Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Transcription activation by the resistance protein AlbA as a tool to evaluate derivatives of the antibiotic albicidin.

The rising numbers of fatal infections with resistant pathogens emphasizes the urgent need for new antibiotics. Ideally, new antibiotics should be able to evade or overcome existing resistance mechanisms. The peptide antibiotic albicidin is a highly potent antibacterial compound with a broad activity spectrum but also with several known resistance mechanisms. In order to assess the effectiveness of novel albicidin derivatives in the presence of the binding protein and transcription regulator AlbA, a resistance mechanism against albicidin identified in Klebsiella oxytoca, we designed a transcription reporter assay. In addition, by screening shorter albicidin fragments, as well as various DNA-binders and gyrase poisons, we were able to gain insights into the AlbA target spectrum. We analysed the effect of mutations in the binding domain of AlbA on albicidin sequestration and transcription activation, and found that the signal transduction mechanism is complex but can be evaded. Further demonstrating AlbA's high level of specificity, we find clues for the logical design of molecules capable of avoiding the resistance mechanism.

Acetylenic Replacement of Albicidin's Methacrylamide Residue Circumvents Detrimental E/Z Photoisomerization and Preserves Antibacterial Activity.

The natural product albicidin is a highly potent inhibitor of bacterial DNA gyrase. Its outstanding activity, particularly against Gram-negative pathogens, qualifies it as a promising lead structure in the search for new antibacterial drugs. However, as we show here, the N-terminal cinnamoyl moiety of albicidin is susceptible to photochemical E/Z isomerization. Moreover, the newly formed Z isomer exhibits significantly reduced antibacterial activity, which hampers the development and biological evaluation of albicidin and potent derivatives thereof. Hence, we synthesized 13 different variants of albicidin in which the vulnerable para-coumaric acid moiety was replaced; this yielded photostable analogues. Biological activity assays revealed that diaryl alkyne analogues exhibited virtually undiminished antibacterial efficacy. This promising scaffold will therefore serve as a blueprint for the design of a potent albicidin-based drug.

Extensive Structure-Activity Relationship Study of Albicidin's C-Terminal Dipeptidic p-Aminobenzoic Acid Moiety.

Albicidin is a recently described natural product that strongly inhibits bacterial DNA gyrase. The pronounced activity, particularly against Gram-negative bacteria, turns it into a promising lead structure for an antibacterial drug. Hence, structure-activity relationship studies are key for the in-depth understanding of structural features/moieties affecting gyrase inhibition, antibacterial activity and overcoming resistance. The 27 newly synthesized albicidins give profound insights into possibilities for variations of the C-terminus. Furthermore, in the present study, a novel derivative has been identified as overcoming resistance posed by the Klebsiella-protease AlbD. Structural modifications include, for example, azahistidine replacing the previous instable cyanoalanine as the central amino acid, as well as a triazole amide bond isostere between building blocks D and E.

Molecular insights into antibiotic resistance - how a binding protein traps albicidin.

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs.

Correction to: Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

[This corrects the article DOI: 10.1186/s40694-018-0048-3.].

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

BACKGROUND: Fungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus Aspergillus niger was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in A. niger, and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives. RESULTS: The beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350-600 mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective A. niger expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products. CONCLUSION: In this study, we show that the product portfolio of A. niger can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of A. niger to produce a range of different peptide natural products in titres high enough to become industrially relevant.

Total Synthesis and Biological Assessment of Novel Albicidins Discovered by Mass Spectrometric Networking.

Natural products represent an important source of potential novel antimicrobial drug leads. Low production by microorganisms in cell culture often delays the structural elucidation or even prevents a timely discovery. Starting from the anti-Gram-negative antibacterial compound albicidin produced by Xanthomonas albilineans, we describe a bioactivity-guided approach combined with non-targeted tandem mass spectrometry and spectral (molecular) networking for the discovery of novel antimicrobial compounds. We report eight new natural albicidin derivatives, four of which bear a ß-methoxy cyanoalanine or ß-methoxy asparagine as the central α-amino acid. We present the total synthesis of these albicidins, which facilitated the unambiguous determination of the (2 S,3 R)-stereoconfiguration which is complemented by the assessment of the stereochemistry on antibacterial activity.

