Marcos Pires

In vivo detection of natural killer (NK) cell activity against tumours was achieved with an activatable chemiluminescent probe containing a granzyme B‐reactive peptide substrate linked to a phenoxydioxetane scaffold through a p‐aminobenzyl alcohol linker. The rapid and specific chemiluminescence response of the probe was used to detect NK cell activity against breast cancer cells in living mice.

Abstract

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti‐tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B‐mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B‐specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal‐to‐noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

Nature Communications, Published online: 28 January 2021; doi:10.1038/s41467-020-20756-2

In naturally occurring microbial systems, there is a positive relationship between species diversity and productivity of the community. Here the authors perform model selection to find potential amensal interactions that yield robust stable synthetic microbial consortia.

Nature Immunology, Published online: 25 January 2021; doi:10.1038/s41590-020-00856-3

The microbiome can affect susceptibility to developing asthma. Marsland and colleagues show that changes in the microbial population lead to enrichment of an l-tyrosine metabolite, p-cresol sulfate, which can protect mice against allergic inflammation.

by Dani A. C. Heesterbeek, Remy M. Muts, Vincent P. van Hensbergen, Pieter de Saint Aulaire, Tom Wennekes, Bart W. Bardoel, Nina M. van Sorge, Suzan H. M. Rooijakkers

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

ABSTRACT

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS^– colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS^– A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS^– A. baumannii. PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS^– A. baumannii. PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS^– A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS^– A. baumannii.

IMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS^– Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic β-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS^– A. baumannii.

Nature Chemical Biology, Published online: 04 January 2021; doi:10.1038/s41589-020-00703-4

Crystal structures of FEM1C in apo and in complex with a C-degron ending with arginine reveal a binding pocket in FEM1C that recognizes C-degrons and the essential role of C-terminal arginine for recognition.

Nature Chemical Biology, Published online: 04 January 2021; doi:10.1038/s41589-020-00702-5

Single-molecule FRET of mGluR2 shows that the conformations of the ligand-binding domain and the linked cysteine-rich domain are loosely coupled during ligand-induced activation and defines two pre-active states linking inactive and active states.

Accurate assembly of newly synthesized proteins into functional oligomers is crucial for cell activity. In this study, we investigated whether direct interaction of two nascent proteins, emerging from nearby ribosomes (co-co assembly), constitutes a general mechanism for oligomer formation. We used proteome-wide screening to detect nascent chain–connected ribosome pairs and identified hundreds of homomer subunits that co-co assemble in human cells. Interactions are mediated by five major domain classes, among which N-terminal coiled coils are the most prevalent. We were able to reconstitute co-co assembly of nuclear lamin in Escherichia coli, demonstrating that dimer formation is independent of dedicated assembly machineries. Co-co assembly may thus represent an efficient way to limit protein aggregation risks posed by diffusion-driven assembly routes and ensure isoform-specific homomer formation.

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.

IMPORTANCE S. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.

Nature, Published online: 23 December 2020; doi:10.1038/s41586-020-03074-x

A class of compounds with a dual mechanism of action—direct targeting of IspH and stimulation of cytotoxic γδ T cells to enhance pathogen clearance—are active against multidrug-resistant bacteria.

Nature Microbiology, Published online: 21 December 2020; doi:10.1038/s41564-020-00805-8

A non-invasive Streptococcus pneumoniae strain induces a unique NF-κB signature response in epithelial cells that requires the histone demethylase KDM6B. Modulation of KDM6B can interchange the host response to non-invasive and invasive pneumococcal strains, demonstrating the biological role of KDM6B in cellular responses during infection.

An important microbial modification of bile salts is hydrolysis of a conjugated amino acid moiety. We have created synthetic substrates that yield a fluorescent product upon hydrolysis and, with this assay, characterized in vivo bile salt hydrolase activity on all primary and some of the most relevant secondary bile salts that are commonly associated with various human pathologies.

Abstract

Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities. The category and magnitude of bile salt metabolism can have positive or negative effects on the host. A key modification is bile salt hydrolysis, which is a prerequisite for all additional microbial transformations. We have synthesized five different fluorogenic bile salts for simple and continuous reporting of hydrolysis in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host‐produced primary bile salts, but some microbially modified secondary bile salts also display significant turnover.

By integrating a fluorescent D‐amino acid labeling strategy for gut microbiota and hydrophilic tissue‐clearing, we achieved quantitative 3D imaging of the indigenous mouse gut microbiota in their native context with high spatial resolution and imaging depth. Bacteria were unexpectedly found in the small intestine crypts, a region that was previously considered to be bacteria‐free.

