Micromonosporaceae’s Biosynthetic Gene Cluster Diversity Highlights Need for Broad Spectrum Investigation.
Imraan Alas1, Doug R. Braun1, Scott R. Rajski1, Tim S. Bugni1
1School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA
Investigations of the bacterial family Micromonosporaceae (order Actinomycetales) have enabled the development of secondary metabolites critical to human health like gentamicin, and over 740 bioactive compounds from 1974 to 2005. Efforts focused on marine Micromonosporaceae bacteria have continued to increase the number of chemically diverse and bioactive compounds since then. However, comparisons of the 904 Micromonosporaceae genomes published in the National Center for Biotechnology Information (NCBI) to the traditionally terrestrial bacterial family Streptomycetaceae’s 5,110 genomes highlight a significant disparity. In order to highlight the biosynthetic potential of Micromonosporaceae and the associated genus Micromonospora, we analyzed 25 locally assembled marine Micromonospora bacterial strains using antiSMASH v6.0.1, and queried identified biosynthetic gene clusters (BGCs) against a preprocessed dataset of ~1.2 million BGCs sorted into 29,955 gene cluster families (GCFs) using the program Biosynthetic Gene Clusters – Super Linear Clustering Engine (BiG-SLiCE). This pre-processed dataset generated from complete and draft genomes from NCBI Ref-Seq, fungi and archaea genomes from NCBI GenBank, metagenome-assembled genomes (MAGs), and BGCs from Minimum Information about a Biosynthetic Gene cluster (MiBiG), forms a near-comprehensive dataset of publicly available BGCs. Of the 581 total Micromonospora BGCs identified by antiSMASH, an average of 22.67% of BGCs had a % similarity equal to or exceeding 50%. By defining unique BGCs as not clustering into the gene cluster families in BiG-SLiCE’s preprocessed database using a distance-to-centroid threshold of 900, we observed 318 unique Micromonospora BGCs. These results highlight the strength of marine Micromonospora bacteria as an underexplored biosynthetically diverse source of new natural products.
Aspergillus Nidulans Inhibitor of Apoptosis-like Protein, AnBir1, is Essential for Survival and Regulates Fungal Development
Meareg Amare1, Sachin Jain1, Mehdi Kabbage1
1Department of Plant Pathology, University of Wisconsin – Madison, Madison, Wisconsin, USA
All organisms balance the decision of life or death at the cellular level. Programmed cell death (PCD) is the coordinated and organized mechanism of cell death. Studies in mammalian, insects and viruses have shown that PCD is important in regulating early development, immune system maturation, host-pathogen interactions and more. Although PCD is largely conserved across eukaryotic kingdoms, there is significant divergence in the regulatory mechanisms. Inhibitors of apoptosis proteins (IAPs) are negative regulators of PCD and are amongst the most highly conserved regulatory proteins across eukaryotic kingdoms. IAPs are defined by the presence of 1-3 Baculovirus IAP Repeat (BIR) domain(s) on the N-terminus end of the protein. Studies of IAPs in mammalian, insect, and viral systems show that they are crucial in regulating cell death and other fundamental processes, including cell division, inflammation and more. Although IAPs are conserved in fungi, there is very limited research into the processes that IAPs regulate and the mechanism through which they regulate fungal processes. In this study, we identified an IAP-like protein in the model filamentous fungal organism Aspergillus nidulans (AnBir1) and investigated fungal processes it regulates. Bioinformatic analyses revealed that AnBir1 contains two BIR domains, a conserved feature among fungal species.
We found that AnBIR1 is an essential gene. Gene deletion is lethal and when AnBIR1 is placed under the control of a conditional promoter, fungal growth is only observed when the promoter is turned on. Moreover, we found that AnBir1 is critical in regulating fungal development. Constitutive expression of AnBIR1 resulted in a strong push towards sexual reproduction with asexual reproduction almost completely lost. Despite this apparent push towards sexual reproduction, the strain overexpressing AnBIR1 is initially delayed whereby the wild type matures earlier. We are investigating the role that AnBir1 plays in regulating A. nidulans PCD and the biochemical context within which it operates to regulate fungal processes.
