An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

Comparison of four commercial EBV DNA quantitative tests to a new test at an early stage of development.

BACKGROUND: Regular screening for Epstein-Barr virus (EBV) DNA using quantitative RT-PCR is recommended for early intervention in at-risk patients. Harmonization of quantitative RT-PCR assays is critical to avoid misinterpretation of results. Here, we compare quantitative results of the cobas® EBV assay to four commercial RT-qPCR assays. METHODS: The cobas EBV, EBV R-Gene, artus EBV RG PCR, RealStar EBV PCR kit 2.0 and Abbott EBV RealTime assays were compared for analytic performance using a 10-fold dilution series of EBV reference material, normalized to the WHO standard. For clinical performance, their quantitative results were compared using anonymized, leftover EBV-DNA-positive EDTA plasma samples. RESULTS: For analytic accuracy, the cobas EBV deviated -0.0097 log10 from target values. The other tests showed deviations between 0.0037 and -0.12 log10. For clinical performance, accuracy and linearity of cobas EBV data from both study sites were excellent. Bland-Altman bias and Deming regression analyses showed statistical correlation for cobas EBV to both EBV R-Gene and Abbott RealTime assays but an offset of cobas EBV to artus EBV RG PCR and RealStar EBV PCR kit 2.0. CONCLUSION: The cobas EBV showed the closest correlation to the reference material, followed closely by EBV R-Gene and Abbott EBV RealTime. Values obtained are stated in IU/mL, facilitating comparison across testing sites and potentially improving utilization of guidelines for diagnosis, monitoring, and treatment of patients.

Insights into the mode of action of the two-peptide lantibiotic lichenicidin.

Lantibiotics are promising candidates to address the worldwide problem of antibiotic resistance. They belong to a class of natural compounds exhibiting strong activity against clinically relevant Gram-positive bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Lichenicidin is a class II two-peptide lantibiotic. The presence of the two mature peptides, Bliα and Bliß, is necessary for full activity against target bacteria. This work aims at clarifying the synergistic activity of both peptides in their interaction with the target membranes. The effect of lichenicidin was tested against S. aureus cells and large unilamellar vesicles. Lichenicidin increases the net surface charge of S. aureus, as shown by zeta-potential measurements, without reaching electroneutralization. In addition, lichenicidin causes cell surface perturbations that culminate in the leakage of its internal contents, as observed by atomic force microscopy. Bliα seems to have low affinity for S. aureus, however, it contributes to increase the affinity of Bliß, because together they present higher affinity than separately. In contrast, Bliα seems to provide an anchoring site for lichenicidin in lipid II-containing membranes. Interestingly, Bliß alone can induce high levels of membrane leakage, but this effect appears to be faster in the presence of Bliα. Based on this information, we propose a mechanism of action of lichenicidin.

Neoplastic and Non-Neoplastic Causes of Acute Intracerebral Hemorrhage on CT : The Diagnostic Value of Perihematomal Edema.

OBJECTIVE: The aim of this study was to investigate the diagnostic value of perihematomal edema (PHE) volume in non-enhanced computed tomography (NECT) to discriminate neoplastic and non-neoplastic causes of acute intracerebral hemorrhage (ICH). METHODS: In this retrospective study, from 560 patients with acute ICH 91 patients fulfilled the inclusion criteria and were classified into neoplastic and non-neoplastic ICH. For each patient, ICH and total hemorrhage volume (ICH + PHE) were segmented semiautomatically. The PHE volume and relative PHE were further calculated and all parameters were compared between the different groups. Additionally, hematoma density was measured and compared between the groups. RESULTS: The PHE volume and relative PHE on NECT were significantly higher in neoplastic vs. the non-neoplastic ICH (p = 0.003 and p < 0.001, respectively). Absolute ICH volume, symptom time onset to CT and ICH localization showed no significant difference between the two groups (p > 0.1). Univariate receiver operating characteristics (ROC) analysis revealed a high diagnostic performance for relative PHE in the discrimination of neoplastic and non-neoplastic ICH with an optimal cut-off of 0.50 (area under the curve, AUC 0.81, 60.0% sensitivity, 91.8% specificity), followed by PHE (AUC 0.69) and hematoma density (AUC 0.68). CONCLUSION: Relative PHE with a cut-off of >0.50 is a specific and simple indicator for neoplastic causes of acute ICH and a potential tool for clinical implementation. This observation needs to be validated in an independent patient cohort.

AML: high serum ferritin at initial diagnosis has a negative impact on long-term survival.

