Julia E. Bandow

Proteomic Approach to Understanding Antibiotic Action

ABSTRACT We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics.

Here we show that a new class of antibiotics—acyldepsipeptides—has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

iTRAQ experimental design for plasma biomarker discovery.

There is considerable interest in using mass spectrometry for biomarker discovery in human blood plasma. We investigated aspects of experimental design for large studies that require analysis of multiple sample sets using iTRAQ reagents for sample multiplexing and quantitation. Immunodepleted plasma samples from healthy volunteers were compared to immunodepleted plasma from patients with osteoarthritis in eight separate iTRAQ experiments. Our analyses utilizing ProteinPilot software for peptide identification and quantitation showed that the methodology afforded excellent reproducibility from run to run for determining protein level ratios (coefficient of variation 11.7%), in spite of considerable quantitative variances observed between different peptides for a given protein. Peptides with high variances were associated with lower intensity iTRAQ reporter ions, while immunodepletion prior to sample analysis had a negligible affect on quantitative variance. We examined the influence of different reference samples, such as pooled samples or individual samples on calculating quantitative ratios. Our findings are discussed in the context of optimizing iTRAQ experimental design for robust plasma-based biomarker discovery.

Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.

Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies--COPD biomarker discovery study.

2‐D gel electrophoresis has been used for more than three decades to study the protein complement of organisms, tissues, and cells. Three issues are holding back large‐scale proteomics studies: low‐throughput, high technical variation, and study designs lacking statistical power. We identified image analysis as the central factor connecting these three issues. By developing an improved image analysis workflow we shortened project timelines, decreased technical variation, and thus enabled large‐scale proteomics studies that are statistically powered. Rather than detecting protein spots on each gel image and matching spots across gel images, the improved workflow is based on aligning images first, then creating a consensus spot pattern and finally propagating the consensus spot pattern to all gel images for quantitation. This results in a data table without gaps. As an example we show here a study aimed at discovering circulating biomarkers for chronic obstructive pulmonary disease (COPD). Eight candidate biomarkers were identified by comparing plasma from 24 smokers with COPD and 24 smokers without COPD. Among the candidates are proteins such as plasma retinal‐binding protein (RETB) and fibrinogen that had previously been linked to the disease and are frequently monitored in COPD patients, as well as other proteins such as apolipoprotein E (ApoE), inter‐α‐trypsininhibitor heavy chain H4 (ITIH4), and glutathione peroxidase.

Photons and particles emitted from cold atmospheric-pressure plasma inactivate bacteria and biomolecules independently and synergistically

Cold atmospheric-pressure plasmas are currently in use in medicine as surgical tools and are being evaluated for new applications, including wound treatment and cosmetic care. The disinfecting properties of plasmas are of particular interest, given the threat of antibiotic resistance to modern medicine. Plasma effluents comprise (V)UV photons and various reactive particles, such as accelerated ions and radicals, that modify biomolecules; however, a full understanding of the molecular mechanisms that underlie plasma-based disinfection has been lacking. Here, we investigate the antibacterial mechanisms of plasma, including the separate, additive and synergistic effects of plasma-generated (V)UV photons and particles at the cellular and molecular levels. Using scanning electron microscopy, we show that plasma-emitted particles cause physical damage to the cell envelope, whereas UV radiation does not. The lethal effects of the plasma effluent exceed the zone of physical damage. We demonstrate that both plasma-generated particles and (V)UV photons modify DNA nucleobases. The particles also induce breaks in the DNA backbone. The plasma effluent, and particularly the plasma-generated particles, also rapidly inactivate proteins in the cellular milieu. Thus, in addition to physical damage to the cellular envelope, modifications to DNA and proteins contribute to the bactericidal properties of cold atmospheric-pressure plasma.

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains

Significance To date, simple membrane pore formation resulting in cytoplasmic leakage is the prevailing model for how membrane-active antibiotics kill bacteria and also is one of the main explanations for the activity of the membrane-binding antibiotic daptomycin. However, such models, typically derived from model membrane studies, often depict membranes as homogenous lipid bilayers. They do not take into account the complex architecture of biological membranes, with their many different membrane proteins, or the presence of microdomains with different fluidity properties. Here we report that daptomycin perturbs fluid microdomains in bacterial cell membranes, thereby interfering with membrane-bound cell wall and lipid synthesis processes. Our results add a different perspective as to how membrane-active antibiotics can kill bacteria. Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.

Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics

Two hetero-tri-organometallic compounds with potent activity against Gram-positive bacteria including multi-resistant Staphylococcus aureus (MRSA) were identified. The compounds consist of a peptide nucleic acid backbone with an alkyne side chain, substituted with a cymantrene, a (dipicolyl)Re(CO)3 moiety, and either a ferrocene (FcPNA) or a ruthenocene (RcPNA). Comparative proteomic analysis indicates the bacterial membrane as antibiotic target structure. FcPNA accumulation in the membrane was confirmed by manganese tracing with atomic absorption spectroscopy. Both organometallics disturbed several essential cellular processes taking place at the membrane such as respiration and cell wall biosynthesis, suggesting that the compounds affect membrane architecture. Correlating with enhanced antibacterial activity, oxidative stress was induced only by the ferrocene-substituted compound. The organometallics described here target the cytoplasmic membrane, a clinically proven antibacterial target structure, feature a bactericidal but non-bacteriolytic mode of action and limited cytotoxicity within the limits of solubility. Thus, FcPNA represents a promising lead structure for the development of a new synthetic class of antibiotics.

Synthesis and Biological Evaluation of Chromium Bioorganometallics Based on the Antibiotic Platensimycin Lead Structure

The recent discovery of the natural product platensimycin as a new antibiotic lead structure has triggered the synthesis of numerous organic derivatives for structure–activity relationship studies. Herein, we describe the synthesis, characterization and biological evaluation of the first organometallic antibiotic inspired by platensimycin. Two bioorganometallic compounds containing (η6‐pentamethylbenzene)Cr(CO)3 (2) and (η6‐benzene)Cr(CO)3 (3), linked by an amide bond to the aromatic part of platensimycin, were synthesized. Their antibiotic activities were tested against B. subtilis 168 (Gram positive) and E. coli W3110 (Gram negative) bacterial strains. Both compounds were found to be inactive against E. coli but derivative 2 inhibits B. subtilis growth at a moderate MIC value of 0.15 mM. To test the intrinsic toxicity of chromium, several chromium salts along with {η6‐(3‐pentamethylphenyl propionic acid)}Cr(CO)3 (5) and {η6‐(3‐phenyl propionic acid)}Cr(CO)3 (6) were tested against both bacterial strains. No activity was observed against E. coli for any of the compounds; B. subtilis growth was not inhibited by Cr(NO3)3 and only very weakly by 5, K2Cr2O7 and Na2CrO4 at MIC values of 0.5, 0.68 and 1.24 mM, respectively. Compounds 2, 3, 5 and 4 (the pure organic analogue of 2) show similar cytotoxicity against HeLa, HepG2 and HT‐29 mammalian cell lines. Furthermore, the cellular uptake and the intracellular distribution of compounds 2, 3 and Cr(NO3)3 in B. subtilis were studied using atomic absorption spectroscopy to gain insight in to the possible cellular targets. Compound 2 was found to be readily taken up and distributed almost equally among cytosol, cell debris and cell membrane in B. subtilis.

Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

ABSTRACT Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.