Synthesis of Albicidin Derivatives: Assessing the Role of N-terminal Acylation on the Antibacterial Activity.

The peptide antibiotic albicidin, which is synthesized by the plant pathogenic bacterium, Xanthomonas albilineans, represents the most prominent member of a new class of antibacterial gyrase inhibitors. It shows remarkable antibacterial activities against Gram-positive and Gram-negative microorganisms. Its unique structure potentially represents a new lead structure for the development of an antibacterial drug. Here we report the synthesis of 14 albicidin derivatives with structural variations at the N-terminus, primarily investigating the effects of variation of cinnamoyl, phenylpropanoyl, and benzoyl residues. Gyrase inhibition in vitro and determination of minimal inhibitory concentrations were assessed in parallel. Activities in a nanomolar range and the importance of N-acylation were demonstrated.

Synthesis and Antimicrobial Activity of Albicidin Derivatives with Variations of the Central Cyanoalanine Building Block.

To investigate the pharmacophore regions of the antibiotic albicidin, derivatives with variations on the central amino acid were synthesized. Charged as well as uncharged residues were chosen to explore the influence of charge, chirality, and steric bulk. The bioactivity of the newly synthesized derivatives was determined by a microdilution technique to obtain minimum inhibitory concentrations (MIC) values. The compounds were also tested in a cell-free system to obtain information about their ability to inhibit their primary target, DNA gyrase. It was then shown that derivatives with uncharged side chains retain antibacterial activity, whereas incorporation of charged amino acid residues decreases the antibacterial activity dramatically, possibly due to restricted cell penetration of these derivatives. From the newly synthesized derivatives, the threonine derivative shows the most promising results in both tests. The information will help to develop the features of albicidin toward more drug-like structures.

Targeting osteomyelitis with complete [99mTc]besilesomab and fragmented [99mTc]sulesomab antibodies: kinetic evaluations.

BACKGROUND: The aim of this retrospective study was to compare the targeting of "pure" osteomyelitis (i.e., without surrounding soft tissue infection) by directly 99mTc-labelled complete immunoglobulin G (IgG) monoclonal antibody (MAb) ([99mTc]besilesomab) and by directly 99mTc-labelled fragment antigen-binding (FAb) MAb ([99mTc]sulesomab) in relation to their kinetic fate. A total of 73 patients with "pure" osteomyelitis were examined with [99mTc]besilesomab, (Scintimun®, IBA/CIS bio international, Saclay, France; N.=38) and [99mTc]sulesomab (LeukoScan®, Immunomedics Inc., Morris Plains, NJ, USA; N.=35). METHODS: Kinetic data were deduced from whole-body and single-photon emission computed tomographic scans, performed 10 minutes to 24 hour p.i. (region-of-interest technique [ROI]). RESULTS: In targeting "pure" osteomyelitis, sensitivities at 1-4 hours were found to be higher for [99mTc]sulesomab (44% and 80% for [99mTc]besilesomab and [99mTc]sulesomab, respectively) but at significantly lower target/background (T/B) ratios than with [99mTc]besilesomab (1.8±0.3 versus 1.4±0.5 for [99mTc]besilesomab and [99mTc]sulesomab respectively; P<0.01). With [99mTc]besilesomab, there was a continuous osteomyelitis uptake over 24 hours, whereas with [99mTc]sulesomab, the maximal uptake occurred mostly within 1-4 hours, with subsequent clearance being slower for antigen-bound activity than for nonspecific background. Hence, diagnosis was possible mostly after 4h with [99mTc]sulesomab but often not before 24 hours with [99mTc]besilesomab, the later increasing significantly (P<0.01) in sensitivity (87% and 84% for [99mTc]besilesomab and [99mTc]sulesomab, respectively). CONCLUSIONS: These results show that the higher sensitivity of [99mTc]sulesomab in osteomyelitis targeting at earlier p.i. times does not rely on an increased antibody uptake but on a more rapid clearance of nonspecific background activity due to faster metabolism and excretion. Intact [99mTc]besilesomab show a slow, continuous uptake, leading to higher T/B at later p.i. times, often beyond the imaging possibilities of 99mTc.