Increased serum ferritin (SF) is common in hematologic malignancies; however, its prognostic role in acute myeloid leukemia (AML) is not clearly established. We examined the impact of baseline SF on long-term survival in 137 intensively treated AML patients. Patients and baseline characteristics were retrieved from an AML database at Charité University Medical Center Berlin, Campus Virchow Clinic. After c-reactive protein (CRP)-based adjustment for inflammation, patients were grouped according to their baseline SF level. Survival analysis was performed accordingly. A significant decline in overall survival and relapse-free survival was observed in patients with high SF as compared to those with low SF. Furthermore, elevated baseline SF remained an independent poor prognostic factor within the multivariate analysis and was associated with a significant higher risk of relapse and non-relapse mortality (NRM). In conclusion, our data show that elevated baseline SF has a negative impact on long-term survival in intensively treated AML patients.

Radiomics of Brain MRI: Utility in Prediction of Metastatic Tumor Type.

Purpose To investigate the feasibility of tumor type prediction with MRI radiomic image features of different brain metastases in a multiclass machine learning approach for patients with unknown primary lesion at the time of diagnosis. Materials and methods This single-center retrospective analysis included radiomic features of 658 brain metastases from T1-weighted contrast material-enhanced, T1-weighted nonenhanced, and fluid-attenuated inversion recovery (FLAIR) images in 189 patients (101 women, 88 men; mean age, 61 years; age range, 32-85 years). Images were acquired over a 9-year period (from September 2007 through December 2016) with different MRI units, reflecting heterogeneous image data. Included metastases originated from breast cancer (n = 143), small cell lung cancer (n = 151), non-small cell lung cancer (n = 225), gastrointestinal cancer (n = 50), and melanoma (n = 89). A total of 1423 quantitative image features and basic clinical data were evaluated by using random forest machine learning algorithms. Validation was performed with model-external fivefold cross validation. Comparative analysis of 10 randomly drawn cross-validation sets verified the stability of the results. The classifier performance was compared with predictions from a respective conventional reading by two radiologists. Results Areas under the receiver operating characteristic curve of the five-class problem ranged between 0.64 (for non-small cell lung cancer) and 0.82 (for melanoma); all P values were less than .01. Prediction performance of the classifier was superior to the radiologists' readings. Highest differences were observed for melanoma, with a 17-percentage-point gain in sensitivity compared with the sensitivity of both readers; P values were less than .02. Conclusion Quantitative features of routine brain MR images used in a machine learning classifier provided high discriminatory accuracy in predicting the tumor type of brain metastases. © RSNA, 2018 Online supplemental material is available for this article.

Inverse Perfusion Requirements of Supra- and Infratentorial Brain Metastases Formation.

Background and Aims: Vascular border zones and the gray-white matter junction are preferred sites for the development of brain metastases (BM), whereas microvascular lesions are known to be a protective factor. In this proof of concept study, we aim to study the relationship of blood perfusion and the spatial distribution of BM. Materials and Methods: An average CT perfusion atlas of 107 healthy patients was created. Voxel-wise reference perfusion values were extracted from BM-negative and BM-positive regions in a second cohort of 100 untreated patients harboring 809 BM confirmed by MRI. A comparison of regional perfusion values was performed using the independent t-test. Results: In contrast to supratentorial BM that develop preferably in areas with lower CBV/CBF and longer MTT/TTP compared to the average regional perfusion (p < 0.001), infratentorial BM showed a higher CBV/CBF and shorter MTT/TTP (p < 0.001). Conclusion: Our results imply differing pathophysiological mechanisms underlying supra- and infratentorial BM spreading. The inverse perfusion patterns may result from differences in vascular supply, hemodynamic requirements, and/or production of pro-angiogenic factors.

Hemodynamic Forces Tune the Arrest, Adhesion, and Extravasation of Circulating Tumor Cells.

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth.

Edema is not a reliable diagnostic sign to exclude small brain metastases.

No prior systematic study on the extent of vasogenic edema (VE) in patients with brain metastases (BM) exists. Here, we aim to determine 1) the general volumetric relationship between BM and VE, 2) a threshold diameter above which a BM shows VE, and 3) the influence of the primary tumor and location of the BM in order to improve diagnostic processes and understanding of edema formation. This single center, retrospective study includes 173 untreated patients with histologically proven BM. Semi-manual segmentation of 1416 BM on contrast-enhanced T1-weighted images and of 865 VE on fluid-attenuated inversion recovery/T2-weighted images was conducted. Statistical analyses were performed using a paired-samples t-test, linear regression/generalized mixed-effects model, and receiver-operating characteristic (ROC) curve controlling for the possible effect of non-uniformly distributed metastases among patients. For BM with non-confluent edema (n = 545), there was a statistically significant positive correlation between the volumes of the BM and the VE (P < 0.001). The optimal threshold for edema formation was a diameter of 9.4 mm for all BM. The primary tumors as interaction term in multivariate analysis had a significant influence on VE formation whereas location had not. Hence VE development is dependent on the volume of the underlying BM and the site of the primary neoplasm, but not from the location of the BM.