Pangenomics of the ‘Death Cap’ Mushroom, Amanita Phalloides, and of Agaricales Reveal Dynamic Evolution of Toxin-Related Gene Family in an Invasive Range
M.T. Drott1, S.C. Park1, Harrow L.2, Wang Y.W.2, N.P. Keller1, A. Pringle2
1Department of Molecular and Environmental Toxicology, University of Wisconsin, Madison, Wisconsin, USA, 2Department of Botany, University of Wisconsin, Madison, Wisconsin, USA
The deadly-poisonous Amanita phalloides is invading California but the role of toxic secondary metabolites in the invasion is unknown. We developed a bioinformatic pipeline to automate identification of toxin-associated MSDIN genes and probed 88 genomes from California and the native European range, discovering a diverse MSDIN pangenome with both core and accessory elements. Toxin genes are maintained by strong natural selection. Genes are clustered within genomes. MSDIN diversity was generated by ancient and independent gene-family expansions among genera of Agaricales. We report the discovery of an MSDIN in an Amanita outside the clade of “lethal Amanitas”. The dynamic evolution of the Agaricales-MSDIN pangenome underscores the potential role of these compound’s in mediating ecological interactions in A. phalloides‘ ranges and is reshaping our understanding of the genes’ evolutionary history. Our results enable drug-prospecting efforts in a previously-inaccessible class of compound, providing a roadmap to approach metabolites found in Basidiomycete genomes.
Fitness Contribution of Secondary Metabolites on the Ecology of the Apple Pathogen Penicillium Expansum
Justin L. Eagan 1, Evan R. Digman 2, Christina M. Hull 3, and Nancy P. Keller 1 4
1 Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2 School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA, 3Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA, 4Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Penicillium expansum is a postharvest pathogen of apples and a primary producer of two toxic secondary metabolites, patulin and citrinin. Despite co-occurrence of these mycotoxins within infected apples, biosynthesis is not a prerequisite for successful infections. Secondary metabolites often have antimicrobial activity, and we hypothesize patulin and citrinin, as well as other metabolites, are important for P. expansuminteractions with the apple microbiome to outcompete microbiota and efficiently colonize its ecological niche. A comparison of wildtype, ΔpatL and ΔctnA strains supports the antimicrobial role of both mycotoxins against isolated members of the apple microbiome. The P. expansum-apple infection system represents a model for understanding secondary metabolism importance in fungal pathogen colonization within the context of the host microbiome.
Determining the Role of the Spore-Enriched Protein Isp2 in the Maintenance of Dormancy
Anna B. Frerichs1, M Huang1, Christina M. Hull1,2
1Department of Biomolecular Chemistry, University of Wisconsin-Madison, Wisconsin, USA, 2Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Wisconsin, USA
Spores are vital for the long-term survival of many organisms due to their roles in reproduction and stress resistance. Among fungal pathogens of plants and animals, spores are infectious particles that are more resistant to environmental conditions than other cell types. In the human fungal pathogen Cryptococcus, spores cause disease only when they germinate into yeast, initiating vegetative growth in the host. Although little is known about the germination process, it holds the potential to reveal fungus-specific pathways or proteins that could serve as targets for germination inhibitors. We previously discovered a spore-enriched protein, Identified Spore Protein 2 (Isp2), which is required for normal spore biology. Strains lacking ISP2 (isp2∆) exhibit increased sporulation and initiate germination more quickly than wild type spores. This “jump start” is then followed by a stall later in germination, resulting in a net increase in total germination time. These findings suggest that Isp2 plays an active role in maintaining spore dormancy or a repressive role in germination initiation. Our hypothesis is that Isp2 controls a checkpoint that senses germination conditions in the environment and permits germination when growth conditions (e.g. nutrient requirements) are met. To test this hypothesis, we are using genetic, biochemical, and molecular biological approaches to evaluate the function of Isp2. Understanding the full extent of Isp2 function promises to provide valuable insights into germination and how the process is initiated to ultimately cause disease.
