A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Pervasive Protein Thermal Stability Variation during the Cell Cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

Detecting envelope stress by monitoring ß-barrel assembly.

The cell envelope protects bacteria from their surroundings. Defects in its integrity or assembly are sensed by signal transduction systems, allowing cells to rapidly adjust. The Rcs phosphorelay responds to outer membrane (OM)- and peptidoglycan-related stress in enterobacteria. We elucidated how the OM lipoprotein RcsF, the upstream Rcs component, senses envelope stress and activates the signaling cascade. RcsF interacts with BamA, the major component of the ß-barrel assembly machinery. In growing cells, BamA continuously funnels RcsF through the ß-barrel OmpA, displaying RcsF on the cell surface. This process spatially separates RcsF from the downstream Rcs component, which we show is the inner membrane protein IgaA. The Rcs system is activated when BamA fails to bind RcsF and funnel it to OmpA. Newly synthesized RcsF then remains periplasmic, interacting with IgaA to activate the cascade. Thus RcsF senses envelope damage by monitoring the activity of the Bam machinery.

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.

Unravelling the collateral damage of antibiotics on gut bacteria.

Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease1. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species2. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for ß-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal3-5 and human6,7 gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella.

Pathogenic bacteria proliferating inside mammalian host cells need to rapidly adapt to the intracellular environment. How they achieve this and scavenge essential nutrients from the host has been an open question due to the difficulties in distinguishing between bacterial and host metabolites in situ. Here, we capitalized on the inability of mammalian cells to metabolize mannitol to develop a stable isotopic labeling approach to track Salmonella enterica metabolites during intracellular proliferation in host macrophage and epithelial cells. By measuring label incorporation into Salmonella metabolites with liquid chromatography-mass spectrometry (LC-MS), and combining it with metabolic modeling, we identify relevant carbon sources used by Salmonella, uncover routes of their metabolization, and quantify relative reaction rates in central carbon metabolism. Our results underline the importance of the Entner-Doudoroff pathway (EDP) and the phosphoenolpyruvate carboxylase for intracellularly proliferating Salmonella. More broadly, our metabolic labeling strategy opens novel avenues for understanding the metabolism of pathogens inside host cells.

Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, ß-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.

Phenotypic landscape of a bacterial cell.

The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.

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.

Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of peptidoglycan synthesis by outer-membrane proteins.

Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases.

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.

Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli.

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.

Unravelling metabolic cross-feeding in a yeast-bacteria community using 13 C-based proteomics.

Cross-feeding is fundamental to the diversity and function of microbial communities. However, identification of cross-fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13 C isotope tracing in peptides to elucidate cross-fed metabolites in co-cultures of Saccharomyces cerevisiae and Lactococcus lactis. The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13 C. Data analysis allowing for the possible mass-shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross-fed metabolites was further supported by metabolite labelling in the co-culture supernatant, and by diminished fitness of a galactose-negative yeast mutant in the community. Together, our results demonstrate the utility of 13 C-based proteomics for uncovering microbial interactions.

Consistency across multi-omics layers in a drug-perturbed gut microbial community.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities.

Emerging and evolving concepts in gene essentiality.

Gene essentiality is a founding concept of genetics with important implications in both fundamental and applied research. Multiple screens have been performed over the years in bacteria, yeasts, animals and more recently in human cells to identify essential genes. A mounting body of evidence suggests that gene essentiality, rather than being a static and binary property, is both context dependent and evolvable in all kingdoms of life. This concept of a non-absolute nature of gene essentiality changes our fundamental understanding of essential biological processes and could directly affect future treatment strategies for cancer and infectious diseases.

Extensive impact of non-antibiotic drugs on human gut bacteria.

A few commonly used non-antibiotic drugs have recently been associated with changes in gut microbiome composition, but the extent of this phenomenon is unknown. Here, we screened more than 1,000 marketed drugs against 40 representative gut bacterial strains, and found that 24% of the drugs with human targets, including members of all therapeutic classes, inhibited the growth of at least one strain in vitro. Particular classes, such as the chemically diverse antipsychotics, were overrepresented in this group. The effects of human-targeted drugs on gut bacteria are reflected on their antibiotic-like side effects in humans and are concordant with existing human cohort studies. Susceptibility to antibiotics and human-targeted drugs correlates across bacterial species, suggesting common resistance mechanisms, which we verified for some drugs. The potential risk of non-antibiotics promoting antibiotic resistance warrants further exploration. Our results provide a resource for future research on drug-microbiome interactions, opening new paths for side effect control and drug repurposing, and broadening our view of antibiotic resistance.