Analysis of radiation doses incurred during diagnostic cerebral angiography after the implementation of dose reduction strategies.

BACKGROUND: One goal of increasing awareness of radiation dose is to encourage personal and technical modifications in order to reduce the radiation exposure of patients and staff. OBJECTIVE: To analyze the radiation doses incurred during diagnostic cerebral angiography and the angiographic techniques practiced over a 4-year period, in order to demonstrate the effectiveness of implementing radiation dose reduction strategies. METHODS: A retrospective review of the first 50 consecutive adult and pediatric patients undergoing diagnostic cerebral angiography each year from 2010 to 2013 was performed. Angiograms and procedure examination protocols were reviewed for patient age, gender, diagnosis, angiography techniques, fluoroscopy time, reference point air kerma (Ka,r in mGy), and kerma-area product (PKA in µGym2). RESULTS: From January 2010 to June 2013, a total of 231 diagnostic cerebral angiograms were reviewed (200 adults, 31 children). Adult patients were aged from 19 to 94 years and included 77 men and 123 women. Pediatric patients were aged from 2 to 18 years and comprised 11 boys and 20 girls. Median Ka,r and PKA significantly decreased from 2010 to 2013 in adults (1867 mGy; 21 231 µGym2 vs 653 mGy; 7860 µGym2) and children (644 mGy; 6495 µGym2 vs138 mGy; 1465 µGym2), (p<0.001). CONCLUSIONS: Increased awareness and implementation of dose reduction strategies resulted in decreased radiation doses for diagnostic cerebral angiography both in adult and pediatric patients. The use of lower and variable digital subtraction angiography frame rates and tailored examinations contributed significantly to the reduced radiation doses observed during diagnostic cerebral angiography.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

Flow cytometric maturity score as a novel prognostic parameter in patients with acute myeloid leukemia.

The European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). In order to establish immunophenotypic features that predict prognosis, the expression of single AML blast cell antigens has been evaluated with partly conflicting results; however, the influence of immunophenotypic blast maturity is largely unknown. In our study, 300 AML patients diagnosed at our institution between January 2003 and April 2012 were analyzed. A flow cytometric maturity score was developed in order to distinguish "mature" AML (AML-ma) from "immature" AML (AML-im) by quantitative expression levels of early progenitor cell antigens (CD34, CD117, and TdT). AML-ma showed significantly longer relapse-free survival (RFS) and overall survival (OS) than AML-im (p < 0.001). Interestingly, statistically significant differences in RFS and OS were maintained within the "intermediate-risk" group according to ELN (RFS, 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS, 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome. It remains to be clarified whether these results simply reflect an accumulation of favorable molecular phenotypes in the AML-ma subgroup or whether they rely on biological differences such as a higher proportion of leukemia stem cells and/or a higher degree of genetic instability within the AML-im subgroup.

Magnetic resonance imaging findings in patients presenting with (sub)acute cerebellar ataxia.

INTRODUCTION: Acute or subacute cerebellar inflammation is mainly caused by postinfectious, toxic, neoplastic, vascular, or idiopathic processes and can result in cerebellar ataxia. Previous magnetic resonance (MR) studies in single patients who developed acute or subacute ataxia showed varying imaging features. METHODS: Eighteen patients presenting with acute and subacute onset of ataxia were included in this study. Cases of chronic-progressive/hereditary and noncerebellar causes (ischemia, multiple sclerosis lesions, metastasis, bleedings) were excluded. MR imaging findings were then matched with the clinical history of the patient. RESULTS: An underlying etiology for ataxic symptoms were found in 14/18 patients (postinfectious/infectious, paraneoplastic, autoimmune, drug-induced). In two of five patients without MR imaging findings and three of eight patients with minimal imaging features (cerebellar atrophy, slight signal alterations, and small areas of restricted diffusion), adverse clinical outcomes were documented. Of the five patients with prominent MR findings (cerebellar swelling, contrast enhancement, or broad signal abnormalities), two were lost to follow-up and two showed long-term sequelae. CONCLUSION: No correlation was found between the presence of initial MRI findings in subacute or acute ataxia patients and their long-term clinical outcome. MR imaging was more flagrantly positive in cases due to encephalitis.

Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

HDAC inhibition suppresses bladder cancer cell adhesion to collagen under flow conditions.