Identification of PASTA Kinase Substrates that Mediate Intrinsic β-lactam Resistance in Methicillin-ResistantStaphylococcus Aureus
Caroline M. Grunenwald1, John-Demian Sauer1
1Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Methicillin-resistant Staphylococcus aureus (MRSA) infections cause approximately 300,000 hospitalizations and 20,000 deaths in the U.S. annually. These trends underscore the critical need for novel antimicrobial strategies and treatments for MRSA. Stk1, a eukaryotic-like serine/threonine kinase with penicillin-binding-protein and serine/threonine kinase-associated (PASTA) domains plays a central role in regulating intrinsic resistance to b-lactam antibiotics such that deletion of stk1 drastically sensitizes MRSA to b-lactams. However, the Stk1-dependent signaling cascades that mediate intrinsic antibiotic resistance remain undefined. To identify Stk1 substrates required for resistance to b-lactams, we took an orthogonal approach using a forward genetic screen combined with shotgun phosphoproteomics in the context of sub-inhibitory antibiotic stress. Screening of the Nebraska Transposon Mutant Library revealed over 70 oxacillin-sensitive mutants encoding for proteins of diverse functions, including purine metabolism, signal transduction, and cell wall synthesis. In parallel, phosphoproteomics analysis identified 271 phosphopeptides from 187 proteins, revealing distinct populations of Ser/Thr phosphoproteins within WT and ∆stk1. 40 phosphoproteins were specific to WT and included proteins involved in cell wall homeostasis (ReoM, MurZ, GpsB), heme biosynthesis, and membrane transport. Notably, 86 unique phosphoproteins were observed in ∆stk1, including drug and antimicrobial peptide transporters and DNA replication and repair proteins, suggesting other Ser/Thr kinases may play an important role in regulating stress responses when stk1 is absent. 10 phosphoproteins overlapped with the genetic screen and likely represent key nodes of regulation of intrinsic antibiotic resistance. Future work will determine the contribution phosphorylation of these putative substrates to enzymatic function, b-lactam resistance, and pathogenesis.
Roles of Essential Genes in Pseudomonas Aeruginosa Biofilm Formation
William Heelan1, Amy Banta1,3, Warren Rose6, Jason Peters1,2,3,4,5
1Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin; 2Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, Wisconsin, USA, 3Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA, 4Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 5Department of Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 6Pharmacy Practice Division, University of Wisconsin-Madison, Madison, Wisconsin, USA
A biofilm is a collection of surface attached microorganisms that exist in an extracellular matrix that serves as a protective barrier against antibiotics and other environmental stressors. Several studies have identified gene pathways that are important for biofilm formation in Pseudomonas aeruginosa, but these studies lack the ability to assess the roles of essential genes. Here, I propose the use of a P. aeruginosa essential gene knockdown library to discover novel connections between core cellular processes and biofilm formation. My goal is to find essential gene knockdowns that positively or negatively impact biofilm formation in P. aeruginosa. These findings may lead to new therapeutic strategies that can simultaneously disrupt biofilm formation and the viability of P. aeruginosa by perturbing a single pathway.
High Levels of Cyclic Di-Guanylate Interfere with Initiation of a Beneficial Symbiosis
Ruth Y. Isenberg1,2, David G. Christensen3, Karen L. Visick3, and Mark J. Mandel1,2,
1 Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA, 3Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Chicago, Illinois, USA
Bacteria often transition from a motile environmental lifestyle to a biofilm state in the host. Cyclic di-guanylate (c-di-GMP) inhibits motility and promotes biofilm formation in numerous bacteria. Vibrio fischeri is the sole light organ symbiont of the Hawaiian bobtail squid (Euprymna scolopes) and encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP. Deletion of multiple genes encoding enzymes that synthesize or degrade the molecule yielded high and low c-di-GMP strains, respectively, that each had the expected motility and biofilm phenotypes in culture. In the host as early as 18 hours post inoculation, the high c-di-GMP strain was defective in colonization, while the low c-di-GMP strain had no defect. Colonization by the high c-di-GMP strain was rescued by expression of a V. choleraephosphodiesterase, arguing that c-di-GMP levels, not signaling specificity, caused the colonization defect. Elevated c-di-GMP levels resulted in biofilm aggregates in the host mucus that were larger and more numerous than those made by a low c-di-GMP strain. Gene expression in the aggregates revealed a novel regulatory interplay between Syp and cellulose polysaccharides: Syp expression, which is required for squid colonization, is repressed by cellulose that is upregulated by c-di-GMP. These interactions do not occur in culture-grown cells, emphasizing the importance of studying symbiosis signal transduction in vivo. Together, our data show that proper retention of a low c-di-GMP state is critical for successful establishment of the vibrio-squid symbiosis and that even modest increases impact biofilm composition, aggregation, and motility in the host.
