Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

The functional proteome landscape of Escherichia coli.

Recent developments in high-throughput reverse genetics1,2 have revolutionized our ability to map gene function and interactions3-6. The power of these approaches depends on their ability to identify functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out7-9. However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts10. Here we use a high-content biochemical readout, thermal proteome profiling11, to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes12 that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.

Species-specific activity of antibacterial drug combinations.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine1,2. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

Thermal proteome profiling in bacteria: probing protein state in vivo.

Increasing antibiotic resistance urges for new technologies for studying microbes and antimicrobial mechanism of action. We adapted thermal proteome profiling (TPP) to probe the thermostability of Escherichia coli proteins in vivoE. coli had a more thermostable proteome than human cells, with protein thermostability depending on subcellular location-forming a high-to-low gradient from the cell surface to the cytoplasm. While subunits of protein complexes residing in one compartment melted similarly, protein complexes spanning compartments often had their subunits melting in a location-wise manner. Monitoring the E. coli meltome and proteome at different growth phases captured changes in metabolism. Cells lacking TolC, a component of multiple efflux pumps, exhibited major physiological changes, including differential thermostability and levels of its interaction partners, signaling cascades, and periplasmic quality control. Finally, we combined in vitro and in vivo TPP to identify targets of known antimicrobial drugs and to map their downstream effects. In conclusion, we demonstrate that TPP can be used in bacteria to probe protein complex architecture, metabolic pathways, and intracellular drug target engagement.

Origin, diversification and substrate specificity in the family of NCS1/FUR transporters.

NCS1 proteins are H(+)/Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub-families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.

TAC-TIC, a high-throughput genetics method to identify triggers or blockers of bacterial toxin-antitoxin systems.

Toxin-antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation-inhibition conjugation (TAC-TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs4. TAC-TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC-TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC-TIC gene-donor libraries from phages, enabling the use of TAC-TIC for identifying TA triggers and antidefense mechanisms in phage genomes.

Transcriptional and Post-Transcriptional Polar Effects in Bacterial Gene Deletion Libraries.

Single-gene deletions can affect the expression levels of other genes in the same operon in bacterial genomes. Here, we used proteomics for 133 Escherichia coli gene deletion mutants and transcriptome sequencing (RNA-seq) data from 71 mutants to probe the extent of transcriptional and post-transcriptional effects of gene deletions in operons. Transcriptional effects were common on genes located downstream of the deletion and were consistent across all operon members, with nearly 40% of operons showing greater than 2-fold up- or downregulation. Surprisingly, we observed an additional post-transcriptional effect that leads to the downregulation of the gene located directly downstream of the targeted gene. This effect was correlated with their intergenic distance, despite the ribosome binding site of the gene downstream remaining intact during library construction. Overall, the data presented can guide future library construction and highlight the importance of follow-up experiments for assessing direct effects of single-gene deletions in operons. IMPORTANCE Single-gene deletion libraries have allowed genome-wide characterization of gene function and interactions. While each mutant intends to disrupt the function of a single gene, it can unintentionally target other genes, such as those located in the same operon as the deletion. The extent to which such polar effects occur in deletion libraries has not been assessed. In this work, we use proteomics and transcriptomics data to show that transcript level changes lead to nearly 40% of deletions in operons affecting the protein levels of genes located downstream by at least 2-fold. Furthermore, we observed a post-transcriptional effect on the gene located directly downstream of the deletion. These results can guide the design of future gene deletion libraries and emphasizes the importance of follow-up work when linking genotypes to phenotypes.

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.