Bacillus as a Heterologous Platform for Peptide Drug Discovery

The rising incidence of resistance to current antibiotics has become one of the world’s leading health problems. Nationally, at least 2 million people are infected with antibiotic-resistant bacteria, and there are over 23,000 deaths each year from antibiotic resistant infections. Natural products are a rich source of novel compounds for antibiotic development that can help circumvent antibiotic resistance. Bacteria have already selectively developed these molecules for that very purpose. At subinhibitory concentrations, natural product antibiotics can cause developmental and physiological responses in bacteria. This research focuses on harnessing the biosynthetic potential of one bacterial genus, Bacillus, to discover new antibiotic natural products and understand the broader roles these compounds play in bacterial communication. We use a combination of coculture, fluorescent transcriptional reporter assays, and MALDI-TOF IMS to identify and characterize the thiocillins and the broader thiopeptide family as chemical signals that induce biofilm formation in Bacillus subtilis. Biofilm production is an important bacterial defense. We probe into the mechanism of this thiocillin signaling activity in relation to antibiosis. We further pursue an intensive bioinformatics analysis of genus Bacillus to measure its chemical diversity and better understand the role of its specialized metabolites. We uncovered the biosynthetic machinery for a set of highly-conserved compounds across the Bacillus genus that play either known or currently unknown roles in signaling and bacterial development within Bacilli. We ascribe a signaling role to the highly-conserved alkylpyrone biosynthesis pathway in Bacillus. Additionally, we identify a number of unique, weakly conserved natural product biosynthesis pathways scattered across all species. The unique pathways offer leads for identifying new, distinct natural products that could exhibit previously unknown biological activities. To access the new pathways we identify in our bioinformatics analysis, we develop a heterologous expression platform that would allow us to rapidly move entire biosynthetic pathways into the host organism, Bacillus subtilis. Using a Bacillus based platform allows for rapid cloning and expression of peptides, and circumvents many challenges in the field. We develop tools that will enable future efforts to discover, characterize, and modify natural product antibiotics in Bacillus subtilis.Doctor of Philosoph

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

Thiazolyl peptides are known antibiotics produced by diverse bacterial taxa. It has been believed that antibiotics are deployed by bacteria as weapons, providing them with an evolutionary advantage over other microbes. We show here that these weapons can also act as chemical tools that elicit biofilm production in the model bacterium Bacillus subtilis. Importantly, the biofilm-inducing (and therefore signaling) properties of these compounds are independent of their killing activity. We go on to use this biofilm-inducing activity to identify and confirm the presence of thiazolyl peptide gene clusters in other bacteria. These results indicate that thiazolyl peptides, and potentially other antibiotics, have the ability to alter bacterial behavior in ways important both to the environment and to human health

Large-Scale Bioinformatics Analysis of Bacillus Genomes Uncovers Conserved Roles of Natural Products in Bacterial Physiology

ABSTRACT Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these “singleton” BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli , we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCE Bacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus’s metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation

Discovery of a Selective, Substrate-Competitive Inhibitor of the Lysine Methyltransferase SETD8

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure–activity relationships (SAR) of this series

Growing old together: What we know about the influence of diet and exercise on the aging host's gut microbiome

The immune system is critical in defending against infection from pathogenic microorganisms. Individuals with weakened immune systems, such as the elderly, are more susceptible to infections and developing autoimmune and inflammatory diseases. The gut microbiome contains a plethora of bacteria and other microorganisms, which collectively plays a significant role in immune function and homeostasis. Gut microbiota are considered to be highly influential on host health and immune function. Therefore, dysbiosis of the microbiota could be a major contributor to the elevated incidence of multiple age-related pathologies. While there seems to be a general consensus that the composition of gut microbiota changes with age, very little is known about how diet and exercise might influence the aging microbiome. Here, we examine the current state of the literature regarding alterations to the gut microbiome as hosts age, drawing particular attention to the knowledge gaps in addressing how diet and exercise influence the aging microbiome. Further, we will demonstrate the need for more controlled studies to investigate the roles that diet and exercise play driving the composition, diversity, and function of the microbiome in an aging population

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called “secondary” metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin’s antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects—acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes

Interception of the Bycroft–Gowland Intermediate in the Enzymatic Macrocyclization of Thiopeptides

Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide-iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis