Marcos Pires

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics here, we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer, which moreover allows also characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli , OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only a few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles as a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

Advances in immunotherapy have revolutionized the treatment of multiple cancers. Unfortunately, tumors usually have impaired blood perfusion, which limits the delivery of therapeutics and cytotoxic immune cells to tumors and also results in hypoxia—a hallmark of the abnormal tumor microenvironment (TME)—that causes immunosuppression. We proposed that normalization of TME using...

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved...

Matched isolate sequencing and shotgun metagenomics reconstruct Staphylococcus epidermidis spatiotemporal strain diversity, demonstrating how strain admixture can affect virulence, evolution, and metabolism within the human skin microbiome.

Nature, Published online: 29 January 2020; doi:10.1038/d41586-020-00201-6

Modified bacteria and carefully formulated microbial communities could form the basis of new living treatments.

A tip of the hap: A time‐resolved chemical proteomics strategy enables host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of bacteria into host cell.

Abstract

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time‐resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host‐cell proteins at different time points initiated by UV for label‐free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella‐containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.

Nature Chemical Biology, Published online: 23 January 2020; doi:10.1038/s41589-019-0452-x

The native stress-tolerant phenotypes of various non-conventional microbes have the potential to enable more efficient bioprocessing for chemical production, addressing certain design challenges encountered when using model production hosts.

Human gut microbiota development has been associated with healthy growth but understanding the determinants of community assembly and composition is a formidable challenge. We cultured bacteria from serially collected fecal samples from a healthy infant; 34 sequenced strains containing 103,102 genes were divided into two consortia representing earlier and later...

ABSTRACT

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.

IMPORTANCE Listeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1–based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1–based internalization event can exceed the theoretical size limit for this endocytic pathway.

Burslem and Crews provide a guide to the design, implementation, and therapeutic and basic research applications of PROTACs, small molecules designed to induce protein degradation.

Nature, Published online: 15 January 2020; doi:10.1038/s41586-019-1894-8

Understanding the dynamics between bacteria and bacteriophages could enable the development of phage-based therapies and biotechnological tools and provide insights into the ecology and evolution of these microorganisms.

Nature, Published online: 15 January 2020; doi:10.1038/s41586-019-1915-7

Mycobacterium tuberculosis suppresses the production of inflammatory cytokines by host cells through the host-mediated ubiquitination of a mycobacterial protein, enhancing the interaction of a host signalling inhibitor with another signalling molecule.

How the gut microbiome affects Parkinson’s disease remains unclear. Goya et al. show that the probiotic B. subtilis strain PXN21 inhibits and clears α-synuclein aggregation in a C. elegans model. The bacterium acts via metabolites and biofilm formation to activate protective pathways in the host, including DAF-16/FOXO and sphingolipid metabolism.

Nature Biotechnology, Published online: 13 January 2020; doi:10.1038/s41587-019-0390-x

Cancer vaccines that self-assemble into uniform nanoparticles improve tumor clearance.

Nature Microbiology, Published online: 13 January 2020; doi:10.1038/s41564-019-0632-1

Bacterial cell wall amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so they can separate. An amidase that cleaves uncrosslinked peptidoglycan and its regulator are identified here and shown to regulate cell growth, rather than separation. This enzyme regulates the density of peptidoglycan assembly sites, ensuring coordination between cell expansion and cell division.

Caught in a trap: The antibiotic colistin targets lipopolysaccharide (LPS) in the Gram‐negative bacterial (GNB) membrane. This interaction is disrupted by free LPS released during infection (path a). A methylated gold nanosheet (SAuM) binds to free LPS, preventing free LPS from binding colistin and reducing endotoxemia (path b). This increases the antibacterial efficacy of colistin, decreasing both the required dose and the risk of resistance.

Abstract

A strong interaction between colistin, a last‐resort antibiotic of the polymyxin family, and free lipopolysaccharide (LPS, also referred to as endotoxin), released from the Gram‐negative bacterial (GNB) outer membrane (OM), has been identified that can decrease the antibacterial efficacy of colistin, potentially increasing the dose of this antibiotic required for treatment. The competition between LPS in the GNB OM and free LPS for the interaction with colistin was prevented by using a supramolecular trap to capture free LPS. The supramolecular trap, fabricated from a subnanometer gold nanosheet with methyl motifs (SAuM), blocks lipid A, preventing the interaction between lipid A and colistin. This can minimize endotoxemia and maximize the antibacterial efficacy of colistin, enabling colistin to be used at lower doses. Thus, the potential crisis of colistin resistance could be avoided.

