Antiseptic 9-Meric Peptide with Potency against Carbapenem-Resistant Acinetobacter baumannii Infection

Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.

Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: further Insights into Their Cooperative Action

Lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics. Although the membrane-associated lantibiotic biosynthesis machinery has long been proposed to exist, the isolation of such a complex has not been reported yet, which limits the elucidation of the processive mechanism of lantibiotic biosynthesis.

Lactococcus lactis resistance to aureocin A53- and enterocin L50-like bacteriocins and membrane-targeting peptide antibiotics relies on the YsaCB-KinG-LlrG four-component system

Resistance to non-ribosomally synthesized peptide antibiotics affecting the cell envelope is well-studied and mostly associated with the action of peptide-sensing and detoxification (PSD) modules which consist of a two-component system (TCS) and an ATP-binding cassette (ABC) transporter. In contrast, the resistance mechanisms to ribosomally synthesized bacterial toxic peptides (bacteriocins), which also affect the cell envelope, are studied to lesser extent, and possible cross-resistance between them and antibiotics is still poorly understood. In the present study, we investigated the development of resistance of Lactococcus lactis to aureocin A53- and enterocin L50-like bacteriocins and cross-resistance with antibiotics. First, 19 spontaneous mutants resistant to their representatives were selected and displayed changes in the sensitivity also to peptide antibiotics acting on the cell envelope (bacitracin, daptomycin, and gramicidin). Sequencing of their genomes revealed mutations in genes encoding ABC transporter YsaCB and TCS KinG-LlrG, the emergence of which induced upregulation of the dltABCD and ysaDCB operons. The ysaB mutations were either nonsense or frameshift and led to the generation of truncated YsaB but with the conserved N-terminal FtsX domain intact. Deletions of ysaCB or llrG had a minor effect on the resistance of the obtained mutants to the tested bacteriocins, daptomycin, and gramicidin, indicating that the development of resistance is dependent on the modification of the protein rather than its absence. In further corroboration of the above conclusion, we show that the FtsX domain, which functions effectively when the YsaB is lacking its central and C-terminal parts, is critical for the resistance to these antimicrobials.

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions – their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

Amphipathic Peptide Antibiotics with Potent Activity against Multidrug-Resistant Pathogens

The emergence and prevalence of multidrug-resistant (MDR) bacteria have posed a serious threat to public health. Of particular concern are methicillin-resistant Staphylococcus aureus (MRSA) and blaNDM, mcr-1 and tet(X)-positive Gram-negative pathogens. The fact that few new antibiotics have been approved in recent years exacerbates this global crisis, thus, new alternatives are urgently needed. Antimicrobial peptides (AMPs) originated from host defense peptides with a wide range of sources and multiple functions, are less prone to achieve resistance. All these characteristics laid the foundation for AMPs to become potential antibiotic candidates. In this study, we revealed that peptide WW307 displayed potent antibacterial and bactericidal activity against MDR bacteria, including MRSA and Gram-negative bacteria carrying blaNDM-5, mcr-1 or tet(X4). In addition, WW307 exhibited great biofilm inhibition and eradication activity. Safety and stability experiments showed that WW307 had a strong resistance against various physiological conditions and displayed relatively low toxicity. Mechanistic experiments showed that WW307 resulted in membrane damage by selectively targeting bacterial membrane-specific components, including lipopolysaccharide (LPS), phosphatidylglycerol (PG), and cardiolipin (CL). Moreover, WW307 dissipated membrane potential and triggered the production of reactive oxygen species (ROS). Collectively, these results demonstrated that WW307 represents a promising candidate for combating MDR pathogens.