Marcos Pires

Antimicrobial peptides (AMPs) are mucosal defense effectors of the human innate immune response. In the intestine, AMPs are produced and secreted by epithelial cells to protect the host against pathogens and to support homeostasis with commensals. The inducible nature of AMPs suggests that potent inducers could be used to increase...

RIP2 filament formation is required for NOD2 dependent NF-κB signalling

RIP2 filament formation is required for NOD2 dependent NF-κB signalling, Published online: 02 October 2018; doi:10.1038/s41467-018-06451-3

Binding of bacterial peptidoglycan muramyl dipeptides induces NOD2 activation and signalling via the downstream adaptor kinase RIP2. Here the authors show that RIP2 forms filaments via its CARD domain, analyse the structure of the CARD filaments and demonstrate the requirement of RIP2 polymerisation for the activation of NF-κB by NOD2.

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3β1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients.

High-resolution NMR studies of antibiotics in cellular membranes

High-resolution NMR studies of antibiotics in cellular membranes, Published online: 27 September 2018; doi:10.1038/s41467-018-06314-x

Antibiotics that target the peptidoglycan precursor lipid II are promising templates for next-generation antibiotics. Here authors use solid-state NMR and monitor lipid II-binding antibiotics, such as nisin, directly in cell membranes.

Angewandte Chemie International Edition, EarlyView.

The bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion of bldD or whiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG in Streptomyces.

IMPORTANCE Streptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin, Published online: 21 September 2018; doi:10.1038/s41467-018-06298-8

The bacterial type III secretion system of Gram-negative bacteria uses its core, the needle complex, to penetrate through the infected host cell membrane. Here authors show a near-atomic resolution structure of a needle complex which sheds light on the assembly and function of this nanomachine.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate...

Unnatural presentation: Semisynthetic cyclic peptides containing both non‐proteinogenic building blocks and a genetically encoded sequence amenable to DNA‐based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Our approach allows the generation of cyclic peptide libraries on E. coli cells suitable for selection by fluorescence‐activated cells sorting.

Abstract

Semisynthetic cyclic peptides containing both non‐proteinogenic building blocks, as the synthetic part, and a genetically encoded sequence amenable to DNA‐based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Key to an efficient selection of novel binders to biomacromolecules is a robust method to link their genotype and phenotype. A novel bacterial cell surface display technology has been developed to present cyclic peptides composed of synthetic and genetically encoded fragments in their backbones. The fragments were combined by protein trans‐splicing and intramolecular oxime ligation. To this end, a split intein half and an unnatural amino acid were displayed with the genetically encoded part on the surface of Escherichia coli. Addition of the synthetic fragment equipped with the split intein partner and an aminooxy moiety, as well as the application of a pH‐shift protocol, resulted in the onsurface formation of the semisynthetic cyclic peptide. This approach will serve for the generation of cyclic peptide libraries suitable for selection by fluorescence‐activated cell sorting, and more generally enables chemical modification of proteins on the bacterial surface.

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is...

ABSTRACT

The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as Pseudomonas aeruginosa that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated P. aeruginosa individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for P. aeruginosa. d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes ponA and dacC. Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from P. aeruginosa cells revealed that dadA deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.

IMPORTANCE The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen Pseudomonas aeruginosa. This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of P. aeruginosa cells. We demonstrate that d-Ala—a component of the peptidoglycan—is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential P. aeruginosa targets that may enhance antibacterial chemotherapies or lead to new approaches.

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of Escherichia coli

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of <i>Escherichia coli</i>, Published online: 10 September 2018; doi:10.1038/s41467-018-06143-y

Resistance to one antibiotic can in some cases increase susceptibility to other antibiotics. Here, Podnecky et al. study these collateral responses in E. coli clinical isolates and show that efflux-related resistance mechanisms and relative fitness of the strains are principal contributors to this phenomenon.

Angewandte Chemie International Edition, EarlyView.

Angewandte Chemie International Edition, EarlyView.

TB drug susceptibility is more than MIC

TB drug susceptibility is more than MIC, Published online: 24 August 2018; doi:10.1038/s41564-018-0226-3

Genome-wide association studies and genetic analyses have identified a clinically prevalent alteration in the Mycobacterium tuberculosis genome that rewires bacterial propionate metabolism, conditionally reduces antibiotic killing without affecting minimum inhibitory concentrations, and may drive emergence of drug resistance.

Enterobactin, a siderophore produced by the commensal bacterium E. coli, is a key contributor to iron uptake and homeostasis of host cells via its interaction with host ATP synthase.

CD47 and PD-L1 serve as critical innate and adaptive checkpoints, respectively. Liu et al. show CD47 and PD-L1 coordinate in tumor cells to evade immune response. Furthermore, a bispecific antibody design enables better targeting on tumor cells, but less on non-tumor cells, and enhanced therapeutic efficacy.

Angewandte Chemie International Edition, EarlyView.

A conserved cell-penetrating peptide (CPP) encoded by the HPV genome enables viral protein passage across the endosomal membrane into the cytoplasm and drives non-enveloped viral entry during infection.

Biomaterial-assisted targeted modulation of immune cells in cancer treatment

Biomaterial-assisted targeted modulation of immune cells in cancer treatment, Published online: 13 August 2018; doi:10.1038/s41563-018-0147-9

Immunotherapies have shown significant promise in cancer treatment. This Review discusses how a range of materials have been employed to enhance the effectiveness of these therapies by mediating their delivery and immunomodulatory activity.

Neurotransmitter-containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with SVs that are likely to help form such clusters have been reported, including synapsin. Here we found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could coassemble into this condensate but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II, mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses.