An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli\u27s proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA\u27s chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO$_{2}$H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidA$_{HOCl\u27s}$ chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones

Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology

Influence of Amino Acid Feeding on Production of Calcimycin and Analogs in Streptomyces chartreusis

Streptomyces chartreusis NRRL 3882 produces the polyether ionophore calcimycin and a variety of analogs, which originate from the same biosynthetic gene cluster. The role of calcimycin and its analogs for the producer is unknown, but calcimycin has strong antibacterial activity. Feeding experiments were performed in chemically defined medium systematically supplemented with proteinogenic amino acids to analyze their individual effects on calcimycin synthesis. In the culture supernatants, in addition to known calcimycin analogs, eight so far unknown analogs were detected using LC-MS/MS. Under most conditions cezomycin was the compound produced in highest amounts. The highest production of calcimycin was detected upon feeding with glutamine. Supplementation of the medium with glutamic acid resulted in a decrease in calcimycin production, and supplementation of other amino acids such as tryptophan, lysine, and valine resulted in the decrease in the synthesis of calcimycin and of the known intermediates of the biosynthetic pathway. We demonstrated that the production of calcimycin and its analogs is strongly dependent on amino acid supply. Utilization of amino acids as precursors and as nitrogen sources seem to critically influence calcimycin synthesis. Even amino acids not serving as direct precursors resulted in a different product profile regarding the stoichiometry of calcimycin analogs. Only slight changes in cultivation conditions can lead to major changes in the metabolic output, which highlights the hidden potential of biosynthetic gene clusters. We emphasize the need to further study the extent of this potential to understand the ecological role of metabolite diversity originating from single biosynthetic gene clusters

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides

Fear in your eyes? The importance of the eyes for rapid discrimination of fearful facial expressions

The human face and its profound relevance for social cognition has been a topic of interest in psychology for a long time. Many different aspects of face processing have been investigated and one of these areas of interest is how we perceive and process the facial expressions of other people to recognize their emotions. Another issue that has caused a considerable amount of debate in the past decade is the question of how the eyes are processed and how important a facial feature they are. Hypotheses have been formed about a separate eye processor that coexists in the human brain with a distinct face processor. The present study sought to find out if there is a connection between the above mentioned two issues, and to find answers to the question of whether the eyes are important for the rapid discrimination of fearful and neutral facial expressions as measured with event-related potentials. The participants of this study were healthy volunteer adults (22 participants, mean a! ge 27 years). The participants viewed fearful and neutral faces in three different conditions regarding the eye region of the stimulus. The conditions were intact full faces, full faces with the eyes covered with sunglasses, and isolated eyes. The participants viewed the stimuli from a computer screen while their EEG activity was recorded. ERP waveforms were then averaged from the data, and peak amplitude and latency of the early P100 and N170 components of the ERP were analysed. Based on prior research, a clear difference was expected to be found between the responses to the fearful and neutral stimuli, and also that the isolated eyes would evoke a distinct response from the full faces. The visibility of the eye region was expected to have an effect on the responses to the facial expressions. The results lent support for the eyes being a special facial feature but conclusive evidence was not found for a critical role of the eyes in the discrimination of fearful and neutral expressions. Specifically, the results of the present study partly replicated results of prior studies in showing enhanced N170 amplitude and delayed N170 latency for isolated eyes as compared to full face stimuli (irrespective of facial expression). The results also showed that fearful stimuli delayed the N170 latency across all three conditions, suggesting that fearful expressions may delay responses due to the involvement of the subcortical route in emotional information processing, or that they may hold the attention of the observer more effectively and thus take longer to process than the neutral faces. The fact that the latency difference was observed when the eye region alone was presented supports the hypothesis that the eye region is important for the discrimination of fearful and neutral facial expressions. The P100 component reflected only the difference between the isolated eyes and the full face stimuli. Contrary to the expectations, no effects of emotional expression were found on the P100 component or the N170 amplitude. There are several possible explanations as to why the results of the present study were inconclusive. One of these includes the possibility that openness of the eyes (i.e., whether the expression fearful or not) could be read from all the stimuli because the eyebrows were in plain sight in all three conditions. Further investigation is needed to find more solid evidence for the importance of the eyes. KEY WORDS: emotion, fear, facial expressions, eye detector, ERP

Supplementary Figures S1-S3 from The cold atmospheric pressure plasma-generated species superoxide, singlet oxygen and atomic oxygen activate the molecular chaperone Hsp33

Cold atmospheric pressure plasmas are used for surface decontamination or disinfection, e.g. in clinical settings. Protein aggregation has been shown to significantly contribute to the antibacterial mechanisms of plasma. To investigate the potential role of the redox-activated zinc-binding chaperone Hsp33 in preventing protein aggregation and thus mediating plasma resistance, we compared the plasma sensitivity of wild-type E. coli to that of an hslO deletion mutant lacking Hsp33 as well as an over-producing strain. Over-production of Hsp33 increased plasma survival rates above wild-type levels. Hsp33 was previously shown to be activated by plasma in vitro. For the PlasmaDerm source applied in dermatology, reversible activation of Hsp33 was confirmed. Thiol oxidation and Hsp33 unfolding, both crucial for Hsp33 activation, occurred during plasma treatment. After prolonged plasma exposure, however, unspecific protein oxidation was detected, the ability of Hsp33 to bind zinc ions was decreased without direct modifications of the zinc-binding motif, and the protein was inactivated. To identify chemical species of potential relevance for plasma-induced Hsp33 activation, reactive oxygen species were tested for their ability to activate Hsp33 in vitro. Superoxide, singlet oxygen and potentially atomic oxygen activate Hsp33, while no evidence was found for activation by ozone, peroxynitrite or hydroxyl radicals