The Albicidin Resistance Factor AlbD Is a Serine Endopeptidase That Hydrolyzes Unusual Oligoaromatic-Type Peptides.

The para-aminobenzoic acid-containing peptide albicidin is a pathogenicity factor synthesized by Xanthomonas albilineans in infections of sugar cane. Albicidin is a nanomolar inhibitor of the bacterial DNA gyrase with a strong activity against various Gram-negative bacteria. The bacterium Pantoea dispersa expresses the hydrolase AlbD, conferring natural resistance against albicidin. We show that AlbD is a novel type of endopeptidase that catalyzes the cleavage of albicidin at a peptide backbone amide bond, thus abolishing its antimicrobial activity. Additionally, we determined the minimal cleavage motif of AlbD with substrates derived by chemical synthesis. Our results clearly identify AlbD as a unique endopeptidase that is the first member of a new subfamily of peptidases. Our findings provide the molecular basis for a natural detoxification mechanism, potentially rendering a new tool in biological chemistry approaches.

Total synthesis of albicidin: a lead structure from Xanthomonas albilineans for potent antibacterial gyrase inhibitors.

The peptide antibiotic albicidin, which is synthesized by the plant pathogenic bacterium Xanthomonas albilineans, displays remarkable antibacterial activity against various Gram-positive and Gram-negative microorganisms. The low amounts of albicidin obtainable from the producing organism or through heterologous expression are limiting factors in providing sufficient material for bioactivity profiling and structure-activity studies. Therefore, we developed a convergent total synthesis route toward albicidin. The unexpectedly difficult formation of amide bonds between the aromatic amino acids was achieved through a triphosgene-mediated coupling strategy. The herein presented synthesis of albicidin confirms the previously determined chemical structure and underlines the extraordinary antibacterial activity of this compound. The synthetic protocol will provide multigram amounts of albicidin for further profiling of its drug properties.

On the influence of surfactants on the adsorption of polysaccharide-based polymers on cotton studied by means of fluorescence spectroscopy.

In this study, we examined the influence of surfactants on the adsorption of polymers on cotton fibers. The extent of polymer adsorption on cotton was determined directly by means of fluorescence spectroscopy using fluorescently labeled polymers. The investigation of polymer adsorption in the presence of different types of surfactants and for a large range of differently structured polymers allows us to obtain a rather general picture of this important issue. Systematic relationships between the presence of surfactant and the type of polymer can be deduced but cannot be cast in simple terms such as electrostatic interaction but instead depend on the detailed interaction between the surfactant and polymer both in solution and adsorbed on the cotton surface. A particularly complex situation arises for the case of oppositely charged surfactant and polymer because of the possibility of precipitate formation. The study of such complex systems not only is of scientific interest but also is of great commercial interest because both polymers and surfactants are parts of detergent formulations and cotton is one of the most abundantly used materials for fabrics.

Unexpected 99mTc-tetrofosmin findings during myocardial perfusion scintigraphy: intraindividual comparison with PET/computed tomography.

OBJECTIVE: 99mTc-tetrofosmin single photon emission computed tomography (SPECT) is routinely used in the evaluation of coronary artery disease. A variety of different tumors, however, also demonstrate 99mTc-tetrofosmin uptake. We report six patients found with unexpected mediastinal and thoracic tumor uptake during Tc-tetrofosmin myocardial perfusion scintigraphy (MPS). MATERIALS AND METHODS: We investigated 2,155 patients with Tc-tetrofosmin MPS during 2006-2007. One thousand four hundred and eighty-six of these patients had no coronary history and were sent to our department due to newly developed thoracic complaint such as chest pain, dyspnea and others. Six hundred and sixty-nine patients had coronary history. All patients underwent 99mTc-tetrofosmin exercise study. Patients with unexpected extracardiac Tc-tetrofosmin findings during MPS were referred to PET/CT for further diagnostic investigation. Region of interest (ROI; 99mTc-tetrofosmin) and SUVmax (2-[F]fluoro-2-deoxy-D-glucose, F-FDG) were estimated and the results were compared with histological findings. RESULTS: Abnormal mediastinal and/or thoracic activities were visualized in six of the 2,155 patients with 99mTc-tetrofosmin images. Subsequently, the patients underwent resection of a thymoma (n=2), nonsmall cell lung cancer (n=1) and breast cancer (n=3). In the patients with breast cancer one was a male patient with ductal, invasive breast cancer. Benign thymomas showed high 99mTc-tetrofosmin ROI >4.0 and low F-FDG SUVmax <2.0, whereas low 99mTc-tetrofosmin ROI <2.0 were found in nonsmall cell lung cancer and breast cancer and high F-FDG SUVmax >2.5 in these malignant tumors. CONCLUSION: During Tc-tetrofosmin SPECT exercise stress tests performed in patients with suspected coronary artery disease, much more attention must be given to unexpected extracardiac uptakes. With 99mTc-tetrofosmin a large variety of different unknown tumors can be detected during MPS.

