Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides.

Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents.

Modular Fragment Synthesis and Bioinformatic Analysis Propose a Revised Vancoresmycin Stereoconfiguration.

Elaborate fragments of the proposed stereostructure of the complex polyketide antibiotic vancoresmycin have been synthesized in a stereoselective fashion based on a modular and convergent approach. Significant nuclear magnetic resonance differences in one of these subunits compared with the natural product question the proposed stereoconfiguration. Consequently, an extensive bioinformatics analysis of the biosynthetic gene cluster was carried out, leading to a revised stereoconfigurational proposal for this highly potent antibiotic.

The genus Micromonospora as a model microorganism for bioactive natural product discovery.

This review covers the development of the genus Micromonospora as a model for natural product research and the timeline of discovery progress from the classical bioassay-guided approaches through the application of genome mining and genetic engineering techniques that target specific products. It focuses on the reported chemical structures along with their biological activities and the synthetic and biosynthetic studies they have inspired. This survey summarizes the extraordinary biosynthetic diversity that can emerge from a widely distributed actinomycete genus and supports future efforts to explore under-explored species in the search for novel natural products.

Applied evolution: phylogeny-based approaches in natural products research.

Covering: up to 2019Phylogenetic methods become increasingly important in natural product research. The growing amount of genetic data available today is enabling us to infer the evolutionary history of secondary metabolite gene clusters and their encoded compounds. We are starting to understand patterns and mechanisms of how the enormous diversity of chemical compounds produced by nature has evolved and are able to use phylogenetic inference to facilitate functional predictions of involved enzymes. In this review, we highlight how phylogenetic methods can aid natural product discovery and predictions and demonstrate several examples how these have been used in the past. We are featuring a number of easy to use tools that aid tree building and analysis and are providing a short overview how to create and interpret a phylogenetic tree.

The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor.

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.

Recovery of the Peptidoglycan Turnover Product Released by the Autolysin Atl in Staphylococcus aureus Involves the Phosphotransferase System Transporter MurP and the Novel 6-phospho-N-acetylmuramidase MupG.

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-ß-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-ß-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.

Comparative genomics reveals phylogenetic distribution patterns of secondary metabolites in Amycolatopsis species.

BACKGROUND: Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. RESULTS: Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis' strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. CONCLUSIONS: Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds.

The Antibiotic Resistant Target Seeker (ARTS), an exploration engine for antibiotic cluster prioritization and novel drug target discovery.

With the rise of multi-drug resistant pathogens and the decline in number of potential new antibiotics in development there is a fervent need to reinvigorate the natural products discovery pipeline. Most antibiotics are derived from secondary metabolites produced by microorganisms and plants. To avoid suicide, an antibiotic producer harbors resistance genes often found within the same biosynthetic gene cluster (BGC) responsible for manufacturing the antibiotic. Existing mining tools are excellent at detecting BGCs or resistant genes in general, but provide little help in prioritizing and identifying gene clusters for compounds active against specific and novel targets. Here we introduce the 'Antibiotic Resistant Target Seeker' (ARTS) available at https://arts.ziemertlab.com. ARTS allows for specific and efficient genome mining for antibiotics with interesting and novel targets. The aim of this web server is to automate the screening of large amounts of sequence data and to focus on the most promising strains that produce antibiotics with new modes of action. ARTS integrates target directed genome mining methods, antibiotic gene cluster predictions and 'essential gene screening' to provide an interactive page for rapid identification of known and putative targets in BGCs.

Mining Bacterial Genomes for Secondary Metabolite Gene Clusters.