PASTA Kinase-Dependent Control of Peptidoglycan Synthesis is Required for Cell Wall Stress Responses, Cytosolic Survival, and Virulence in Listeria Monocytogenes
Jessica L. Kelliher1, McKenzie E. Daanen1, John-Demian Sauer1
1Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Penicillin-binding protein and serine/threonine-associated (PASTA) kinases sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. These kinases are conserved in single copy in Firmicutes and Actinobacteria, and we have shown that genetic or pharmacologic disruption of the PASTA kinase in two major pathogens, Listeria monocytogenes and Staphylococcus aureus, potentiates the effects of β-lactam antibiotics. The PASTA kinase in the cytosolic pathogen L. monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. Using orthogonal phosphoproteomic and genetic approaches, we found that during β-lactam exposure PrkA phosphorylates 23 proteins with functions in cell wall homeostasis, central metabolism, stress responses, and more. We further show that PrkA-dependent regulation of one substrate, ReoM, is required to increase peptidoglycan synthesis during cell wall stress. PrkA-mediated regulation of ReoM is also critical for cytosolic survival and virulence of L. monocytogenes. ReoM is conserved in PASTA kinase-containing organisms, and we find that the PASTA kinase-ReoM pathway is important for the response to cell wall stress in methicillin-resistant S. aureus. Current work is focusing on the importance of PrkA-mediated phosphoregulation of GpsB, another conserved protein identified as a putative PrkA substrate in our phosphoproteomics screen. GpsB is a spatiotemporal regulator of peptidoglycan synthesis and in L. monocytogenes controls activity of the major bifunctional penicillin-binding protein.Altogether, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating peptidoglycan synthesis to facilitate cytosolic survival and virulence.
Culturing Bacterial Isolates from the Skin of Pediatric Atopic Dermatitis Subjects
N. M. Lane Starr1,2, O. R. Steidl1, J. M. Smith1, S. Sandstrom2, M.H., Swaney2, J. Gern1, A. M. Singh1, L. Kalan2
1Deparment of Pediatrics, University of Wisconsin–Madison, Madison, Wisconsin, USA, 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin,USA
Atopic dermatitis (AD), characterized by skin barrier dysfunction and inflammation, is associated with food allergy and asthma development. Underlying mechanisms for disease co-expression remain poorly understood. The skin microbiome’s role in modulating skin disorders, including AD, and systemic immunity, especially epithelial function and immune education, remains under investigation. Certain microbial products have been shown to affect not only the surrounding microbial community, but also the host itself. One such product, Staphylococcal enterotoxin B, has previously been associated with AD. We hypothesize that metabolic output of skin-associated bacteria in AD contributes to barrier dysfunction and atopic march. Skin swabs were collected from the anterior forearms of 20 pediatric subjects aged 1-14 (9 control, 11 AD—sampled at lesional and non-lesional sites). Samples were processed for microbial metagenomic analysis and bacterial isolation. Isolates confirmed to be S. aureus were tested for enterotoxin production. Metagenomic sequencing identified 64 bacterial genera >0.1% relative abundance, with Streptococcus being most abundant, followed by Cutibacterium, Micrococcus, and Staphylococcus. Isolates representing 11 of the 64 genera from metagenomics were cultured. The most abundantly represented genera were Micrococcus, Staphylococcus, Kocuria, and Rothia. In vitro analysis of confirmed S. aureus isolates (all from AD subjects) revealed strain-specific differences in enterotoxin production. The strain from the severe AD subject produced enterotoxin B levels >100-fold higher than the strains from subjects with mild-moderate AD (p<0.0001). Enterotoxin E levels also varied significantly between strains (p<0.0001). In conclusion, we confirmed elevated levels of viable, potentially pathogenic bacteria on AD skin and were able to culture isolates representing a range of genera. Furthermore, S. aureus isolates varied in their in vitro enterotoxin production capabilities. Future analysis will expand the bacterial products investigated and explore how these products may interact with the immune system and contribute to the TH2 phenotype associated with the atopic march.
