In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters ...(BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:
Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAF
-driven cancers, effective targeted ...therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRAS
). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.
Systemic therapies for metastatic biliary tract cancers are few, and patients have a median overall survival of less than 1 year. MyPathway evaluates the activity of US Food and Drug ...Administration-approved therapies in non-indicated tumours with potentially actionable molecular alterations. In this study, we present an analysis of patients with metastatic biliary tract cancers with HER2 amplification, overexpression, or both treated with a dual anti-HER2 regimen, pertuzumab plus trastuzumab, from MyPathway.
MyPathway is a non-randomised, multicentre, open-label, phase 2a, multiple basket study. Patients aged 18 years and older with previously treated metastatic biliary tract cancers with HER2 amplification, HER2 overexpression, or both and an Eastern Cooperative Oncology Group performance status of 0–2 were enrolled from 23 study sites in the USA and received intravenous pertuzumab (840 mg loading dose, then 420 mg every 3 weeks) plus trastuzumab (8 mg/kg loading dose, then 6 mg/kg every 3 weeks). The primary endpoint was investigator-assessed objective response rate according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The primary outcome and adverse events were analysed in all patients who received at least one dose of pertuzumab and trastuzumab. This trial is registered with ClinicalTrials.gov, NCT02091141, and is ongoing.
39 patients enrolled in the MyPathway HER2 biliary tract cancer cohort between Oct 28, 2014, and May 29, 2019, were evaluable for anti-tumour activity by the March 10, 2020, data cutoff date. Median follow-up was 8·1 months (IQR 2·7–15·7). Nine of 39 patients achieved a partial response (objective response rate 23% 95% CI 11–39). Grade 3–4 treatment-emergent adverse events were reported in 18 (46%) of 39 patients, most commonly increased alanine aminotransferase and increased aspartate aminotransferase (each five 13% of 39). Treatment-related grade 3 adverse events were reported in three (8%) of 39 patients, including increased alanine aminotransferase, aspartate aminotransferase, blood alkaline phosphatase, and blood bilirubin. Serious treatment-emergent adverse events were observed in ten (26%) of 39 patients, of which only abdominal pain occurred in more than one patient (two 5% of 39). There were no treatment-related serious adverse events, treatment-related grade 4 events, or deaths.
Treatment was well tolerated in patients with previously treated HER2-positive metastatic biliary tract cancer. The response rate is promising for the initiation of randomised, controlled trials of pertuzumab plus trastuzumab in this patient population.
F Hoffmann-La Roche–Genentech.
Plants benefit from associations with a diverse community of root-colonizing microbes. Deciphering the mechanisms underpinning these beneficial services are of interest for improving plant ...productivity. We report a plant-beneficial interaction between Arabidopsis thaliana and the root microbiota under iron deprivation that is dependent on the secretion of plant-derived coumarins. Disrupting this pathway alters the microbiota and impairs plant growth in iron-limiting soil. Furthermore, the microbiota improves iron-limiting plant performance via a mechanism dependent on plant iron import and secretion of the coumarin fraxetin. This beneficial trait is strain specific yet functionally redundant across phylogenetic lineages of the microbiota. Transcriptomic and elemental analyses revealed that this interaction between commensals and coumarins promotes growth by relieving iron starvation. These results show that coumarins improve plant performance by eliciting microbe-assisted iron nutrition. We propose that the bacterial root microbiota, stimulated by secreted coumarins, is an integral mediator of plant adaptation to iron-limiting soils.
Display omitted
•Coumarins alter the root microbiota and improve plant growth in iron-limiting soil•The microbiota improves plant iron nutrition via a coumarin-dependent mechanism•The iron-beneficial commensal trait is taxonomically widespread but strain specific•Coumarin-microbiota interaction resolves iron starvation and regulates immune response
Iron-limiting soils are widespread, causing significant losses in plant growth and productivity. Harbort et al. show that under iron limitation, plant-secreted coumarin compounds are mediators of a beneficial plant-microbiota interaction. These specialized metabolites alter root microbiota composition and are required for microbiota-mediated plant iron uptake and immune regulation.
Despite significant advances in antimalarial chemotherapy over the past 30 years, development of resistance to frontline drugs remains a significant challenge that limits efforts to eradicate the ...disease. We now report the discovery of a new class of antimalarials, salinipostins A–K, with low nanomolar potencies and high selectivity indices against mammalian cells (salinipostin A: Plasmodium falciparum EC50 50 nM, HEK293T cytotoxicity EC50 > 50 μM). These compounds were isolated from a marine-derived Salinospora sp. bacterium and contain a bicyclic phosphotriester core structure, which is a rare motif among natural products. This scaffold differs significantly from the structures of known antimalarial compounds and represents a new lead structure for the development of therapeutic targets in malaria. Examination of the growth stage specificity of salinipostin A indicates that it exhibits growth stage-specific effects that differ from compounds that inhibit heme polymerization, while resistance selection experiments were unable to identify parasite populations that exhibited significant resistance against this compound class.
Abstract The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using ...preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway–dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. Significance: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway–driven cancers.
The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct ...stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.
Display omitted
•Several BAM complexes sit together in an assembly precinct•Assembly precincts are distributed right across the bacterial cell surface•The BamB and BamA subunits mediate BAM-BAM interactions•Assembly precincts potentiate porin trimerization by neigboring BAM complexes
Bacteria grow and divide by assembling new material into their surface membranes. Gunasinghe et al. used super-resolution microscopy and in situ crosslinking in live bacterial cells in order to visualize intimate contacts between BAM complexes suggesting a model whereby bacteria use highly organized precincts to drive membrane protein assembly.
Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. ...S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.
Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe-microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 ...binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic
R401. Microbiota reconstitution with a core of
root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.
Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial ...communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.
PDF
