Actinorhodin is a redox-active antibiotic with a complex mode of action against Gram-positive cells.

Actinorhodin is a blue-pigmented, redox-active secondary metabolite that is produced by the bacterium Streptomyces coelicolor. Although actinorhodin has been used as a model compound for studying secondary metabolism, its biological activity is not well understood. Indeed, redox-active antibiotics in general have not been widely investigated at the mechanistic level. In this work, we have conducted a comprehensive chemical genetic investigation of actinorhodin's antibacterial effect on target organisms. We find that actinorhodin is a potent, bacteriostatic, pH-responsive antibiotic. Cells activate at least three stress responses in the presence of actinorhodin, including those responsible for managing oxidative damage, protein damage and selected forms of DNA damage. We find that mutations in the Staphylococcus aureus walRKHI operon can confer low-level resistance to actinorhodin, indicating possible targeting of the cell envelope. Our study indicates a complex mechanism of action, involving multiple molecular targets, that is distinct from other antibiotics.

Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs.

Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.

Tetramers are the activation-competent species of the HOCl-specific transcription factor HypT.

Hypochlorous acid (HOCl) is an important component of the immune system and is produced by neutrophils to kill invading microorganisms. The transcription factor HypT is specifically activated by HOCl by methionine oxidation and protects Escherichia coli cells from the detrimental effects of HOCl. HypT forms dodecameric ring-like oligomers. Binding of HypT to DNA induces dissociation of the dodecamers into dimers and tetramers, thus forming the DNA-binding species. To dissect HypT dissociation, binding to DNA, and activation, we aimed to dissociate the dodecamers independently of DNA and to analyze HOCl-dependent activation in vitro. We found that HypT dodecamers dissociated into tetramers in the presence of l-arginine and NaCl, which was reversible upon dilution of the additive. Making use of the reversible dissociation, we generated mixed assemblies consisting of wild-type and mutant HypT subunits and determined that mutant subunits with reduced thermal stability were stabilized by wild-type HypT in the mixed assembly. HypT tetramers, as present at high NaCl concentrations, were stabilized against thermal unfolding and aggregation triggered by high HOCl concentrations. Importantly, in vitro activation by HOCl of HypT tetramers was completed within 1 min, whereas activation of dodecamers required 1 h for completion. Furthermore, activation of HypT tetramers required stoichiometric amounts of HOCl instead of an excess of HOCl, as observed for dodecamers. This supports the idea that small HypT oligomers are the activation-competent species, whereas the dodecamers are a storage form. Our study reveals the importance of the dynamic oligomeric state for HypT activation by HOCl.

The expression of antibiotic resistance genes in antibiotic-producing bacteria.

Antibiotic-producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.

Kinetic analysis of ternary and binary binding modes of the bispecific antibody emicizumab.

The binding properties of bispecific antibodies (bsAb) are crucial for their function, especially when two antigens are targeted on the same cell surface. Dynamic interactions between each of the antibody's arms and its cognate target cause the formation and decay of a biologically functional ternary complex. How association and dissociation processes work cooperatively, and how they influence the avidity of the ternary complex, is still poorly understood. Here, we present a biosensor assay for the simultaneous measurement of the binding kinetics of the therapeutic bsAb emicizumab (Hemlibra®) and its two targets, the blood coagulation factors IX and X (FIX, FX). We describe an automated workflow to characterize binary and ternary-binding modes, utilizing a Y-shaped DNA nanostructure to immobilize the antigens on a sensor and to emulate conditions on a cell or platelet surface by presenting the antigens with optimal accessibility for the bsAb flown over the sensor as analyte. We find that emicizumab binds FX much stronger than FIX (Kd = 0.05 µM vs. 5 µM, t1/2 = 20 s vs. 1 s) with profound consequences on the avidity of the ternary complex, which is dominated by FX's binding properties and a hand-off mechanism from FX to FIX. Moreover, formation and decay of the ternary complex depend on the bsAb concentration during the association phase. Emicizumab's in-vivo mode of action and the catalytic activation of FX can be rationalized from the analyzed binding kinetics. The assay and workflow are well suited for the screening of bispecific binders in drug discovery and provide valuable new kinetic information.Abbreviations: bsAb: bispecific antibody; FVIII/FIX/FX: coagulation factors VIII/IX/X; SPR: surface plasmon resonance; kon: association rate constant; koff: dissociation rate constant; KD: equilibrium dissociation constant; t1/2: dissociation half-life.

