cases, arrest requires the presence of a small molecule and alters the expression of downstream genes in a metabolite-dependent manner. The aim of this project was to decipher the arrest code that dictates whether a nascent protein blocks translation. In practice, this involved addressing the following questions.
First, what types of small molecules can arrest peptides sense? Having a system where you can control translation in a ligand-dependent manner allows you to build biological sensors or metabolite-controlled gene circuitry for synthetic biology or biotechnology applications. But to build them, one must first have the means to identify new amino acid motifs that arrest translation.
Second, what is the prevalence of arrest peptides in nature? Fewer than a dozen arrest sequences have been found to date, most of them by chance. And yet, it is increasingly evident that short functional peptides are widespread in all kingdoms of life. This suggests that an entire layer of gene regulation is simply hidden in plain sight, waiting to be discovered.
Third, can arrest peptides lead us to new antibiotics? Arrest peptides interact with the same sites on the ribosome as several classes of clinically important drugs. Deciphering the arrest code in bacteria will therefore make it easier for us to design or evolve peptides with antimicrobial properties. In fact, antimicrobial peptides produced by the innate immune response are known to kill harmful bacteria by binding to the very sites that are targeted by arrest peptides.
Fourth and last, how do nascent and free peptides block the ribosome? Data available at the onset of the project pointed to several modes of inhibition, but it is important to get the whole picture in order to fully decipher the arrest code. This includes understanding how small molecules are sensed by arrest peptides.
To summarize, the NascenTomiX project has sought to decipher the arrest code in bacteria. This meant discovering nascent peptides that arrest translation in response to small molecules (L-ornithine, Erythromycin, Tetracenomycin X) and control gene expression in pathogenic bacteria (SpeFL, MsrDL). It meant developing tools (iTP-seq) to facilitate the study and future development of ribosome-targeting antibiotics, and the identification and characterization of arrest peptides. It also meant identifying unexpected modes of ribosome inhibition by nascent (ErmDL, TnaC, SpeFL, MsrDL) and free (Tur1A) peptides. This project has had an impact on a fundamental level, by revealing the mechanisms through which arrest peptides sense small metabolites. It will also have a practical impact by spearheading the development of next-generation antimicrobials and small molecule sensors.
First, what types of small molecules can arrest peptides sense? Having a system where you can control translation in a ligand-dependent manner allows you to build biological sensors or metabolite-controlled gene circuitry for synthetic biology or biotechnology applications. But to build them, one must first have the means to identify new amino acid motifs that arrest translation.
Second, what is the prevalence of arrest peptides in nature? Fewer than a dozen arrest sequences have been found to date, most of them by chance. And yet, it is increasingly evident that short functional peptides are widespread in all kingdoms of life. This suggests that an entire layer of gene regulation is simply hidden in plain sight, waiting to be discovered.
Third, can arrest peptides lead us to new antibiotics? Arrest peptides interact with the same sites on the ribosome as several classes of clinically important drugs. Deciphering the arrest code in bacteria will therefore make it easier for us to design or evolve peptides with antimicrobial properties. In fact, antimicrobial peptides produced by the innate immune response are known to kill harmful bacteria by binding to the very sites that are targeted by arrest peptides.
Fourth and last, how do nascent and free peptides block the ribosome? Data available at the onset of the project pointed to several modes of inhibition, but it is important to get the whole picture in order to fully decipher the arrest code. This includes understanding how small molecules are sensed by arrest peptides.
To summarize, the NascenTomiX project has sought to decipher the arrest code in bacteria. This meant discovering nascent peptides that arrest translation in response to small molecules (L-ornithine, Erythromycin, Tetracenomycin X) and control gene expression in pathogenic bacteria (SpeFL, MsrDL). It meant developing tools (iTP-seq) to facilitate the study and future development of ribosome-targeting antibiotics, and the identification and characterization of arrest peptides. It also meant identifying unexpected modes of ribosome inhibition by nascent (ErmDL, TnaC, SpeFL, MsrDL) and free (Tur1A) peptides. This project has had an impact on a fundamental level, by revealing the mechanisms through which arrest peptides sense small metabolites. It will also have a practical impact by spearheading the development of next-generation antimicrobials and small molecule sensors.
Addressing the objectives defined for the NascenTomiX project required us to develop new methodologies. We first established iTP-seq, a scalable and versatile tool to identify peptide-encoding transcripts that induce translational arrest in vitro (Seip et al., 2018). While it is similar to Ribo-seq, a technique that is widely used to monitor patterns of gene expression in living cells, iTP-seq can be used on transcript libraries of any size, composition or complexity, making it ideal to analyze translational landscapes at a fraction of the cost.
We have used iTP-seq to study how bacteria harboring an ermD resistance gene under the control of an ErmDL arrest peptide become resistant to macrolides upon exposure to these antibiotics (Beckert et al., 2021). Coupled to a study on the drug-dependent arrest peptide MsrDL (Fostier et al., 2023), our work shed light on the mechanisms used by certain bacteria to control the expression of resistance genes. Moreover, we found that iTP-seq is ideally suited to the study of ribosome-targeting antibiotics, such as the aromatic polyketide Tetracenomycin X (Leroy et al., 2023). Identifying detailed mechanisms of action for widely-used, recently discovered or forgotten antibiotics should facilitate the development of improved therapeutics.