A real‐time in vivo imaging method to fulfill the unmet need for gut microbiota visualization was developed by integrating the strategy of d‐amino acid‐based metabolic labeling and NIR‐II fluorescence imaging. The versatility of this technique was demonstrated in imaging different bacterial species, and in the study of gastrointestinal motility‐induced gut microbiota biogeography changes.

Abstract

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real‐time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near‐infrared (NIR‐II)‐based method for in vivo imaging of gut bacteria. Using d‐propargylglycine in gavage and then click reaction with an azide‐containing NIR‐II dye, gut microbiota of a donor mouse was strongly labeled with NIR‐II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR‐II‐based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut “dark matters”.

The disulfide bond is frequently present in biologically important peptide hormones and toxins, playing pivotal roles in defining their activity, stability and biophysical properties. Chemical synthesis is essential for studying their structure–function relationships. This minireview will focus on the regiospecific, stepwise construction of multiple disulfides, summarizing the historical achievements and recent developments in this field.

Abstract

The disulfide bond plays an important role in biological systems. It defines global conformation, and ultimately the biological activity and stability of the peptide or protein. It is frequently present, singly or multiply, in biologically important peptide hormones and toxins. Numerous disulfide‐containing peptides have been approved by the regulatory agencies as marketed drugs. Chemical synthesis is one of the prerequisite tools needed to gain deep insights into the structure–function relationships of these biomolecules. Along with the development of solid‐phase peptide synthesis, a number of methods of disulfide construction have been established. This minireview will focus on the regiospecific, stepwise construction of multiple disulfides used in the chemical synthesis of peptides. We intend for this article to serve a reference for peptide chemists conducting complex peptide syntheses and also hope to stimulate the future development of disulfide methodologies.

Drug combinations are widely used in clinical practice to prevent the evolution of resistance. However, little is known about the effect of tolerance, a different mode of survival, on the efficacy of drug combinations for preventing the evolution of resistance. In this work, we monitored Staphylococcus aureus strains evolving in patients under treatment. We detected the rapid emergence of tolerance mutations, followed by the emergence of resistance, despite the combination treatment. Evolution experiments on the clinical strains in vitro revealed a new way by which tolerance promotes the evolution of resistance under combination treatments. Further experiments under different antibiotic classes reveal the generality of the effect. We conclude that tolerance is an important factor to consider in designing combination treatments that prevent the evolution of resistance.

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell−cell communication and behavioral changes, as well as attenuate...

Nature, Published online: 08 January 2020; doi:10.1038/s41586-019-1898-4

Neonatal mice are protected against infection with the enteric pathogen enterotoxigenic Escherichia coli by maternally derived natural antibodies as well as by maternal commensal microbiota that induce antibodies that recognize antigens expressed by Enterobacteriaceae.

Upon infection, Vibrio cholerae must alter its surface profile to evade host defenses and adapt to the gastrointestinal environment. Zingl et al. show that increased release of outer membrane vesicles upon host entry allows the bacteria to rapidly modify their cell surface, thus increasing in vivo adaptation and colonization fitness.

Host-directed therapy is a strategy to improve the lengthy treatment of tuberculosis (TB). Puyskens et al. find that the aryl hydrocarbon receptor (AhR) binds to several TB drugs, resulting in altered host defense and drug metabolism. Modulation of the AhR in infected zebrafish embryos leads to improved treatment efficacy.

Cryo-electron tomography allows structure determination of the bacterial S-layer in its cellular context at near-atomic resolution, providing insights into how it’s assembled and organized.

Nature Chemistry, Published online: 16 December 2019; doi:10.1038/s41557-019-0378-7

Screening commercial kinase inhibitors for antibacterial activity identified the anticancer drug sorafenib as a major hit. Subsequent structure–activity optimization created a new antibacterial analogue with high potency against methicillin-resistant Staphylococcus aureus, including challenging persisters and biofilms, as well as demonstrating efficacy in an in vivo mouse model. The mode of action involves stimulation of protein secretion and inhibition of menaquinone biosynthesis.

Nature Microbiology, Published online: 23 December 2019; doi:10.1038/s41564-019-0620-5

The lipopolysaccharide of the intracellular pathogen Shigella, in particular its O antigen, interacts with caspases and blocks their activation to prevent apoptosis.