Prospecting Novel Microbiomes for Antibiotic Compounds Using Metagenomics and Genome Mining
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UCL SCHOOL OF PHARMACY BRUNSWICK SQUARE Prospecting Novel Microbiomes for Antibiotic Compounds using Metagenomics and Genome Mining Tim Ali Charles Walker Thesis submitted in fulfilment of the requirements of the UCL degree of Doctor of Philosophy 2020 I, Tim Walker, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: --------------------- 2 Abstract 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 3 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. 4 Impact Statement Discovery of new antibiotic classes has stalled over the past four decades. This has been caused in part by the ‘traditional’ model of bioprospecting becoming less economical from high rediscovery rates and resource costs. During this time, antimicrobial resistant infections have continued to proliferate and are a major global public health concern. Leading to warnings from public health authorities of the risk of a ‘post-antibiotic age’ where current antibiotics have lost all effectiveness. The potential costs of such a situation to the global economy, public health, and quality of life are estimated to be high. Therefore, the need for the development of new antibiotic classes with novel modes of action is urgent. To fill the discovery void more novel antibiotic lead compounds need to be discovered. By utilising new technologies and approaches that may mitigate the issues that led to the decline in bioprospecting then more research groups and industry may begin searching again. The work in this thesis has detailed attempts to discover novel bacterial isolates producing antibiotic compounds and develop strategies to improve the bioprospecting pathway using Oxford Nanopore long-read sequencing and genome-mining for dereplication. To this end, five bacterial isolates (four from honey and one from the hot springs of the Roman Baths) with antibiotic activity were characterised. The analysis identified aspects of the isolates phylogenetics and secondary metabolism profiles. Which revealed that the isolates may have biosynthetic gene clusters for potentially novel compounds. The impact of this is that it forms the basis for further isolation of these compounds. A pipeline for the whole-genome sequencing and annotation of isolates using Oxford Nanopore technology was developed and validated in the analysis of a novel isolate from the Roman Baths, UK. This pipeline can allow researchers to produce accurate genome assembles to dereplicate and prioritise lead isolates quickly and efficiently, and thus make bioprospecting an economical and viable strategy for more researchers to perform. The results of the analysis revealed the discovery of known antibiotic compounds and highlighted how taxonomy alone is not a marker of expected secondary metabolite potential. Genome-mining also revealed the presence of gene pathways for AS-48 bacteriocin in the Bacillus isolates from honey. This is the first report of this compound in this genus and further highlights the disconnect between taxonomy and biosynthetic potential. This is an important finding for bioprospectors to ensure that their dereplication strategies do not discount lead isolates wrongly. 5 In addition, the microbiome of the Roman Baths, UK was profiled using Oxford Nanopore sequencing. This is a UNESCO world heritage site of significant cultural and geological importance, and this was the first survey of its kind to be performed on this site. The findings revealed microbial taxa not previously reported to be present in the waters and gives a basis by which researchers can plan studies into the site from both a bioprospecting and ecological point of view. The Oxford Nanopore sequencing pipeline used to profile the microbiome also revealed evidence for the presence of metal resistance genes, which demonstrates the potential of the pipeline for functional gene profiling of the site. 6 Table of Contents Title Page 1 Declaration of Authenticity 2 Abstract 3 Impact Statement 5 Table of Contents 7 List of Figures 12 List of Tables 15 Abbreviations 17 Chapter 1. Introduction 20 1.1. Antibiotics and their Modes of Action and Resistance Mechanisms 21 1.1.1. Inhibition of Cell Wall Synthesis 21 1.1.2. Membrane Disruption 22 1.1.3. Inhibition of Nucleic Acid Synthesis 22 1.1.4. Inhibition of Protein Synthesis 23 1.1.5. Inhibition of Folic Acid Synthesis 23 1.1.6. Biochemical Mechanisms of Antibiotic Resistance 23 1.1.6.1. Reduction in Intracellular Concentrations 24 1.1.6.2. Modification of Antibiotic Target 25 1.1.6.3. Inactivation of Antibiotic 25 1.2 Microbially-Derived Antibiotics 26 1.3. Principles of Bioactivity-Guided Isolation 32 1.4. Biosynthetic Classes of Major Microbially-derived Antibiotics 35 1.4.1. Polyketide Synthases and Non-Ribosomal Synthases 35 1.4.2. Ribosomally Synthesized and Post-Translationally Modified Peptides 37 1.4.2.1. Lantipeptides 39 1.4.2.2. Thiopeptides 40 1.4.2.3. Lasso Peptides 41 1.4.2.4. Bottromycins 42 1.4.2.5. Cyanobactins 42 1.4.2.6. Glycocins 42 1.4.2.7. Bacterial Head-to-Tail Cyclised Peptides 43 1.4.2.8. Linear azole(in)e-containing peptides (LAPs) 44 1.4.2.9. Proteusins 44 1.4.3. Terpenes 44 1.5. The Decline in Antibiotic Discovery and the Need for New Antibiotics 47 1.6. New Approaches