In order to identify naturally occurring arrest peptides in bacteria, we developed retapamulin-assisted inverse toeprinting (RET-iTP), a genome-wide profiling method for the identification of translation initiation sites in vitro that is independent of transcript abundance (to be published). During the course of the project, we discovered and characterized SpeFL, an arrest peptide that functions as an L-ornithine sensor to activate polyamine biosynthesis in γ-proteobacteria (Herrero del Valle et al., 2020). Using a combined structural and biochemical approach, we determined that activation of the putrescine biosynthesis gene speF relies upon ribosome stalling resulting from the capture of L-ornithine by a ribosome translating speFL. Moreover, we showed structurally how the ribosome and SpeFL form a highly selective binding pocket for L-ornithine. Similarly, we determined the mechanism by which the arrest peptide TnaC senses the amino acid L-tryptophan to trigger indole production in γ-proteobacteria (van der Stel et al., 2021). Together, these studies revealed the basic principles underlying the detection of small molecules with low intrinsic affinity for the ribosome by arrest peptides.
Although iTP-seq is a key tool in our search for metabolite-dependent arrest peptides, evolving peptides that target ribosomes in trans required us to develop a different kind of methodology. We therefore devised a droplet-based microfluidics approach that compartmentalizes an in vitro translation reaction, such that a single peptide variant is produced per droplet (to be published). The effect of each peptide on the production of a fluorescent reporter inside live bacteria is monitored and fluorescence-activated droplet sorting is used to retain droplets that contain inhibitory peptides. We have established that this approach can be used to select peptides that inhibit translation and plan to incorporate it into a novel pipeline to develop ribosome-targeting antimicrobial peptides.
We have used iTP-seq to study how bacteria harboring an ermD resistance gene under the control of an ErmDL arrest peptide become resistant to macrolides upon exposure to these antibiotics (Beckert et al., 2021). Coupled to a study on the drug-dependent arrest peptide MsrDL (Fostier et al., 2023), our work shed light on the mechanisms used by certain bacteria to control the expression of resistance genes. Moreover, we found that iTP-seq is ideally suited to the study of ribosome-targeting antibiotics, such as the aromatic polyketide Tetracenomycin X (Leroy et al., 2023). Identifying detailed mechanisms of action for widely-used, recently discovered or forgotten antibiotics should facilitate the development of improved therapeutics.
In order to identify naturally occurring arrest peptides in bacteria, we developed retapamulin-assisted inverse toeprinting (RET-iTP), a genome-wide profiling method for the identification of translation initiation sites in vitro that is independent of transcript abundance (to be published). During the course of the project, we discovered and characterized SpeFL, an arrest peptide that functions as an L-ornithine sensor to activate polyamine biosynthesis in γ-proteobacteria (Herrero del Valle et al., 2020). Using a combined structural and biochemical approach, we determined that activation of the putrescine biosynthesis gene speF relies upon ribosome stalling resulting from the capture of L-ornithine by a ribosome translating speFL. Moreover, we showed structurally how the ribosome and SpeFL form a highly selective binding pocket for L-ornithine. Similarly, we determined the mechanism by which the arrest peptide TnaC senses the amino acid L-tryptophan to trigger indole production in γ-proteobacteria (van der Stel et al., 2021). Together, these studies revealed the basic principles underlying the detection of small molecules with low intrinsic affinity for the ribosome by arrest peptides.
Although iTP-seq is a key tool in our search for metabolite-dependent arrest peptides, evolving peptides that target ribosomes in trans required us to develop a different kind of methodology. We therefore devised a droplet-based microfluidics approach that compartmentalizes an in vitro translation reaction, such that a single peptide variant is produced per droplet (to be published). The effect of each peptide on the production of a fluorescent reporter inside live bacteria is monitored and fluorescence-activated droplet sorting is used to retain droplets that contain inhibitory peptides. We have established that this approach can be used to select peptides that inhibit translation and plan to incorporate it into a novel pipeline to develop ribosome-targeting antimicrobial peptides.
Elucidating the mechanism by which a ribosome translating a specific arrest peptide can be turned into a small molecule sensor represents a breakthrough in our understanding of an important yet often overlooked aspect of ribosome function. Arrest peptides were first discovered over forty years ago, but their detailed mechanisms of action have remained a mystery, with the exception of a few drug-dependent arrest peptides whose antibiotic ligands bind to the ribosome with high affinity even in the absence of a nascent peptide. In contrast, this ERC project has shown that the sensing of small molecules with low intrinsic affinity for the ribosome is a dynamic process, in which the kinetics of translation and ligand binding must be finely tuned. With this knowledge and with the tools developed during the course of this project, it will be possible to develop novel biological sensors, to better understand certain forms of gene regulation (e.g. related to antibiotic resistance or the onset of virulence) and, perhaps in the long run, to design context-dependent inhibitors of translation that block the synthesis of individual proteins.