Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

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 ß-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 ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-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 pathogens1. 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.

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell walls and are refractory to resistance development; thus, they have promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk among a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and thus support their further characterization in animal models.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.

An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.