The influence of the histone deacetylase (HDAC)-inhibitor, valproic acid (VPA), on bladder cancer cell adhesion in vitro was investigated in this paper. TCCSUP and RT-112 bladder cancer cells were treated with VPA (0.5 or 1 mM) twice or thrice weekly for 14 days. Controls remained untreated. Tumour cell interaction with immobilized collagen was evaluated by a flow-based adhesion assay using a shear force of 2 or 4 dyne/cm(2). The effects of VPA on the integrin adhesion receptors α3, α5, ß1, ß3 and ß4 were assessed by flow cytometry to determine integrin surface expression and by western blotting to determine the cytoplasmic integrin level. VPA of 0.5 mM and 1 mM significantly prevented binding of both RT-112 and TCCSUP cells to collagen, compared with the untreated controls. Adhesion was reduced to a higher extent when RT-112 (subjected to 2 dyne/cm(2)) or TCCSUP (subjected to 2 or 4 dyne/cm(2)) tumour cells were treated with VPA three times a week, compared to the two times a week protocol. VPA caused a significant up-regulation of the integrin α3, α5, ß1, ß3 and ß4 subtypes on the TCCSUP cell surface membrane. In RT-112 cells, only integrin α5 was elevated on the cell surface following VPA exposure. Western blotting revealed an up-regulation of α3, α5, ß3 and ß4 integrins and down-regulation of the integrin ß1 protein by VPA in TCCSUP. VPA also up-regulated α5 and down-regulated ß1 integrin in RT-112 cells, but also reduced α3 and ß3 in TCCSUP. VPA exerted adhesion-blocking properties on bladder cancer cells under physiologic flow conditions. The effects were accompanied by distinct modifications of the integrin expression profile, which differ depending on the cell lines used. Application of VPA might be an innovative option to prevent bladder cancer dissemination.

The nonantibiotic small molecule cyslabdan enhances the potency of ß-lactams against MRSA by inhibiting pentaglycine interpeptide bridge synthesis.

The nonantibiotic small molecule cyslabdan, a labdan-type diterpene produced by Streptomyces sp. K04-0144, markedly potentiated the activity of the ß-lactam drug imipenem against methicillin-resistant Staphylococcus aureus (MRSA). To study the mechanism of action of cyslabdan, the proteins that bind to cyslabdan were investigated in an MRSA lysate, which led to the identification of FemA, which is involved in the synthesis of the pentaglycine interpeptide bridge of the peptidoglycan of MRSA. Furthermore, binding assay of cyslabdan to FemB and FemX with the function similar to FemA revealed that cyslabdan had an affinity for FemB but not FemX. In an enzyme-based assay, cyslabdan inhibited FemA activity, where as did not affected FemX and FemB activities. Nonglycyl and monoglycyl murein monomers were accumulated by cyslabdan in the peptidoglycan of MRSA cell walls. These findings indicated that cyslabdan primarily inhibits FemA, thereby suppressing pentaglycine interpeptide bridge synthesis. This protein is a key factor in the determination of ß-lactam resistance in MRSA, and our findings provide a new strategy for combating MRSA.

Eurocin, a new fungal defensin: structure, lipid binding, and its mode of action.

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/ß-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

Interaction of type A lantibiotics with undecaprenol-bound cell envelope precursors.

Lantibiotics are a unique group within the antimicrobial peptides characterized by the presence of thioether amino acids (lanthionine and methyllanthionine). These peptides are produced by and primarily act on Gram-positive bacteria exerting multiple activities at the cytoplasmic membrane of susceptible strains. Previously, the cell wall precursor lipid II was identified as the molecular target for the prototype lantibiotic nisin. Binding and sequestration of lipid II blocks the incorporation of the central cell wall precursor into the growing peptidoglycan network, thereby inhibiting the formation of a functional cell wall. Additionally, nisin combines this activity with a unique target-mediated pore formation, using lipid II as a docking molecule. The interaction with the pyrophosphate moiety of lipid II is crucial for nisin binding. We show that, besides binding to lipid II, nisin interacts with the lipid intermediates lipid III (undecaprenol-pyrophosphate-N-acetyl-glucosamine) and lipid IV (undecaprenol-pyrophosphate-N-acetyl-glucosamine-N-acetyl-mannosamine) of the wall teichoic acid (WTA) biosynthesis pathway. Binding of nisin to the precursors was observed at a stoichiometry of 2:1. The specific interaction with WTA precursors further promoted target-mediated pore formation in artificial lipid bilayers. Specific interactions with lipid III and lipid IV could also be demonstrated for related type A lantibiotics, for example, gallidermin, containing the conserved lipid-II-binding motif.

Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II.

Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.