18F-FDG PET, somatostatin receptor scintigraphy, and CT in metastatic medullary thyroid carcinoma: a clinical study and an analysis of the literature.

AIM: To determine the clinical potential of 2-[F]fluoro-2-deoxy-D-glucose positron emission tomography (F-FDG PET) in patients with medullary thyroid carcinoma (MTC), we compared it to computed tomography (CT), and somatostatin receptor scintigraphy (SRS). PATIENTS AND METHODS: Blinded evaluation of PET, CT and SRS images obtained from 26 patients with histologically proven metastatic MTC was done by nuclear medicine and radiology specialists. Sites of tumour involvement were classified as "sure" or "suspicious". The data were analysed in comparison to two different standards. Either those sites classified as "sure" by at least one of the methods were defined as the standard or those sites of involvement which were classified as "sure" by at least two methods. RESULTS: Dependent on the type of data analysis performed, PET was able to demonstrate 56.8%/80.6% of the tumour sites, CT showed 64.5%/79.6%, and SRS showed 47.5%/69.9% of the tumour sites. CONCLUSION: Overall, CT is similar or better than PET in our patients (dependent on the standard) while SRS is inferior to both other techniques. Our data are in agreement with publications that consider CT superior to PET in the diagnosis of metastatic MTC while other studies show superiority of PET. However, a combination of CT and PET seems to be the most appropriate non-invasive diagnostic approach in patients with MTC.

99mTc-HMPAO-labeled autologous versus heterologous leukocytes for imaging infection.

UNLABELLED: Radiolabeled autologous leukocytes are the gold standard for imaging infectious foci in patients. Good results have also been reported for radiolabeled heterologous leukocytes from noninfected donors. Until now, the 2 methods have not been directly compared. In this study, we compared the infection-imaging potential of 99mTc-hexamethylpropyleneamine oxime (HMPAO)-labeled autologous granulocytes with that of 99mTc-HMPAO-labeled granulocytes from either infected or noninfected donors in rabbits with Escherichia coli infection. METHODS: The radiolabeled granulocyte preparations were studied in rabbits with an E. coli infection in the left calf muscle. The soft-tissue infections were scintigraphically visualized after injection of 18 MBq of either 99mTc-HMPAO purified autologous granulocytes or radiolabeled purified heterologous granulocytes from infected or noninfected donor rabbits. Gamma camera images were acquired at 2 min and at 1, 2, and 4 h after injection. After the last image, the rabbits were killed and uptake of the radiolabel in the dissected tissues was determined. RESULTS: The 99mTc-HMPAO autologous granulocytes and heterologous granulocytes from infected donors accurately revealed the infectious focus in the calf muscle at 2 h after injection. At 4 h after injection, a significantly better (P < 0.05) delineation of the infection was established with the 99mTc-HMPAO autologous granulocytes and 99mTc-HMPAO heterologous granulocytes from the infected rabbits than with the heterologous granulocytes from noninfected donors. With both cell preparations, the intensity of uptake in the infected calf muscle continuously increased until 4 h after injection. The 99mTc-HMPAO heterologous granulocytes from noninfected donors showed no significant increase in contrast after 2 h after injection. Absolute uptake in the infected calf muscle was much higher for 99mTc-HMPAO autologous granulocytes (7.81 +/- 1.21 percentage injected dose [%ID]) and 99mTc-HMPAO heterologous infected granulocytes (8.91 +/- 1.92 %ID) than for the radiolabeled heterologous noninfected granulocytes (2.32 +/- 0.75 %ID) (P < 0.04) at 4 h after injection. The ratio of infected muscle to noninfected contralateral muscle was significantly higher for 99mTc-HMPAO autologous granulocytes and 99mTc-HMPAO heterologous granulocytes from infected donors than for 99mTc-HMPAO heterologous granulocytes from noninfected donors (5.53 +/- 1.09, 3.86 +/- 0.75, and 1.86 +/- 0.31, respectively; P < 0.05). CONCLUSION: For nuclear medicine imaging of infection, purified granulocytes derived from infected rabbits are superior to purified granulocytes derived from noninfected donor rabbits. In addition, autologous granulocytes gave similar results to heterologous granulocytes from infected donor rabbits, suggesting the need for intrinsic cell activation for specific granulocyte migration.

