Emily P. Balskus

Bacteria engage in microbial warfare by secreting toxic molecules. These toxins can serve as therapeutics or drug discovery starting points, motivating investigations of their production. Bacteria that produce these toxins must protect themselves, and a common strategy is to synthesize an inactive prodrug that is activated upon secretion. Some prodrugs are capped with N -acyl-ᴅ-asparagine motifs, which are removed by two types of prodrug peptidases. Type I peptidases are membrane-bound S12 hydrolases; type II peptidases have an additional C-terminal half-ABC transporter module. Recent structures of a type I peptidase revealed how its transmembrane domain completes the substrate-binding site, but little is known about type II peptidases. Here, we show that the B. cereus type II peptidase ZmaM is responsible for both the export of prezwittermicin and its activation into zwittermicin. The structure of homodimeric ZmaM reveals a central type IV ABC transporter flanked by peptidase modules. The periplasmic hydrolase domains interact over the transporter’s open outer vestibule. Mutations to intermodule interfaces impact both peptidase and ATP hydrolysis activities, indicating the peptidase and transporter modules are interdependent. Together, our findings reveal an evolutionary strategy to coordinate toxin activation with its export and inform future efforts to harness these systems in biotechnology.

Background: The human aryl hydrocarbon receptor (AHR) integrates chemical signals derived from the environment, gut microbes, and endogenous sources to regulate processes ranging from intestinal barrier integrity to xenobiotic detoxification. Despite strong evidence that dysregulation of AHR signaling is a causal factor in metabolic and autoimmune disorders, we currently lack a comprehensive understanding of the factors that regulate AHR activity in human cells. Results: Here, we use genome-scale CRISPR screening to systematically identify regulators of AHR signaling in a model of human hepatocytes. The resulting datasets recapitulate the core AHR signaling pathway and identify a large network of regulators. Many of these factors have roles beyond AHR signaling, reflecting that AHR signaling is deeply integrated into human cell biology. We further dissect this network to reveal novel modes of regulation of AHR expression, protein levels, and signaling. For example, we find that the E3 ubiquitin ligase UBR5 sustains AHR signaling by counteracting degradation of ligand-bound AHR. Finally, we identify components of the AHR regulatory network that are specific to cell types and ligands as potential nodes to manipulate AHR signaling in a targeted manner for therapeutic benefit. Conclusions: Overall, our results define the regulatory network that underpins AHR activation, with implications for our understanding of host-microbe interactions and integrative chemosensation and the etiology of metabolic and inflammatory disorders.

Glycyl radical enzymes (GREs) catalyze challenging chemical reactions using a posttranslationally installed glycyl radical cofactor. One such enzyme, indoleacetate decarboxylase (IAD), performs the radical-based decarboxylation of indole-3-acetate (I3A) to form the malodorant molecule skatole. In addition to being an odor nuisance, skatole is a human and livestock lung toxin, a suspected carcinogen, and a mosquito attractant, all of which impact human health, agriculture, food production, and wastewater treatment. Here, we use cryogenic electron microscopy to solve a 2.45-Å resolution structure of IAD from the gut bacterium Olsenella uli . We observe IAD in a homotetrameric form with the substrate I3A bound in all four protomers. The positioning of I3A in the active site is unexpected and is more consistent with a Kolbe-type decarboxylation mechanism, i.e., a decarboxylation initiated by a 1-electron oxidation of the carboxylate moiety rather than being initiated by hydrogen atom transfer (HAT). Previously, a high deuterium content in skatole from IAD assays in D ₂ O was used to support a HAT mechanism over a Kolbe-type mechanism. However, we show here that deuterium content does not necessarily inform on mechanism as IAD can catalyze the exchange of skatole’s 3′-methyl hydrogens postturnover. Structural comparisons show that both IAD and hydroxyphenylacetate decarboxylase display structural features that are not found in other characterized GREs, suggesting that they represent a distinct GRE-subclass. Collectively, these insights will inform IAD inhibitor design aimed at decreasing skatole production.

Colibactin is a bacterial genotoxin that damages DNA and is linked to colorectal cancer. It is implicated in interbacterial competition but its role in natural microbiomes is unclear. Here we show that Frischella perrara, a colibactin-producing symbiont restricted to honeybee guts, lowers bee lifespan but increases survivorship following challenge with the opportunistic bee pathogen Serratia marcescens. F. perrara mutants lacking colibactin production fail to improve survivorship following challenge. The symbiont reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. Colibactin is toxic to the pathogen under anoxic conditions, reflecting the bee gut. clbS, a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but absent from opportunistic colonizers, including pathogens. Indeed, F. perrara colonization representing colibactin pressure had limited impact on the bee gut microbiome where strains encode clbS. Our findings suggest that colibactin functions in interbacterial competition and protects against pathogens while permitting co-evolved symbionts to persist.