Biofilm Inhibitor BinK is a Key Regulator Throughout the Vibrio Fischeri Host Colonization Process
Denise A. Ludvik1, Natalia Rosario-Meléndez1, Mark J. Mandel1
1Department of Medical Microbiology & Immunology, University of Wisconsin–Madison
The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the symbiosis polysaccharide Syp. Previously, we identified the hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore BinK function. Using a syp’-gfp+ reporter and conducting controlled colonization assays, we demonstrate that BinK functions to inhibit biofilm gene expression at multiple stages of host colonization. In a ΔbinK background, RscS is no longer necessary for colonization or the initial aggregation phenotype, providing evidence that BinK—and not RscS—senses a signal from the host to regulate proper symbiotic development. To determine how BinK interprets such a signal, we used differential scanning fluorimetry which identified ethanolamine as a putative ligand of BinK, and we are evaluating the effect of this compound and its derivatives on biofilm formation and host colonization. Overall, this study provides evidence for opposing activities of RscS and BinK and suggests that BinK interprets a host signal during the colonization process.
A Shelter from the Elements: Understanding Conditions Required for Fungal Chlamydospore Formation and Bacterial Invasion
Isabelle Ludwikoski1, Nandhitha Venkatesh, Nancy Keller1
1Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bacterial-fungal interactions (BFIs) drive microbiome dynamics from environmental to healthcare settings, impacting survival and dispersal of the interacting partners. Previous work from our lab established that the plant pathogen Ralstonia solanecearum induces formation of swollen, overwintering spores in Aspergillus spp. through production of a cyclic lipopeptide called ralsolamycin. With deletion of rmyA, the polyketide synthase in the ralsolamycin biosynthetic cluster, production of ralsolamycin is diminished and fungal chlamydospores are not induced in co-culture. Further, in co-culture R. solanecearum can invade the chlamydospores. Recently we have identified a survival benefit for bacterial invaders under nutrient starvation and cold stress conditions compared to mutants unable to invade chlamydospores, proposing a new mechanism for bacterial overwintering inside of fungi. Additionally, several Gram-negative bacteria unable to invade chlamydospores independently and previously not known to be endofungal, can invade when co-cultures are supplemented with ralsolamycin, whereas the Gram-positives tested could not. We also observed simultaneous colonization of a chlamydospore by bacteria of two different species, suggesting the potential for chlamydospores to harbor communities of bacteria inside. Current and future work is focused on gaining understanding of the mechanisms driving chlamydospore formation and bacterial invasion and investigating the potential for bacterial DNA exchange inside of fungal chlamydospores.
