High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials

Characterising the production of novel antimicrobials in Streptomyces formicae

Antibiotic resistance poses a major risk to modern medicine, therefore finding new antimicrobial compounds is vital. Most currently used antibiotics originate from actinomycetes discovered more than half a century ago. Previous work from the Hutchings laboratory led to the isolation of a new Streptomyces species named S. formicae from the African fungus-farming plant-ant, Tetraponera penzigi. S. formicae produces novel pentacyclic polyketides, the formicamycins, that have potent antibacterial activity against drug-resistant pathogens including methicillinresistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). During this work, the genes responsible for formicamycin biosynthesis in the native producer were identified and characterised in detail using CRISPR/Cas9-mediated genome editing. In addition, we used cappable RNA- and ChIP-sequencing to determine the transcriptional organisation of the pathway and study the regulatory cascade controlling the production of- and host resistance to- these potent antimicrobials. We exploited this information to generate multiple mutants of S. formicae that overproduce formicamycins as well as various biosynthetic intermediates and shunt metabolites, some of which also have bioactivity. Attempts to understand the evolutionary origins of the biosynthetic pathway and the mode of action of these novel compounds are also presented. Furthermore, the potentiall for novel chemistry from S. formicae is not limited to the formicamycin pathway; antiSMASH analysis shows this talented strain contains at least 45 secondary metabolite biosynthetic gene clusters (BGCs). Under standard laboratory conditions, wild-type S. formicae also exhibits antifungal activity against the drug-resistant Lomentospora prolificans, and when the formicamycin BGC is deleted, the strain produces additional compounds with potent antibacterial activity against MRSA. Overall, this work demonstrates that searching under-explored environments for new species combined with genome editing is a promising route towards finding new anti-infectives

Prospecting Novel Microbiomes for Antibiotic Compounds using Metagenomics and Genome Mining

There has been a void in the discovery and development of new antibiotic classes over the past four decades due, in part, to the traditional bioprospecting pipeline becoming inefficient from high compound rediscovery rates and high costs. The need for new antibiotic classes is urgent as antimicrobial drug resistant infections are now a major public health concern. Strategies such as exploring novel environments, use of next-generation sequencing, and metagenomics may reduce rediscovery rates and costs which could help accelerate lead discovery and encourage greater participation in bioprospecting. Whole genome-sequencing and analysis was used to characterise four bacterial strains (Y1-4) isolated from raw honey that were shown to have antibiotic activity. The isolates were identified as Bacillus and were closely related but distinctive strains with variations amongst their secondary metabolite profiles. All isolates contained a gene cluster homologous to AS- 48, a circular bacteriocin produced by Enterococcus faecalis, which has broad-spectrum antibiotic activity. To date, no example of this bacteriocin has been reported in Bacillus. This work demonstrated the value of whole-microbial genome sequencing for dereplication. A pipeline for the low-cost sequencing and assembly of bacterial genomes using Oxford Nanopore MinION was developed in order to produce contiguous and accurate genome assemblies for taxonomic and bioprospecting analysis. The pipeline developed used a combination of Nanopore draft assembly by Canu and polishing with RACON and Nanopolish, with final polishing with Illumina reads using Pilon. The Nanopore-only assembly of Streptomyces coelicolor A3(2) produced was contiguous and covered 98.9 % of reference. AntiSMASH analysis identified the full secondary metabolite profile of the genome through homology searches. However, indel rates were high (66.82 per 100 kbp) causing fragmented gene annotations which limited secondary metabolite structure prediction. Illumina read polishing reduced indels (2.03 per 100 kbp) and enabled accurate structure prediction from the identified biosynthetic pathways. This demonstrates that Nanopore sequencing can provide a viable dereplication strategy by detection of known biosynthetic pathways. Additionally, supplementation with Illumina sequencing can allow for structure prediction of biosynthetic pathways which could inform chemical extraction strategies for novel pathways. Nanopore sequencing was further utilised to characterise an antibiotic producing isolate (KB16) active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus from the hot spring of the Roman Baths, UK. Genomic analysis showed KB16 to be highly related to Streptomyces canus and to contain 26 putative secondary metabolite gene clusters - some of which were potentially novel. One of the gene clusters was identified as encoding the antibiotic albaflavenone. Attempts to chemically identify the antibiotic produced by KB16 showed that it may produce multiple antimicrobial compounds. These findings demonstrate the value in prospecting underexplored environments such as the Roman Baths for microbially-derived antimicrobial leads. A PCR screen was used to amplify NRPS and PKS gene fragments from a human oral metagenome. Analysis of the fragments suggested that some are from uncharacterised gene clusters. Nanopore shotgun metagenomic sequencing was used to profile the water of the Roman Baths which revealed a diverse microbiome of species with reported metabolic characteristics that are in keeping with the known geochemistry of the waters and aligned with 16S rRNA analysis. Further analysis also identified putative heavy metal resistance genes which can be a co-marker for their metabolism and aligned with the chemical properties of the water. These findings demonstrate the potential value in these sites for bioprospecting whilst also giving insight that can inform bioprospecting strategies. The investigations also highlight the utility of Nanopore sequencing for taxonomic and functional gene profiling of environmental microbiomes. In combination these findings have all contributed information on novel environments, potential isolate leads, and cost-efficient methodologies to accelerate the discovery of microbially-derived antibiotics