Abstract

Owing to the challenges to acquire detailed spatial information of gut bacteria in situ, three‐dimensional (3D) microbiota distributions in the gut remain largely uncharted. Here, we propose a tissue clearing‐based and D‐amino acid labeling‐facilitated (TiDaL) strategy that combines a novel microbiota in vivo labeling protocol, CUBIC‐based tissue clearing and whole‐mount tissue imaging, to achieve 3D imaging of indigenous gut microbiota. We demonstrate high‐resolution 3D acquisition of their biogeography in different gut sections, and present quantitative spatial details in relation to the host epithelium. We unexpectedly observe microbiota in the small intestine crypts, which were thought to be bacteria‐free. Significant bacterial overgrowth in the first two‐thirds of the small intestine is detected in an enteritis model. We expect that this quantitative 3D imaging strategy for native gut microbiota will provide insightful information into the host–microbiota interactions.

Nature Chemistry, Published online: 07 December 2020; doi:10.1038/s41557-020-00584-z

A method for the covalent labelling of proteins by installing a biostable peptide nucleic acid (PNA) tag has now been developed. The PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization and fluorophore removal by toehold-mediated strand displacement. Imaging of cell surface receptors, including internalized receptors, has been demonstrated using this approach.

Nature Chemical Biology, Published online: 07 December 2020; doi:10.1038/s41589-020-00696-0

Using synthetic ubiquitins with non-natural acceptor site, the authors revealed that the length of lysine side chain in acceptor ubiquitins affects ubiquitin chain linkage specificity with native lysine as the preferred geometry.

Nature Communications, Published online: 07 December 2020; doi:10.1038/s41467-020-19970-9

Most chemotherapeutic agents, including gemcitabine, do not elicit immunogenic cell death, a phenomenon associated with the release of damage-associated molecule patterns (DAMPs). Here, the authors show that gemcitabine-treated dying cancer cells express hallmark DAMPs but their immunogenic properties are hindered by the concomitant release of the inhibitory DAMP PGE2.

Isoform‐specific targeting: The synthesis and comparison of selective, bioorthogonal, activity‐based probes for penicillin‐binding proteins (PBPs) is reported. We demonstrate the expanded functional utility of bioorthogonal probes compared to fluorescent analogues and explore design considerations for the development of bioorthogonal probes that are applicable beyond the probes described in this work.

Abstract

Penicillin‐binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell‐wall biosynthesis and the target of β‐lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal β‐lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity‐based probes for PBPs will facilitate the activity‐based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Hydroxamic acids immobilized on resins (HAIRs) were developed and utilized for the library synthesis of DNA‐alkylating HDAC inhibitors and a proof‐of‐concept HDAC degrader (PROTAC). A hybrid compound based on the pharmacophores of chlorambucil and panobinostat was identified as the most promising chimeric HDAC inhibitor and demonstrated improved anticancer properties compared to the sum of the activities of either pharmacophore alone.

Abstract

Inhibition of more than one cancer‐related pathway by multi‐target agents is an emerging approach in modern anticancer drug discovery. Here, based on the well‐established synergy between histone deacetylase inhibitors (HDACi) and alkylating agents, we present the discovery of a series of alkylating HDACi using a pharmacophore‐linking strategy. For the parallel synthesis of the target compounds, we developed an efficient solid‐phase‐supported protocol using hydroxamic acids immobilized on resins (HAIRs) as stable and versatile building blocks for the preparation of functionalized HDACi. The most promising compound, 3 n, was significantly more active in apoptosis induction, activation of caspase 3/7, and formation of DNA damage (γ‐H2AX) than the sum of the activities of either active principle alone. Furthermore, to demonstrate the utility of our preloaded resins, the HAIR approach was successfully extended to the synthesis of a proof‐of‐concept proteolysis‐targeting chimera (PROTAC), which efficiently degrades histone deacetylases.

Peptide to oligourea replacements have been employed to generate high‐affinity foldamer ligands targeted to specific proteins. X‐ray structure analysis of several peptide–oligourea hybrids bound to their respective protein targets confirms the high degree of α‐helix mimicry that can be achieved with oligoureas and reveals general principles enabling the design of more stable peptide‐based inhibitors of protein–protein interactions.

Abstract

Efficient optimization of a peptide lead into a drug candidate frequently needs further transformation to augment properties such as bioavailability. Among the different options, foldamers, which are sequence‐based oligomers with precise folded conformation, have emerged as a promising technology. We introduce oligourea foldamers to reduce the peptide character of inhibitors of protein–protein interactions (PPI). However, the precise design of such mimics is currently limited by the lack of structural information on how these foldamers adapt to protein surfaces. We report a collection of X‐ray structures of peptide–oligourea hybrids in complex with ubiquitin ligase MDM2 and vitamin D receptor and show how such hybrid oligomers can be designed to bind with high affinity to protein targets. This work should enable the generation of more effective foldamer‐based disruptors of PPIs in the context of peptide lead optimization.

Nature, Published online: 25 November 2020; doi:10.1038/s41586-020-2971-8

Influence of the gut microbiome on the human immune system is revealed by systems analysis of vast clinical data from decades of electronic health records paired with massive longitudinal microbiome sequencing.