Marcos Pires

Abstract

Abstract

The mechanisms by which two different anti-CRISPR proteins inhibit Cas9 may provide CRISPR-based regulatory tools to allow DNA binding while preventing cleavage.

Abstract

Abstract

The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility...

Coulson et al. show that Mycobacterium tuberculosis displays enhanced sensitivity to thiol stress in acidic environments. Their small molecule, AC2P36, potently depletes free thiol pools, promotes accumulation of reactive oxygen species at acidic pH, and efficiently synergizes with the first-line antibiotic isoniazid.

Nature Chemistry. doi:10.1038/nchem.2826

Authors: Stephan M. Hacker, Keriann M. Backus, Michael R. Lazear, Stefano Forli, Bruno E. Correia & Benjamin F. Cravatt

A chemical proteomic strategy has now been reported for the global profiling of lysine reactivity and ligandability. Using this approach, >9000 lysines in the human proteome were evaluated, leading to the discovery of hyper-reactive lysines, and lysines that can be targeted by electrophilic small molecules to perturb enzyme function and protein–protein interactions.

N-Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related N-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.

ABSTRACT

The rapid increase of carbapenem resistance in Gram-negative bacteria has resurrected the importance of the polymyxin antibiotics. The recent discovery of plasmid-mediated polymyxin resistance (mcr-1) in carbapenem-resistant Enterobacteriaceae serves as an important indicator that the golden era of antibiotics is under serious threat. We assessed the bacterial killing of 15 different FDA-approved antibiotics alone and in combination with polymyxin B in time-killing experiments against Escherichia coli MCR1_NJ, the first reported isolate in the United States to coharbor mcr-1 and a New Delhi metallo-β-lactamase gene (bla_NDM-5). The most promising regimens were advanced to the hollow-fiber infection model (HFIM), where human pharmacokinetics for polymyxin B, aztreonam, and amikacin were simulated over 240 h. Exposure to polymyxin B monotherapy was accompanied by MCR1_NJ regrowth but not resistance amplification (polymyxin B MIC from 0 to 240 h [MIC_0h to MIC_240h] of 4 mg/liter), whereas amikacin monotherapy caused regrowth and simultaneous resistance amplification (amikacin MIC_0h of 4 mg/liter versus MIC_240h of >64 mg/liter). No MCR1_NJ colonies were observed for any of the aztreonam-containing regimens after 72 h. However, HFIM cartridges for both aztreonam monotherapy and the polymyxin B-plus-aztreonam regimen were remarkably turbid, and the presence of long, filamentous MCR1_NJ cells was evident in scanning electron microscopy, suggestive of a nonreplicating persister (NRP) phenotype. In contrast, the 3-drug combination of polymyxin B, aztreonam, and amikacin provided complete eradication (>8-log₁₀ CFU/ml reduction) with suppression of resistance and prevention of NRP formation. This is the first comprehensive pharmacokinetic/pharmacodynamic study to evaluate triple-drug combinations for polymyxin- and carbapenem-resistant E. coli coproducing MCR-1 and NDM-5 and will aid in the preparation for a so-called "postantibiotic" era.

IMPORTANCE A global health crisis may be on the horizon, as the golden era of antibiotics is under serious threat. We recently reported the first case in the United States of a highly resistant, Escherichia coli so-called "superbug" (MCR1_NJ), coharboring two of the most worrying antibiotic resistance genes, encoding mobile colistin resistance (mcr-1) and a New Delhi metallo-β-lactamase (bla_NDM-5). Worryingly, the medical community is vulnerable to this emerging bacterial threat because optimal treatment strategies are undefined. Here, we report the activity of an optimized combination using simulated human doses of commercially available antibiotics against MCR1_NJ. A unique triple combination involving a cocktail of polymyxin B, aztreonam, and amikacin eradicated the MCR-1- and NDM-5-producing E. coli. Each antimicrobial agent administered as monotherapy or in double combinations failed to eradicate MCR1_NJ at a high inoculum. To our knowledge, this is the first study to propose 3-drug therapeutic solutions against superbugs coharboring mcr-1 and bla_NDM, seeking to prepare clinicians for future occurrences of these pathogens.