Protection strategies for biocatalytic proteins under plasma treatment

In plasma-driven biocatalysis, enzymes are employed to carry out reactions using species generated by non-thermal plasmas as the precursors. We have previously demonstrated that this is feasible in principle, but that the approach suffers from the short lifetime of the biocatalyst under operating conditions. In this work, protection strategies were investigated to prevent the dielectric barrier discharge plasma-induced inactivation of biocatalysts, using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), one of the most promising enzymes for plasma-driven biocatalysis. Treatment in oxygen-free atmospheres did not provide any advantage over treatment in synthetic air, indicating that the detrimental reactive species did not originate from oxygen in the plasma phase. Chemical scavengers were employed to eliminate undesired reactive species, without any long-term effect on enzyme lifetime. Similarly, chaperones, including the known stress response proteins Hsp33, CnoX, and RidA did not increase the lifetime of rAaeUPO. Immobilization of the biocatalyst proved effective in preserving enzyme activity. The residual activity of rAaeUPO after plasma treatment strongly depended on the specific immobilization support. Essentially complete protection for at least 15 min of plasma exposure was achieved with an epoxy-butyl-functionalized carrier. This study presents new insights into plasma-protein interactions and plots a path forward for protecting biocatalytic proteins from plasma-mediated inactivation. </p

An Organometallic Structure-Activity Relationship Study Reveals the Essential Role of a Re(CO)3 Moiety in the Activity Against Gram-Positive Pathogens Including MRSA

The worrying appearance of microbial resistance to antibiotics is a worldwide problem which needs to be tackled urgently. Microbial resistance to the common classes of antibiotics involving purely organic compounds unfortunately develops very rapidly and in most cases, resistance was detected soon after or even before release of the antibiotic to the market. Therefore, novel concepts for antibiotics must be investigated, and metal-containing compounds hold particular promise in that area. Taking a trimetallic complex (1a) which contains a ferrocenyl (Fc), a CpMn(CO)3 (cymantrene) and a [(dpa)Re(CO)3] residue as the lead structure, a systematic structure–activity relationship (SAR) study against various grampositive pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA) strains was performed. The [(dpa)Re(CO)3] moiety was discovered to be the essential unit for the observed antibacterial activity of 1a. The ferrocenyl and CpMn(CO)3 units can be replaced one by one or both together by organic moieties such as a phenyl ring without loss of antibacterial activity. The most potent mono-metallic complex (9c0) has an antibacterial activity comparable to the well-established organic drugs amoxicillin and norfloxacin and importantly, only moderate cytotoxicity against mammalian cells. Microbiological studies on membrane potential, membrane permeabilization, and cell wall integrity revealed that 9c0 targets the bacterial membrane and disturbs cell wall integrity, but shows more efficient membrane permeabilization than the lead structure 1a

Bacterial Metabolites Produced Under Iron Limitation Kill Pinewood Nematode and Attract Caenorhabditis elegans

Pine Wilt Disease (PWD) is caused by Bursaphelenchus xylophilus, the pinewood nematode, and affects several species of pine trees worldwide. The ecosystem of the Pinus pinaster trees was investigated as a source of bacteria producing metabolites affecting this ecosystem: P. pinaster trees as target-plant, nematode as disease effector and its insect-vector as shuttle. For example, metals and metal-carrying compounds contribute to the complex tree-ecosystems. This work aimed to detect novel secondary metabolites like metallophores and related molecules produced under iron limitation by PWD-associated bacteria and to test their activity on nematodes. After screening 357 bacterial strains from Portugal and United States, two promising metallophore-producing strains Erwinia sp. A41C3 and Rouxiella sp. Arv20#4.1 were chosen and investigated in more detail. The genomes of these strains were sequenced, analyzed, and used to detect genetic potential for secondary metabolite production. A combinatorial approach of liquid chromatography-coupled tandem mass spectrometry (LC-MS) linked to molecular networking was used to describe these compounds. Two major metabolites were detected by HPLC analyses and described. One HPLC fraction of strain Arv20#4.1 showed to be a hydroxamate-type siderophore with higher affinity for chelation of Cu. The HPLC fraction of strain A41C3 with highest metal affinity showed to be a catecholate-type siderophore with higher affinity for chelation of Fe. LC-MS allowed the identification of several desferrioxamines from strain Arv20#4.1, in special desferrioxamine E, but no hit was obtained in case of strain A41C3 which might indicate that it is something new. Bacteria and their culture supernatants showed ability to attract C. elegans. HPLC fractions of those supernatant-extracts of Erwinia strain A41C3, enriched with secondary metabolites such as siderophores, were able to kill pinewood nematode. These results suggest that metabolites secreted under iron limitation have potential to biocontrol B. xylophilus and for management of Pine Wilt Disease