Bioactivity and genome guided isolation of a novel antimicrobial protein from Thalassomonas viridans

>Magister Scientiae - MScThe continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment

Genome-guided bioprospecting for novel antibiotic lead compounds.

Antimicrobial resistance continues to pose a threat to health and wellbeing. Unmitigated, it is predicted to be the leading cause of death by 2050. Hence, the sustained development of novel antibiotics is crucial. As over 60% of licensed antibiotics are based on scaffolds derived from less than 1% of all known bacterial species, bacterial secondary metabolites constitute an untapped source of novel antibiotics. The aim of this project therefore was to expand the chemical space of bacteria-derived antibiotic lead compounds, using genomics approach. To that end, a topsoil sample was collected from the rhizosphere in which antibiosis occurs naturally. Using starvation stress, sixty-five isolates were recovered from the sample, out of which four were selected based on morphology and designated A13BB, A23BA, A13AA and A23AA. A13BB was identified by 16S rRNA gene sequence comparison as a Pseudomonas spp. and the other three isolates as Hafnia/Obesumbacterium spp. A database search showed that species belonging to these genera have genomes larger than the 3 Mb size above which an increasing proportion of a bacterial genome is dedicated to secondary metabolism. Given their ecological origin, expected genome size and ability to withstand starvation stress, these four isolates were presumed to harbour antibiotic-encoding gene clusters. Isolates A13BB and A23BA were therefore selected for genome mining in the first instance. Illumina and GridION/MinION sequencing data were obtained for both isolates and assembled into high-quality genomes. Isolates' identities were confirmed by FastANI analysis as strains of P. fragi and H. alvei, with 4.94 and 4.77 Mb genomes, respectively. Assembled genomes were mined with antiSMASH. Amongst other secondary metabolite biosynthetic gene clusters (smBGCs) detected, the β-lactone smBGCs in both genomes were selected for activation as their end products bear the hallmarks of an 'ideal antibiotic' that can inhibit several bacteria-specific enzymes simultaneously. Analysis of these smBGCs revealed genes encoding two core enzymes: 2-isopropylmalate synthase (2-IPMS) and acyl CoA ligase homologues. In the biosynthetic pathway, 2-IPMS catalyses the condensation of acetyl CoA with the degradation product of valine or isoleucine to form 2-IPM. 2-IPM is isomerised to 3-IPM which then forms the β-lactone warhead through reactions catalysed by acyl CoA ligase. It was speculated that the β-lactone compound is biosynthesised to efficiently rid the organism of potentially harmful metabolic intermediates as it grows on poor carbon and nitrogen sources. Strain fermentation was therefore performed with 10.8 mM acetate as the main carbon source, and 5 mM L-valine or L-isoleucine as the nitrogen source. Fermentation extracts were analysed by LC-MS with at least thirty-seven metabolite ions detected. Many of these ions have masses in the range m/z 230-750, which is an ideal mass range for antibiotic molecules. As β-lactone compounds are difficult to identify in crude extracts, especially when utilising single-stage mass spectrometry, reactivity-guided screening of extracts with cysteine thiol probe was performed as the probe forms UV- and MS-visible adducts with β-lactone compounds. However, complete dimerization of probe at a faster-than-expected rate in extract matrices hindered successful screening. This meant that it was not possible to determine if any crude extract components were β-lactone compounds without further analysis. Measures to limit or eliminate probe dimerization are proposed, together with molecular networking strategies that can afford global visualisation and rapid dereplication of extract components, using tandem mass spectrometry fragmentation patterns of parent ions. This project provides an original and robust workflow that serves as a strong starting point in the isolation of novel β-lactone compounds from crude extracts, followed by structural optimisation and bioactivity profiling. The hitherto unrecognised potential of β-lactone natural compounds as 'ideal antibiotics' is highlighted, and several structural optimisation strategies required to harness this potential are proposed. The genomes assembled here, and associated data have been deposited in the repositories of the International Nucleotide Sequence Database Collaboration for repurposing by other researchers. Likewise, the hidden metabolic and biosynthetic potentials of P. fragi and H. alvei species uncovered by RASTtk and antiSMASH analyses have been catalogued and placed in the public domain, with many of these attributes reported for the first time

