Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

A Well-Mixed E. coli Genome: Widespread Contacts Revealed by Tracking Mu Transposition.

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.

Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

Pathogenic E. coli Extracts Nutrients from Infected Host Cells Utilizing Injectisome Components.

Microbiota and intestinal epithelium restrict pathogen growth by rapid nutrient consumption. We investigated how pathogens circumvent this obstacle to colonize the host. Utilizing enteropathogenic E. coli (EPEC), we show that host-attached bacteria obtain nutrients from infected host cell in a process we termed host nutrient extraction (HNE). We identified an inner-membrane protein complex, henceforth termed CORE, as necessary and sufficient for HNE. The CORE is a key component of the EPEC injectisome, however, here we show that it supports the formation of an alternative structure, composed of membranous nanotubes, protruding from the EPEC surface to directly contact the host. The injectisome and flagellum are evolutionarily related, both containing conserved COREs. Remarkably, CORE complexes of diverse ancestries, including distant flagellar COREs, could rescue HNE capacity of EPEC lacking its native CORE. Our results support the notion that HNE is a widespread virulence strategy, enabling pathogens to thrive in competitive niches.

Microbial Siderophore Enterobactin Promotes Mitochondrial Iron Uptake and Development of the Host via Interaction with ATP Synthase.

Elucidating the benefits of individual microbiota-derived molecules in host animals is important for understanding the symbiosis between humans and their microbiota. The bacteria-secreted enterobactin (Ent) is an iron scavenging siderophore with presumed negative effects on hosts. However, the high prevalence of Ent-producing commensal bacteria in the human gut raises the intriguing question regarding a potential host mechanism to beneficially use Ent. We discovered an unexpected and striking role of Ent in supporting growth and the labile iron pool in C. elegans. We show that Ent promotes mitochondrial iron uptake and does so, surprisingly, by binding to the ATP synthase α subunit, which acts inside of mitochondria and independently of ATP synthase. We also demonstrated the conservation of this mechanism in mammalian cells. This study reveals a distinct paradigm for the "iron tug of war" between commensal bacteria and their hosts and an important mechanism for mitochondrial iron uptake and homeostasis.

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

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.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.

Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection.

Toxin-antitoxin (TA) systems are widespread in bacteria, but their activation mechanisms and bona fide targets remain largely unknown. Here, we characterize a type III TA system, toxIN, that protects E. coli against multiple bacteriophages, including T4. Using RNA sequencing, we find that the endoribonuclease ToxN is activated following T4 infection and blocks phage development primarily by cleaving viral mRNAs and inhibiting their translation. ToxN activation arises from T4-induced shutoff of host transcription, specifically of toxIN, leading to loss of the intrinsically unstable toxI antitoxin. Transcriptional shutoff is necessary and sufficient for ToxN activation. Notably, toxIN does not strongly protect against another phage, T7, which incompletely blocks host transcription. Thus, our results reveal a critical trade-off in blocking host transcription: it helps phage commandeer host resources but can activate potent defense systems. More generally, our results now reveal the native targets of an RNase toxin and activation mechanism of a phage-defensive TA system.

Widespread Protection of RNA Cleavage Sites by a Riboswitch Aptamer that Folds as a Compact Obstacle to Scanning by RNase E.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency.

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode C. elegans as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

Metabolomics-Driven Exploration of the Chemical Drug Space to Predict Combination Antimicrobial Therapies.

Alternative to the conventional search for single-target, single-compound treatments, combination therapies can open entirely new opportunities to fight antibiotic resistance. However, combinatorial complexity prohibits experimental testing of drug combinations on a large scale, and methods to rationally design combination therapies are lagging behind. Here, we developed a combined experimental-computational approach to predict drug-drug interactions using high-throughput metabolomics. The approach was tested on 1,279 pharmacologically diverse drugs applied to the gram-negative bacterium Escherichia coli. Combining our metabolic profiling of drug response with previously generated metabolic and chemogenomic profiles of 3,807 single-gene deletion strains revealed an unexpectedly large space of inhibited gene functions and enabled rational design of drug combinations. This approach is applicable to other therapeutic areas and can unveil unprecedented insights into drug tolerance, side effects, and repurposing. The compendium of drug-associated metabolome profiles is available at https://zampierigroup.shinyapps.io/EcoPrestMet, providing a valuable resource for the microbiological and pharmacological communities.

Spatiotemporal Organization of the E. coli Transcriptome: Translation Independence and Engagement in Regulation.

RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

Systematic analysis of nonprogrammed frameshift suppression in E. coli via translational tiling proteomics.

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Predictive phage therapy for Escherichia coli urinary tract infections: Cocktail selection for therapy based on machine learning models.

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.