With the emergence of bacterial resistance against frequently used antibiotics, novel antibacterial compounds are urgently needed. Traditional bioactivity-guided drug discovery strategies involve laborious screening efforts and display high rediscovery rates. With the progress in next generation sequencing methods and the knowledge that the majority of antibiotics in clinical use are produced as secondary metabolites by bacteria, mining bacterial genomes for secondary metabolites with antimicrobial activity is a promising approach, which can guide a more time and cost-effective identification of novel compounds. However, what sounds easy to accomplish, comes with several challenges. To date, several tools for the prediction of secondary metabolite gene clusters are available, some of which are based on the detection of signature genes, while others are searching for specific patterns in gene content or regulation.Apart from the mere identification of gene clusters, several other factors such as determining cluster boundaries and assessing the novelty of the detected cluster are important. For this purpose, comparison of the predicted secondary metabolite genes with different cluster and compound databases is necessary. Furthermore, it is advisable to classify detected clusters into gene cluster families. So far, there is no standardized procedure for genome mining; however, different approaches to overcome all of these challenges exist and are addressed in this chapter. We give practical guidance on the workflow for secondary metabolite gene cluster identification, which includes the determination of gene cluster boundaries, addresses problems occurring with the use of draft genomes, and gives an outlook on the different methods for gene cluster classification. Based on comprehensible examples a protocol is set, which should enable the readers to mine their own genome data for interesting secondary metabolites.

A fast and simple method for detecting and quantifying donor-derived cell-free DNA in sera of solid organ transplant recipients as a biomarker for graft function.

BACKGROUND: Timely detection of graft rejection is an important issue in the follow-up care after solid organ transplantation. Until now, biopsy has been considered the "gold standard" in the diagnosis of graft rejection. However, non-invasive tests such as monitoring the levels of cell-free DNA (cfDNA) as a sensitive biomarker for graft integrity have attracted increasing interest. The rationale of this approach is that a rejected organ will lead to a significant release of donor-derived cfDNA, which can be detected in the serum of the transplant recipient. METHODS: We have developed a novel quantitative real-time PCR (qPCR) approach for detecting an increase of donor-derived cfDNA in the recipient's serum. Common insertion/deletion (InDel) genetic polymorphisms, which differ between donor and recipient, are targeted in our qPCR assay. In contrast to some other strategies, no specific donor/recipient constellations such as certain gender combinations or human leukocyte antigen (HLA) discrepancies are required for the application of our test. RESULTS: The method was first validated with serial dilutions of serum mixtures obtained from healthy blood donors and then used to determine donor-derived cfDNA levels in patients' sera within the first 3 days after their kidney transplantation had been performed. CONCLUSIONS: Our method represents a universally applicable, simple and cost-effective tool which can potentially be used to detect graft dysfunction in transplant recipients.

IFNγ+ Treg in-vivo and in-vitro represent both activated nTreg and peripherally induced aTreg and remain phenotypically stable in-vitro after removal of the stimulus.

BACKGROUND: IFNγ-producing CD4+CD25+Foxp3+CD127- Treg represent the first line of Treg during an immune response. In the present study we determined whether IFNγ+ Treg in-vivo and in-vitro are Helios-positive representing activated natural (nTreg) or Helios-negative representing adaptive Treg (aTreg) and whether they originate from CD4+CD25+ and/or CD4+CD25- PBL. Furtheron, we investigated whether they are inducible by recombinant IFNγ (rIFNγ) as a single stimulus, decrease in-vitro after elimination of the stimulus, and have a demethylated Foxp3 Treg-specific demethylated region (TSDR) which is associated with stable Foxp3 expression. METHOD: Subsets of IFNγ+ Treg were determined in peripheral blood of healthy controls using eight-color flow cytometry and were further investigated in-vitro. Foxp3 TSDR methylation status was determined using bisulphite polymerase chain reaction (PCR) and high resolution melt (HRM) analysis. RESULTS: Nearly all Treg in the peripheral blood were Helios+IFNγ- (1.9 ± 1.1/µl) and only few were Helios+IFNγ+ or Helios-IFNγ+ Treg (both 0.1 ± 0.1/µl). Enriched IFNγ+ Treg subsets showed in part strong Foxp3 TSDR demethylation. In-vitro, rIFNγ was unable to induce Treg. CD4+CD25+ enriched PBL stimulated with PMA/Ionomycin in the presence of rIFNγ were rather resistant to the effect of rIFNγ, in contrast to CD4+CD25- enriched PBL which showed increasing total Treg with Helios+ Treg switching from IFNγ- to IFNγ+ and increasing Helios-IFNγ+ Treg. The data indicate that rIFNγ, in combination with a polyclonal stimulus, activates nTreg and induces aTreg. When phorbol 12-myristate 13-acetate (PMA)/Ionomycin was washed out from the cell culture after 6 h stimulation, Treg induction continued for at least 96 h of cell culture, contradicting the hypothesis that removal of the stimulus results in significant decrease of IFNγ- and IFNγ+ CD4+CD25+Foxp3+CD127- Treg due to loss of Foxp3 expression. CONCLUSIONS: IFNγ+Helios- aTreg as well as IFNγ+Helios+ nTreg are detectable in the blood of healthy individuals, show in part strong Foxp3 TSDR demethylation and are inducible in-vitro. The present data provide further insight concerning the in-vivo and in-vitro characteristics of IFNγ+ Treg and help to understand their role in immunoregulation. Alloantigen-specific demethylated IFNγ+Helios+ nTreg might represent a suitable marker for monitoring graft-specific immunosuppression in renal transplant recipients.