Cystic fibrosis (CF) alters the gastrointestinal microbiome from an early age, leading to significant changes in microbial composition and functionality. This study explores the physiological and microbiological factors contributing to dysbiosis in children with cystic fibrosis (cwCF), characterized by an increase in potentially pathogenic Escherichia coli and a decrease in beneficial anaerobes, such as Bacteroides . We employed an in vitro medium representative of the nutritional environment of the CF colon to test the role of factors, including mucin, fat, bile, pH, antibiotics, and features associated with inflammation (e.g., nitrate, sulfate, formate, and reactive oxygen species), on the growth of clinical isolates of E. coli and Bacteroides spp., with a focus on Bacteroides vulgatus . We further examined interactions between these two microbes under CF-like conditions and identified glycerol, a surrogate of increased fat, as a significant driver of altered microbial competition. Finally, we investigated genetic determinants influencing these microbial interactions by performing a transposon mutagenesis screen in E. coli . The results pointed to the role of colibactin, a DNA-damaging genotoxin associated with an increased risk of colorectal cancer (CRC), in mediating this microbial competition. This work enhances our understanding of the mechanisms of microbial competition in the CF gut and potential CRC risk in persons with CF through the identification of early-life microbial biomarkers. IMPORTANCE: The risk of CRC development in CF populations is significantly increased. This in vitro study examines the interplay of altered intestinal physiology in the microbial dysbiosis common in the CF gut, implicating the high-fat/glycerol environment in a competition-mediated depletion of immune-modulating Bacteroides vulgatus . This work identifies candidate features of the young CF intestine and gut microbiome that may contribute to dysbiosis, development of inflammation, and CRC in these populations, informing potential prognostic and therapeutic approaches.

Glycyl radical enzymes (GREs) catalyze challenging chemical reactions using a post-translationally installed glycyl radical cofactor. One such enzyme, indoleacetate decarboxylase (IAD), performs the radical-based decarboxylation of indole-3-acetate (I3A) to form the malodorant molecule skatole. In addition to being an odor nuisance, skatole is a human and livestock lung toxin, a suspected carcinogen, and a mosquito attractant, all of which impact human health, agriculture, food production, and wastewater treatment. Here, we use cryogenic electron microscopy to solve a 2.45-Å resolution structure of indoleacetate decarboxylase from the gut bacterium Olsenella uli . We observe IAD in a homotetrameric form with the substrate I3A bound in all four protomers. The positioning of the I3A in the active site is unexpected and is more consistent with a Kolbe-type decarboxylation mechanism, i.e. a decarboxylation initiated by a 1-electron oxidation of the carboxylate moiety rather than being initiated by hydrogen atom transfer (HAT). Previously, a high deuterium content in skatole from IAD assays in D ₂ O was used to support a HAT mechanism over a Kolbe-type mechanism. However, we show here that deuterium content does not necessarily inform on mechanism as IAD can catalyze the exchange of skatole’s 3′-methyl hydrogens post-turnover. Structural comparisons show that both IAD and HPAD display structural features that are not found in other characterized GREs, suggesting that they represent a distinct GRE-subclass. Collectively, these insights will inform IAD inhibitor design aimed at decreasing skatole production. Significance Statement: Glycyl radical enzymes (GREs) are a superfamily of enzymes that catalyze challenging chemical reactions in anaerobic environments. One such enzyme, indoleacetate decarboxylase (IAD) performs a C–C bond cleavage and decarboxylation reaction on the tryptophan metabolite indole-3-acetate (I3A). Decarboxylation of I3A by IAD forms the malodorant molecule skatole which negatively impacts human health, livestock health, odor emissions, the environment, and food production. Here, we present the first structure of IAD with I3A bound, revealing the substrate binding mode and active site architecture. This work provides new insight into the catalytic mechanism of the enzyme and illuminates new avenues for control of the odor nuisance skatole.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinson’s disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, α-fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinson’s phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

The pks (or clb) gene cluster, which produces the genotoxic natural product colibactin, is encoded by human gut Enterobacteriaceae, including many commensal strains of E. coli. Colibactin cross-links DNA and is implicated in colorectal cancer development, highlighting the importance of identifying colibactin-producing gut bacteria within biological samples. In this study, we develop phenoxy-dioxetane chemiluminescent probes that selectively react with a critical colibactin biosynthetic enzyme, the serine peptidase ClbP. We show that these chemiluminescent probes have superior sensitivity, speed, and detection capabilities compared with previously reported fluorescent ClbP probes. Furthermore, we employ these chemiluminescent probes to detect pks⁺ E. coli directly in complex stool suspensions. These probes will enable multiple applications requiring the detection of colibactin-producing bacteria, including the identification of ClbP inhibitors and the screening of clinical samples.

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine- or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions. All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine. This knowledge has guided predictions and study of BSH activity in the gut microbiome as well as the development of BSH inhibitors. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.