A Novel Role for Microbial Rhodopsins in Building the Lipid Bilayer
Zachary Maschmann1, Indu Menon2, Anant K. Menon2 and Katrina T. Forest1
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2 Department of Biochemistry, Weill Cornell Medical College, New York City, New York, USA
The abundant actinobacterium acI, and other freshwater marine bacteria, encode opsins (actinorhodopsins or ActRs) that, like archaeal bacteriorhodopsin (BR), pump protons across the cytoplasmic membrane in a green light- and retinal-dependent manner to form a gradient that can be harnessed for ATP synthesis3–5. However, despite robust transcription of ActR in native environments, many sequenced acI genomes lack genes encoding the enzymatic machinery to synthesize retinal, strongly suggesting that ActR may “moonlight” and exhibit a second, retinal-independent function. In a cellular context, phospholipids are synthesized in the inner leaflet of the cytoplasmic membrane and must reorient to the outer leaflet to construct the bilayer characteristic of biological membranes. However, as spontaneous reorientation is slow because it involves the energetically unfavorable transport of a charged headgroup across the hydrophobic lipid bilayer it is facilitated in cells by a diffusion channel termed scramblase. Phospholipid scramblases have been identified in archaea and in eukaryotes, however none have been identified in bacteria although their activity has been described. Menon and colleagues used a fluorescence-based assay to show that the visual pigment rhodopsin exhibits scramblase activity when reconstituted into large unilamellar vesicles, accounting for the observation that lipids scramble rapidly across photoreceptor disc membranes. Using this assay and the fluorescent lipid C6-NBD-PC as reporter, we now show that ActR, purified and reconstituted in proteoliposomes following cell-free translation, catalyzes the equilibration of phospholipids across the bilayer, making it the first ever identified bacterial phospholipid scramblase. ActR moonlighting as both a proton pump and scramblase emphasizes the versatility of photoreceptor function and may provide a molecular connection between photoheterotrophy and membrane biogenesis. These results are of immense importance for the fundamental question of how cells build themselves, with implications for understanding the origins of cellular life and the basic biology of bacteria.
Macrolide Glycosyltransferases Render Antibiotics Harmless to Streptomyces Strains but Lethal for E.coli
Daniel S. May1, Shukria Akbar1,2, Tim S. Bugni2, Cameron R. Currie1
1Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA
Diverse Streptomyces strains were grown on sub-inhibitory concentrations of antibiotics to induce the production of new natural products. Out of 100 Streptomyces strains grown in the presence of the macrolide antibiotic erythromycin, 10 strains demonstrated new Gram-negative inhibition against Escherichia coli. Comparative metabolomics and molecular networking of the strains revealed new compounds related to erythromycin when the strains were grown with erythromycin. This suggested that erythromycin was modified by the Streptomyces strains. The difference in mass between the new compound and erythromycin was equivalent to an additional glucose, indicating that erythromycin had been glycosylated. The oleD gene encodes a macrolide glycosyltransferase that modifies diverse macrolides, rendering them inactive. The 10 Streptomyces strains were sequenced, and each strain contained an oleD homolog. Additionally, three previously sequenced Streptomyces strains were predicted to contain oleDhomologs and were selected for further studies. The 13 oleD-encoding Streptomyces strains were tested for their ability to glycosylate four macrolide antibiotics: erythromycin, spiramycin, tylosin, and josamycin. The strains varied in their ability to glycosylate each of the four macrolides, and this variability was consistent with the phylogeny of the oleD homologs. The glycosylated erythromycin inhibited growth of E. coli, but not other Gram-negative pathogens, suggesting a role for these inactivated macrolides as targeted antibiotics.
Establishing the Fundamental Principles of Spore Germination in Spores of Human Fungal Pathogens
Megan McKeon1, Christina Hull1,2
Departments of 1Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Spores are a dormant, stress-resistant cell type used by fungi to spread to new environments. To survive, these cells must remain dormant until they encounter favorable conditions for germination, an essential differentiation process in which dormant spores transition into vegetatively growing cells. Despite the importance of spore germination in the life cycles of the majority of fungi, the molecular networks governing this fundamental process remain poorly understood in any system. To decipher the molecular mechanisms controlling germination, we carried out a time course transcriptomic analysis of Cryptococcus spores, assessing 11 time points over the 10 hour process. This analysis showed a clear global increase in transcript levels after the introduction of a germination trigger. The most dynamic differences between two consecutive timepoints occurred between dormant spores and 20 minutes into germination, displaying a dynamic increase in expression as well as a significant drop of a cohort of genes at 20 minutes. This finding suggests that spores are primed to respond quickly to both initiate new transcription and degrade dormant spore transcripts given appropriate germination signals. We hypothesized that the initiation of the germination program is controlled by master transcription factors, that work to coordinate the transition out of dormancy. We identified two high mobility group transcription factors with significant germination phenotypes, HGR1 and HGR2. Using a high-resolution, quantitative germination assay, we observed that spores produced by both hgr1- and hgr2- strains germinate at a faster rate than wild type spores, suggesting a repressive regulatory role during germination. We will continue to define the roles of these potential master regulators of spore germination and dormancy using multi-omic approaches and high-resolution, quantitative germination assays. Together, these data establish the foundation for understanding the fundamental principles of spore germination.
