The public health threat posed by a looming ‘post-antibiotic’ era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature’s control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the ‘vancapticins’, possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to ‘revitalise’ antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity.

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Citropin 1.1, maculatin 1.1, and caerin 1.1 are short antibacterial cationic peptides from the skin glands of the Australian tree frog Litoria species. Several analogues have been synthesized to give a better insight into the relationship between the structure of the peptides and their antibacterial and haemolytic activity. Binding studies using a surface plasmon resonance (SPR) biosensor together with a vesicle-capture sensor chip have been used to investigate selectivity of the peptides and their analogues for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, as well as for vesicles made from lipid extracts from Escherichia coli and bovine brain. Data obtained for membrane selectivity using natural lipid extracts show better correlation with minimum inhibitory concentration (MIC) values against Gram-positive bacteria and haemolytic activity than that obtained using synthetic DMPG and DMPC. Electron microscopy and membrane leakage studies using Gram-positive bacteria gave further insight into the membrane disruption properties of the peptides. For maculatin 1.1, it was found that the central proline residue, which is responsible for a bend in the alpha-helical structure, is essential not only for the antibacterial activity but also for binding, and perturbation of membranes. The caerin analogues showed only small variations in their MIC values and membrane binding. In contrast, for citropin 1.1, the analogue replacing the aspartate with a lysine showed the lowest MIC against Gram-positive bacteria and best membrane binding to E. coli lipid extracts, coinciding with an increased hydrophobic moment of the peptide. These data give further insight into these antimicrobial natural products, toward the development and evaluation of these and other analogues as potential antibiotics.

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae. By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro. In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 μg/mouse. Moreover, a ClyJ-3 dose of 2 μg/mouse had the same efficacy as a ClyJ dose of 40 μg/mouse, suggesting a 20-fold improvement in vivo. Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

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Summary Bacillus thuringiensis is the most widely used eco‐friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2‐methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl‐CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2‐methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2‐methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2‐methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2‐methylcitrate, the intermediate metabolite of the 2‐methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2‐methylcitrate cycle but also identified 2‐methylcitrate as a novel regulator of sporulation in B. thuringiensis.