High-dose myeloablative radioimmunotherapy of mantle cell non-Hodgkin lymphoma with the iodine-131-labeled chimeric anti-CD20 antibody C2B8 and autologous stem cell support. Results of a pilot study.

BACKGROUND: CD20 has been used successfully as a target molecule for conventional low-dose, as well as high-dose, myeloablative radioimmunotherapy (RIT) of B-cell non-Hodgkin lymphoma (NHL). Mantle cell lymphoma (MCL) is an especially aggressive, prognostically unfavorable subtype of B-cell NHL, associated with an overall 5-year survival rate of less than 20%. Recent evidence has failed to show convincing therapeutic efficacy of conventional, nonmyeloablative RIT in patients with MCL. The aim of this pilot study was to investigate whether high-dose, myeloablative RIT with the iodine-131 ((131)I)-labeled chimeric anti-CD20 antibody C2B8 (rituximab, obtained as Mabthera from Roche Pharma, Reinach, Switzerland) is therapeutically effective in MCL patients. METHODS: A total of seven patients with chemorefractory or relapsed MCL were studied in this pilot trial. All had relapsed after high-dose chemotherapy with autologous stem cell transplantation (four of them combined with 12 grays (Gy) total-body irradiation). A diagnostic-dosimetric study was performed with approximately 10 mCi of (131)I-labeled C2B8 at a protein dose of 2.5 mg per kg body weight, in order to assess its biodistribution and dosimetry. If splenic pooling was observed, as is typically the case in patients with splenomegaly, the protein dose was doubled in additional studies until a "favorable" biodistribution was obtained. Therapy was performed with myeloablative doses of 261-495 mCi of (131)I-labeled C2B8 at the previously optimized protein dose, aiming at lung doses less-than-or-equal 27 Gy. Homologous stem cell support was provided. Clinical follow-up was obtained at 3-month intervals for up to 38 months (median observation time, 25 months). Overall, in six patients the 2.5 mg/kg protein dose was used, whereas in one patient with splenomegaly, 10 mg/kg was necessary to overcome the splenic antigenic sink. RESULTS: Blood cell nadirs were reached at 2-3 weeks after therapy infusion, but all patients reengrafted at 7-10 days after stem cell reinfusion. Nonhematologic toxicity was restricted to mild-to-moderate nausea, fever, transient bilirubin, or liver enzyme elevations. One patient with preexisting alcoholic cirrhosis experienced a deterioration of liver function. Despite thyroid blocking, 5 of 7 patients developed hypothyroidism, requiring thyroxine substitution at 6-18 months after RIT. Six patients experienced a complete and one a good partial remission. Five patients were still in CR at the time this article was written, and six are still alive for more than 3 years; one patient relapsed locally at 3 months and one systemically at 26 months after RIT. CONCLUSIONS: High-dose myeloablative radioimmunotherapy with (131)I-labeled anti-CD20 antibodies seems to be associated with a high response rate and moderate toxicity in patients with MCL. Further follow-up to monitor the long-term outcome as well as systematic prospective clinical studies are indicated.