Computational Advances in the Discovery of a New Class of Fungal Natural Products
Grant Nickles1, Milton Drott1, Nancy Keller1,2
1Department of Medical Microbiology and Immunology, University of Wisconsin—Madison, Madison, Wisconsin, USA, 2 Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA
Fungal secondary metabolites (SMs) are major sources of antimicrobial (e.g. penicillin, griseofulvin) and therapeutic (e.g. cyclosporine, mycophenolate) compounds. Ecologically, they provide important fitness adaptations that are finely tailored to the niche of an organism. The fungal biosynthetic genes responsible for SM synthesis and transportation are uniquely arranged in contiguous clusters within the genome, termed biosynthetic gene clusters (BGCs). Current genome mining algorithms capable of identifying putative BGCs are limited to what is considered ‘canonical’ (BGCs defined by biochemically characterized synthetases and synthases, i.e. nonribosomal peptide synthetases or polyketide synthases). SMs synthesized by BGCs lacking canonical structure are difficult to incorporated into current predictive software (i.e. AntiSmash) and thus preclude informative analysis such as extensive phylogenetic studies. One such example of a noncanonical BGC class blind to existing genome mining software is the isocyanide (N-ºC+) metabolite producing BGCs. Isocyanides have been a major interest of organic and synthetic chemists since the 1920s due to their unique divalent carbon, and high reactivity. While numerous bioactive isocyanide metabolites have been extracted from bacteria and fungi, the genes responsible for their synthesis were largely unknown prior to our laboratory publishing the first examples of isocyanide synthase (ICS) containing BGCs in the fungus A. fumigatus. We have developed paradigm shifting software and computational approach that allows detection of diverse ICS BGCs across the fungal kingdom, and offer initial predictions of functionality in specific ecological settings.
Discovery, Characterization, and Synthesis of Small Molecule Inhibitors of Staphylococcus Aureus Quorum Sensing and Associated Virulence
Thomas J. Polaske1 and Helen E. Blackwell1
1Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Many pathogenic bacteria regulate virulence behavior through quorum sensing (QS). Staphylococcus aureus, the causative agent of methicillin-resistant S. aureus (MRSA) infection, utilizes the accessory gene regulator (agr) QS system to upregulate the production and secretion of a large suite of proteases, lipases, biosurfactants, and pore-forming toxins to damage and invade host tissues. Accordingly, potent and synthetically tractable chemical inhibitors of agr-type QS are of high interest as chemical probes to researchers studying agr systems and as potential adjuvant or replacement therapies to conventional antibiotics. In this study, we have discovered two potent “best-in-class” small molecule inhibitors of S. aureus QS using existing screening technology developed in-part by our lab. Using a variety of cell-based assays, we have shown that these compounds likely target the AgrC histidine kinase receptor with unique specificity yet completely abrogate agr activity in several S. aureus strains tested (including MRSA). Using our organic synthesis capability, we have shown that both compounds are synthetically tractable, developed a novel synthesis, and have begun work on understanding the structure-activity relationship(s) of these exciting new compounds.