Natural Product from the Deep Sea

After the long history of screening, it is becoming difficult to find novel compounds from microorganisms and plants anywhere in the world. Until now, more than about 30,000 marine natural products have been reported. However, with the development of marine natural products research, the hit rate of new compounds is also decreasing. Scientists are now turning their attention to the deep sea, where a high hit rate of novel compounds is expected. Many small compounds and peptides from microorganisms and sponges are with therapeutic activity are shown in this book. This Special Issue Book, “Natural Products from the Deep Sea”, should be useful for the screening of novel and useful compounds from nature

Antimicrobial potential of Clostridium and closely related species derived from farm environmental samples : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand

The exploration of antimicrobial compounds from natural sources such as bacteria, has been fast tracked by the development of antimicrobial resistance to existing antimicrobials and the increasing consumer demand for natural food preservatives. So far, antimicrobial discovery has been biased towards aerobic and facultative anaerobic bacteria and fungi. Strict anaerobes such as Clostridium species have not been thoroughly investigated for their antimicrobial potential. The objective of the current study was to evaluate the antimicrobial potential of Clostridium and closely related species against bacteria associated with food spoilage, food safety, and human health. Tests on culture media inoculated with Clostridium and closely related species from farm samples (conditioned media/CMs) showed various degrees of antimicrobial activity. Farm 4 soil conditioned medium (F4SCM) showed potential for further investigation in the search for potent antimicrobials with its promising antimicrobial activity. Bacterial isolates (FS01, FS2.2, FS03, and FS04) belonging to Clostridium and closely related spp. associated with F4SCM showed antimicrobial potential as evident by culture-based and genome-based methods. F4SCM and FS03CM (CM prepared from FS03) metabolomes showed the presence of several putative antimicrobial metabolites. Among them, 2-hydroxyisocaproic acid (HICA) showed antimicrobial activity against a wide range of bacteria associated with food spoilage and safety indicating its potential as a bio-preservative agent in food products. The cell cytoplasmic membrane is a likely target of the HICA’s antimicrobial activity. Overall, this study demonstrates that anaerobic bacterial species, Clostridium, and closely related species can produce antimicrobial metabolites, that have potential applications in food preservation and human health. The knowledge obtained in this study will help future investigations to identify and characterize antimicrobials from these Clostridium and closely related bacteria and expands the understanding of the potential to produce antimicrobial compounds from the genus Clostridium and closely related species

Multi-approach analysis of the metagenome of a marine sponge containing latrunculin A