Seven novel HLA alleles reflect different mechanisms involved in the evolution of HLA diversity: description of the new alleles and review of the literature.

The human leukocyte antigen (HLA) loci are among the most polymorphic genes in the human genome. The diversity of these genes is thought to be generated by different mechanisms including point mutation, gene conversion and crossing-over. During routine HLA typing, we discovered seven novel HLA alleles which were probably generated by different evolutionary mechanisms. HLA-B*41:21, HLA-DQB1*02:10 and HLA-DQA1*01:12 likely emerged from the common alleles of their groups by point mutations, all of which caused non-synonymous amino acid substitutions. In contrast, a deletion of one nucleotide leading to a frame shift with subsequent generation of a stop codon is responsible for the appearance of a null allele, HLA-A*01:123N. Whereas HLA-B*35:231 and HLA-B*53:31 were probably products of intralocus gene conversion between HLA-B alleles, HLA-C*07:294 presumably evolved by interlocus gene conversion between an HLA-C and an HLA-B allele. Our analysis of these novel alleles illustrates the different mechanisms which may have contributed to the evolution of HLA polymorphism.

Virulence genes in clinical and environmental Stenotrophomas maltophilia isolates: a genome sequencing and gene expression approach.

The rate of nosocomial infections with the opportunistic pathogen Stenotrophomonas maltophilia has remarkably increased in the last decade. To determine S. maltophilia virulence genes, the complete genome sequences of two S. maltophilia isolates were compared. The clinical strain SKK35 was proved virulent in an amoeba host-pathogen model, and wastewater strain RA8 was determined as non-virulent in the amoeba model. The genome sequences of three additional S. maltophilia strains, K279a (clinical, non-virulent against amoeba), R511-3 and SKA14 (both environmental, non-virulent against amoeba) were taken into account as reference strains. We were able to show that all clinical and environmental S. maltophilia strains presented comparable distribution of so far identified potential virulence genes, regardless to their virulence potential against amoebae. Aside from that, strain SKK35 was found harboring a putative, strain specific pathogenicity island, encoding two proteins from the RTX (repeats-in-toxin) family. The actual expression of the RTX genes was verified in growth experiments in different culture media containing blood or blood components and in co-cultures with amoeba.

Genotyping of environmental and clinical Stenotrophomonas maltophilia isolates and their pathogenic potential.

Stenotrophomonas maltophilia is a highly versatile species with useful biotechnological potential but also with pathogenic properties. In light of possible differences in virulence characteristics, knowledge about genomic subgroups is therefore desirable. Two different genotyping methods, rep-PCR fingerprinting and partial gyrB gene sequencing were used to elucidate S. maltophilia intraspecies diversity. Rep-PCR fingerprinting revealed the presence of 12 large subgroups, while gyrB gene sequencing distinguished 10 subgroups. For 8 of them, the same strain composition was shown with both typing methods. A subset of 59 isolates representative for the gyrB groups was further investigated with regards to their pathogenic properties in a virulence model using Dictyostelium discoideum and Acanthamoeba castellanii as host organisms. A clear tendency towards accumulation of virulent strains could be observed for one group with A. castellanii and for two groups with D. discoideum. Several virulent strains did not cluster in any of the genetic groups, while other groups displayed no virulence properties at all. The amoeba pathogenicity model proved suitable in showing differences in S. maltophilia virulence. However, the model is still not sufficient to completely elucidate virulence as critical for a human host, since several strains involved in human infections did not show any virulence against amoeba.