A contractile injection system is required for developmentally regulated cell death in Streptomyces coelicolor.

Diverse bacterial species produce extracellular contractile injection systems (eCISs). Although closely related to contractile phage tails, eCISs can inject toxic proteins into eukaryotic cells. Thus, these systems are commonly viewed as cytotoxic defense mechanisms that are not central to other aspects of bacterial biology. Here, we provide evidence that eCISs appear to participate in the complex developmental process of the bacterium Streptomyces coelicolor. In particular, we show that S. coelicolor produces eCIS particles during its normal growth cycle, and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Furthermore, eCIS-deficient mutants display reduced levels of cell death and altered morphology during growth in liquid media. Our results suggest that the main role of eCISs in S. coelicolor is to modulate the developmental switch that leads to aerial hyphae formation and sporulation, rather than to attack other species.

The pathogenic Escherichia coli type III secreted protease NleC degrades the host acetyltransferase p300.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) are attaching and effacing bacterial pathogens that cause devastating diarrhoeal disease worldwide. These pathogens depend on a type III secretion system, which functions as a molecular syringe to translocate bacterial effector proteins directly into infected host cells. One of these effectors, NleC, was recently described as a zinc metalloprotease that targets NF-κB Rel-A (p65) and thus contributes to dampening of inflammatory signalling during EPEC and EHEC infection. We have identified the acetyltransferase p300 as an additional target of NleC. Several biochemical techniques were employed to demonstrate specific binding of p300 by NleC. We also show that NleC causes decreased abundance of p300 in cellular nuclei and that the metalloprotease domain of NleC is responsible for this phenotype. Furthermore, we demonstrate that overexpression of p300 can antagonize repression of IL-8 secretion by EPEC and that siRNA knock-down of p300 dampens IL-8 secretion by EPEC ΔnleC-infected cells. We have therefore identified a second target of NleC and provided the first example of a bacterial virulence factor targeting the acetyltransferase p300.

Nanodiscs Incorporating Native ß1 Adrenergic Receptor as a Novel Approach for the Detection of Pathological Autoantibodies in Patients with Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a common cause of heart failure with high morbidity and mortality rates. The association of anti-ß1 adrenergic receptor (ß1AR) autoantibodies with disease progression was shown by various studies and in vivo animal experiments. The prevalence of these disease-driving autoantibodies was estimated as 25% to 75% in DCM. The removal of autoantibodies or the interruption of their action leads to a prolonged improvement of heart function. However, presence and impact of the autoimmune aspect in DCM patients must be examined for targeted treatment. METHODS: We developed a heterogeneous immunoassay to support the diagnosis of anti-ß1AR autoantibody-induced DCM. The presentation of the native conformational epitope was enabled by reconstitution of human ß1AR into lipid bilayer nanodiscs, which stabilize the incorporated receptor in aqueous solution for measurements with standard immunological techniques. RESULTS: The incorporation of ß1AR into nanodiscs was verified by chromatographic, spectroscopic, and immunological methods. The functionality was shown by interaction assays with appropriate binding partners. Furthermore, ß1AR nanodiscs were applied to immunoassays for the detection of anti-ß1AR in human sera. Surface plasmon resonance spectroscopy and ELISA were developed, optimized, and validated. The optimized ß1AR nanodisc ELISA enabled a simultaneous measurement of 40 samples in duplicate. An interassay variance of 24% and an intraassay variance of 5% were determined. The limit of detection and the limit of quantification were determined as 0.64 ng/mL and 1.26 ng/mL, respectively (related to a monoclonal anti-ß1AR). CONCLUSIONS: Nanodisc technology is suitable as a novel biomimetic membrane system to stabilize and present ß1AR for detection of autoantibodies with immunological methods in DCM patients.