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017Marine natural products, such as those encountered in sponges, are a prolific source of drug leads. In cancer treatment, several sponges’ secondary metabolites have shown potent cytotoxic activities. One of those is latrunculin A, a macrolide whose biosynthetic production relying on a hybrid pathway involving polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) is still unknown. Considering the amounting evidence that the sponges’ symbionts are the true producers of these compounds, it is reasonable to search for the biosynthetic gene cluster of latrunculin A in the sponges’ metagenome. To achieve this goal, a multi-approach analysis was conducted in marine sponges’ samples, some of them containing latrunculin A. A sponge’s metagenomic DNA library of 3500 clones was produced. Obtaining high molecular weight metagenomic DNA was a critical point which seems to have conditioned successful vector packaging in λ phage particles. Instead, transformation of the vector into the bacterial host was conducted through electroporation and heat-shock. A preliminary PCR screen of the transformants revealed the presence of PKS genes. In parallel, PCR screening of metagenomic DNA of diverse sponge samples was conducted. Due to the hybrid PKS/NRPS origin of latrunculin A, focus was directed towards the presence of PKS and NRPS genes. Several PCR-amplified sequences exhibited homology to the ketosynthase (KS) domain of PKSs. Specific KS primers were designed to latter screening of the produced metagenomic library. Microbiome profiling of three different sponge samples was achieved through 16S rRNA gene sequencing. Even though it was not possible to identify probable latrunculin A’ producers, an analysis of the biosynthetic potential of some of the most abundant symbionts was performed. This demonstrated that further investigation of these sponges for the discovery of novel compounds is promising.Os produtos naturais marinhos, como os encontrados nas esponjas marinhas, são uma prolífica fonte de fármacos. Em particular, no tratamento de cancro, vários metabolitos secundários de esponjas têm mostrado potente atividade citotóxica. Um deles é a latrunculina A, um composto de origem híbrida policetídica e peptídica não ribossomal (PKS/NRPS), cuja via biossintética é ainda desconhecida. Considerando a evidência crescente de que os verdadeiros produtores destes compostos são os simbiontes das esponjas, é lógico procurar o cluster de genes biossintéticos da latrunculina A no metagenoma das esponjas. Para atingir esse objetivo, foi planeada uma abordagem múltipla de análise de amostras de esponjas marinhas, algumas delas contendo latrunculina A. A partir de uma esponja marinha, foi produzida uma biblioteca de ADN metagenómico contendo 3500 clones. A obtenção de ADN metagenómico de alto tamanho molecular foi um ponto crítico que parece ter condicionado o sucesso do empacotamento do vetor no fago λ. Em vez disso, a transformação do vetor no hospedeiro bacteriano foi efetuada por eletroporação e choque térmico. A presença de genes para sintases de polipéptidos na biblioteca foi confirmada por rastreio inicial dos transformantes por PCR. Em paralelo, realizou-se o rastreio por PCR do ADN metagenómico de diversas amostras de esponjas. Devido à origem híbrida PKS/NRPS da latrunculina A, o foco foi direcionado para a presença de genes PKS e NRPS. Diversas sequências amplificadas por PCR demonstraram homologia para com o domínio ketosintase (KS) de sintases de policétidos Primers específicos para KS foram desenhados para posterior uso no rastreio da biblioteca metagenómica produzida. O perfil microbiano de três diferentes amostras de esponjas foi obtido através de sequenciação do gene ARN 16S ribossomal. Apesar de não ter sido possível identificar prováveis produtores de latrunculina A, foi realizada uma análise do potencial biossintético dos simbiontes mais abundantes. Esta demonstrou que parecem promissoras futuras investigações destas esponjas para descoberta de novos compostos

Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy

Filarial nematode parasites are responsible for a number of devastating diseases in humans and animals. These include lymphatic filariasis and onchocerciasis that afflict 150 million people in the tropics and threaten the health of over one billion. The parasites possess intracellular bacteria, Wolbachia, which are needed for worm survival. Clearance of these bacteria with certain antibiotics leads to parasite death. These findings have pioneered the approach of using antibiotics to treat and control filarial infections. In the present study, we have investigated the cell division process in Wolbachia for new drug target discovery. We have identified the essential cell division protein FtsZ, which has a GTPase activity, as an attractive Wolbachia drug target. We describe the molecular characterization and catalytic properties of the enzyme and demonstrate that the GTPase activity is inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. We also found that berberine was effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel antibiotic approach for controlling filarial infection