In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication. There exists a short time lag of about 10–20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate. We show here that TopoIV localizes to the origin proximal side of the fork trailing protein SeqA and follows the movement pattern of the replication machinery in the cell.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

Fluoroquinolones induce double-strand breaks in bacterial DNA, triggering the SOS response, a major DNA damage response that ensures the expression of repair proteins but also promotes the emergence and spread of antibiotic resistance. Fluoroquinolone resistance, particularly in Escherichia coli, is a growing global health concern. Understanding bacterial responses to these antibiotics is critical for developing preventive strategies and novel treatments to combat resistance development. This study investigates DNA morphology in E. coli following exposure to ciprofloxacin (CIP), a fluoroquinolone antibiotic. We show that CIP induces a stepwise DNA reorganization, culminating in a highly dense nucleoid structure at midcell—a process we term DNA supercompaction. This phenomenon occurred also with other genotoxic agents. Live-cell imaging revealed that RecN, a structural maintenance of chromosomes (SMC)-like protein, is required for DNA supercompaction, and that RecN’s dynamics and activity in this response depend on RecA. Additionally, RecN and RecA frequently colocalized at nucleoid-associated positions. We suggest that RecN and RecA play active roles in DNA supercompaction following severe DNA damage, that their interplay is part of a prompt universal survival response to DNA double-strand breaks in E. coli, and that the extent of the compaction response depends on the DNA damage severity.

Antibiotic resistance is an escalating global concern, necessitating the development of novel antibiotics with unique mechanisms of action, and preferably also with a lowered propensity for resistance development. Type-I Toxin-Antitoxin (TA) systems that are ubiquitous in bacterial genomes consist of a genetic toxin element encoding a hydrophobic peptide and an antitoxin element producing an sRNA that inhibits the toxin translation. Although the biological roles of these membrane-associated toxins remain incompletely understood, their inherent lethality upon overexpression suggests a potential as antimicrobial agents. In this study, we explore the ShoB toxin from the shoB-ohsC TA system in Escherichia coli (E. coli) as a basis for designing synthetic antimicrobial peptides for exogenous delivery. We demonstrate that ShoB-derived peptides can retain antimicrobial efficacy when modified into shorter, cationic analogs with enhanced solubility. Our most promising hits exhibit rapid bactericidal action and frequency of resistance within E. coli cultures indicate a limited tendency for resistance development. These findings highlight that type-I TA systems constitute a novel source of potential peptide-based antibiotics, thereby offering an alternative largely unexplored strategy to combat antibiotic-resistant bacterial infections.

In response to the growing threat of antimicrobial resistance, scaffold-based peptidomimetics were explored as potential antibacterial agents.