There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost ...most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a β-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the β-barrel folding complex (BAM) that is required for the folding and insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens
. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.
Research on inflammatory bowel disease (IBD) has produced mounting evidence for the modulation of microRNAs (miRNAs) during pathogenesis. MiRNAs are small, non-coding RNAs that interfere with the ...translation of mRNAs. Their high stability in free circulation at various regions of the body allows researchers to utilise miRNAs as biomarkers and as a focus for potential treatments of IBD. Yet, their distinct regulatory roles at the gut epithelial barrier remain elusive due to the fact that there are several external and cellular factors contributing to gut permeability. This review focuses on how miRNAs may compromise two components of the gut epithelium that together form the initial physical barrier: the mucus layer and the intercellular epithelial junctions. Here, we summarise the impact of miRNAs on goblet cell secretion and mucin structure, along with the proper function of various junctional proteins involved in paracellular transport, cell adhesion and communication. Knowledge of how this elaborate network of cells at the gut epithelial barrier becomes compromised as a result of dysregulated miRNA expression, thereby contributing to the development of IBD, will support the generation of miRNA-associated biomarker panels and therapeutic strategies that detect and ameliorate gut permeability.
The metabolite-rich environment that is the intestinal lumen contains metabolic by-products deriving from microbial fermentation and host cell metabolism, with resident macrophages being constantly ...exposed to this metabolic flux. Succinate, lactate and itaconate are three metabolites secreted by primed macrophages due to a fragmented tri-carboxylic acid (TCA) cycle. Additionally, succinate and lactate are known by-products of microbial fermentation. How these metabolites impact biological functioning of resident macrophages particularly in response to bacterial infection remains poorly understood. We have investigated the potential influence of these metabolites on macrophage phagocytosis and clearance of
Escherichia coli
(
E. coli
) infection. Treatment of murine bone-marrow-derived macrophages (BMDMs) with succinate reduced numbers of intracellular
E. coli
early during infection, while lactate-treated BMDMs displayed no difference throughout the course of infection. Treatment of BMDMs with itaconate lead to higher levels of intracellular
E. coli
early in the infection with bacterial burden subsequently reduced at later time-points compared to untreated macrophages, indicative of enhanced engulfment and killing capabilities of macrophages in response to itaconate. Expression of engulfment mediators MARCKS, RhoB, and CDC42 were reduced or unchanged following succinate or lactate treatment and increased in itaconate-treated macrophages following
E. coli
infection. Nitric oxide (NO) levels varied while pro- and anti-inflammatory cytokines differed in secretory levels in all metabolite-treated macrophages post-infection with
E. coli
or in response to lipopolysaccharide (LPS) stimulation. Finally, the basal phenotypic profile of metabolite-treated macrophages was altered according to marker gene expression, describing how fluid macrophage phenotype can be in response to the microenvironment. Collectively, our data suggests that microbe- and host-derived metabolites can drive distinct macrophage functional phenotypes in response to infection, whereby succinate and itaconate regulate phagocytosis and bactericidal mechanisms, limiting the intracellular bacterial niche and impeding the pathogenesis of infection.
The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the ...generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.
Background: Nanobodies represent a specific means to modulate enzyme function.
Results: HypE-specific nanobodies modulate AMPylation activity and identify HypE as localized to the nuclear envelope.
Conclusion: HypE function can be interfered with and probed for with new tools.
Significance: HypE nanobodies are the first known HypE activators and inhibitors.
The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills ...Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant
and
strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.
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