Nature Chemistry. doi:10.1038/nchem.2811

Authors: Henry D. Herce, Dominik Schumacher, Anselm F. L. Schneider, Anne K. Ludwig, Florian A. Mann, Marion Fillies, Marc-André Kasper, Stefan Reinke, Eberhard Krause, Heinrich Leonhardt, M. Cristina Cardoso & Christian P. R. Hackenberger

Delivery of antibodies into living cells enables the labelling and manipulation of intracellular antigens; however, transporting antibodies into the cytosol in a functional state is difficult. Now, a modular strategy for creating cell-permeable nanobodies capable of targeting intracellular antigens has been developed. The cell-permeable nanobodies are formed by site-specific attachment of cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain antibody fragments.

by Travis J. Kochan, Madeline J. Somers, Alyssa M. Kaiser, Michelle S. Shoshiev, Ada K. Hagan, Jessica L. Hastie, Nicole P. Giordano, Ashley D. Smith, Alyxandria M. Schubert, Paul E. Carlson Jr, Philip C. Hanna

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca²⁺ during C. difficile spore germination and provide direct evidence that intestinal Ca²⁺ coordinates with bile salts to stimulate germination. Endogenous Ca²⁺ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca²⁺. However, environmental Ca²⁺ replaces glycine as a co-germinant and circumvents the need for endogenous Ca²⁺ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca²⁺ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.

Outer membrane vesicles (OMVs) are proteoliposome nanoparticles ubiquitously produced by Gram-negative bacteria. Typically bearing a composition similar to those of the outer membrane and periplasm of the cells from which they are derived, OMVs package an array of proteins, lipids, and nucleic acids. Once considered inconsequential by-products of bacterial growth, OMVs have since been demonstrated to mediate cellular stress relief, promote horizontal gene transfer and antimicrobial activity, and elicit metazoan inflammation. Recently, OMVs have gained appreciation as critical moderators of interorganismal dynamics. In this review, we focus on recent progress toward understanding the functions of OMVs with regard to symbiosis and ecological contexts, and we propose potential avenues for future OMV studies.

Nature advance online publication 05 July 2017. doi:10.1038/nature23002

Authors: Anthony W. P. Fitzpatrick, Benjamin Falcon, Shaoda He, Alexey G. Murzin, Garib Murshudov, Holly J. Garringer, R. Anthony Crowther, Bernardino Ghetti, Michel Goedert & Sjors H. W. Scheres

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Abstract

Illuminating vital surface molecules of symbionts in health and disease

Nature Microbiology, Published online: 26 June 2017; doi:10.1038/nmicrobiol.2017.99

Metabolic labelling can be used to simultaneously tag peptidoglycan, lipopolysaccharide and capsular polysaccharide of live gut bacteria, and to label peptidoglycan in vivo, revealing host–bacteria interactions within the living mammalian host.

Mitochondria localization induced self-assembly of peptide amphiphiles for cellular dysfunction

Nature Communications, Published online: 21 June 2017; doi:10.1038/s41467-017-00047-z

Spatiotemporal control of intracellular molecular self-assembly holds promise for therapeutic applications. Here the authors develop a peptide consisting of a phenylalanine dipeptide with a mitochondrial targeting moiety to form self-assembling fibrous nanostructures within mitochondria, leading to apoptosis.

Nature Chemical Biology 13, 730 (2017). doi:10.1038/nchembio.2376

Authors: Jooyoung Park, Andrew J Gasparrini, Margaret R Reck, Chanez T Symister, Jennifer L Elliott, Joseph P Vogel, Timothy A Wencewicz, Gautam Dantas & Niraj H Tolia