Mechanism of action of pseudopteroxazole and pseudopterosin G: Diterpenes from marine origin.

Pseudopteroxazole (Ptx) and the pseudopterosins are marine natural products with promising antibacterial potential. While Ptx has attracted interest for its antimycobacterial activity, pseudopterosins are active against several clinically relevant pathogens. Both compound classes exhibit low cytotoxicity and accessibility to targeted synthesis, yet their antibacterial mechanisms remain elusive. In this study, we investigated the modes of action of Ptx and pseudopterosin G (PsG) in Bacillus subtilis employing an unbiased approach that combines gel-based proteomics with a mathematical similarity analysis of response profiles. Proteomic responses to sublethal concentrations of Ptx and PsG were compared to a library of antibiotic stress response profiles revealing that both induce a stress response characteristic for agents targeting the bacterial cell envelope by interfering with membrane-bound steps of cell wall biosynthesis. Microscopy-based assays confirmed that both compounds compromise the integrity of the bacterial cell wall without disrupting the membrane potential. Furthermore, LC-MSE analysis showed that the greater potency of PsG against B. subtilis, reflected in a lower MIC and a more pronounced proteomic response, may be rooted in a more effective association with and penetration of B. subtilis cells. We conclude that Ptx and PsG target the integrity of the gram-positive cell wall.

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. In this updated chapter, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation, and proteins are identified and quantified using label-free mass spectrometry.

Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Adaptive Responses of Pseudomonas aeruginosa to Treatment with Antibiotics.

Pseudomonas aeruginosa is among the highest priority pathogens for drug development because of its resistance to antibiotics, extraordinary adaptability, and persistence. Antipseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin-tazobactam. We further investigated the response to CHIR-090, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by two-dimensional polyacrylamide gel electrophoresis was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.

Rational prioritization strategy allows the design of macrolide derivatives that overcome antibiotic resistance.

Antibiotic resistance is a major threat to global health; this problem can be addressed by the development of new antibacterial agents to keep pace with the evolutionary adaptation of pathogens. Computational approaches are essential tools to this end since their application enables fast and early strategical decisions in the drug development process. We present a rational design approach, in which acylide antibiotics were screened based on computational predictions of solubility, membrane permeability, and binding affinity toward the ribosome. To assess our design strategy, we tested all candidates for in vitro inhibitory activity and then evaluated them in vivo with several antibiotic-resistant strains to determine minimal inhibitory concentrations. The predicted best candidate is synthetically more accessible, exhibits higher solubility and binding affinity to the ribosome, and is up to 56 times more active against resistant pathogens than telithromycin. Notably, the best compounds designed by us show activity, especially when combined with the membrane-weakening drug colistin, against Acinetobacter baumanii, Pseudomonas aeruginosa, and Escherichia coli, which are the three most critical targets from the priority list of pathogens of the World Health Organization.

Zwitterionic Peptides Reduce Accumulation of Marine and Freshwater Biofilm Formers.

Zwitterionic peptides are facile low-fouling compounds for environmental applications as they are biocompatible and fully biodegradable as their degradation products are just amino acids. Here, a set of histidine (H) and glutamic acid (E), as well as lysine (K) and glutamic acid (E) based peptide sequences with zwitterionic properties were synthesized. Both oligopeptides (KE)4K and (HE)4H were synthesized in d and l configurations to test their ability to resist the nonspecific adsorption of the proteins lysozyme and fibrinogen. The coatings were additionally tested against the attachment of the marine organisms Navicula perminuta and Cobetia marina as well as the freshwater bacterium Pseudomonas fluorescens on the developed coatings. While the peptides containing lysine performed better in protein resistance assays and against freshwater bacteria, the sequences containing histidine were generally more resistant against marine organisms. The contribution of amino acid-intrinsic properties such as side chain pKa values and hydrophobicity, as well as external parameters such as pH and salinity of fresh water and seawater on the resistance of the coatings is discussed. In this way, a detailed picture emerges as to which zwitterionic sequences show advantages in future generations of biocompatible, sustainable, and nontoxic fouling release coatings.

Evaluation of Ruthenium(II) N-Heterocyclic Carbene Complexes as Antibacterial Agents and Inhibitors of Bacterial Thioredoxin Reductase.

A series of ruthenium(II) complexes with N-heterocyclic carbene (NHC) ligands of the general type (arene)(NHC)Ru(II)X2 (where X = halide) was prepared, characterized, and evaluated as antibacterial agents in comparison to the respective metal free benzimidazolium cations. The ruthenium(II) NHC complexes generally triggered stronger bacterial growth inhibition than the metal free benzimidazolium cations. The effects were much stronger against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) than against Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa), and all complexes were inactive against the fungus Candida albicans. Moderate inhibition of bacterial thioredoxin reductase was confirmed for selected complexes, indicating that inhibition of this enzyme might be a contributing factor to the antibacterial effects.