Investigation of Biosynthetic Gene Clusters in Bacteria Common to Skin Microbiomes Using lsaBGC
Rauf Salamzade1, J.Z. Alex Cheong1, Shelby Sandstrom1, Mary Hannah Swaney1, Nicole Lane-Starr1, Anne Marie Singh2, and Lindsay R. Kalan1,3
1Department of Medical Microbiology & Immunology, University of Wisconsin–Madison, Madison, WI, USA, 2Department of Pediatrics, University of Wisconsin–Madison, Madison, Wisconsin, USA, 3Department of Medicine, Division of Infectious Disease, University of Wisconsin–Madison, Madison, Wisconsin, USA
Bacterial secondary metabolites, encoded by biosynthetic gene clusters (BGCs), can underlie microbiome homeostasis and be developed into high-value commercialized products, which have historically been mined from a select group of taxa. Despite this, dedicated bioinformatics tools for comparative analysis of BGCs are limited. We thus developed lsaBGC to aid exploration of microdiversity and evolutionary trends across BGCs in any bacterial taxa of interest. Application to four taxonomic genera commonly found in skin microbiomes revealed novel insights on both well and less studied taxa and their secondary metabolites. We find that the BGC encoding for the carotenoid staphyloxanthin, a virulence factor in S. aureus, is ubiquitous across the genus of Staphylococcus and exhibits signatures of inter-species horizontal transfer, including mobilization onto plasmids. We experimentally validated the presence of staphyloxanthin in the skin-derived commensals S. epidermidis and S. warneri. Similarly, we identified a BGC with a non-ribosomal peptide synthetase encoding for an unknown metabolite which is highly conserved across several divergent, skin-associated Corynebacterium species and is flanked by transposable elements. Using strain-resolution metagenomics, we further determined that the C. kefirresidentii species complex is the most prevalent clade of Corynebacterium in skin metagenomes. While ubiquitous across skin, there are currently <30 publicly-available genomes for this species complex. Using lsaBGC’s framework for metagenomic mining, we identified 34,545 novel single nucleotide variants (SNVs) in predicted BGCs within metagenomes which were absent in available genomes, including several novel, non-synonymous SNVs within the top 5% of conserved sites of core or adjacent biosynthesis genes for BGCs.
Elucidating the Impact of Inflammation Triggered by Microbes on Wound Healing
Simone Shen1, John-Demian Sauer1, and Anna Huttenlocher1,2
1Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Wisconsin, USA, 2Department of Pediatrics, University of Wisconsin-Madison, Wisconsin, USA
Microbial infection is a major complication in wound healing. Larval zebrafish are a useful model for studying wound healing because the immune system is highly conserved, the tissue is optically transparent, and transgenic lines with cell-specific expression of fluorescent proteins are available, allowing us to visualize immune cells responses to bacterial infections and the damaged epithelial layers. When a zebrafish transected tail wound is infected with Listeria monocytogenes (Lm), wound healing is inhibited, and increased numbers of macrophages and neutrophils are recruited to the wound compared to uninfected zebrafish tail wounds. Early eradication of infection with antibiotics (24 hour-post-wound (hpw)) results in resolution of inflammation and proper wound healing, however delayed eradication 48 or 72 hpw, results in sustained neutrophils and macrophages recruitment at the wound and defective wound healing despite bacterial clearance, suggesting that prolonged inflammation inhibits subsequent wound healing. To determine the impact of prolonged macrophage or neutrophil recruitment on wound healing, we depleted macrophages or neutrophils from the wound site. Surprisingly, we found no difference in wound healing in Lm infected wounds when neutrophils are depleted and worse healing when macrophages are depleted, suggesting that macrophages are essential for wound healing. To modulate the inflammatory response to infection, we utilized Lmstrains genetically engineered to activate either pro- or anti-inflammatory forms of cell deaths. Lm strains that induced inflammatory cell death, including pyroptosis and necrosis, further inhibited wound healing whereas Lm strains that induced anti-inflammatory cell death, apoptosis, did not inhibit wound healing, further supporting the hypothesis that extensive inflammation impairs wound healing. Finally, to investigate the inflammatory mediators that inhibit wound healing we used a variety of pharmacologic and genetic tools to specifically inactivate components of the inflammatory response. Strikingly, we found that treatment of zebrafish infected tail wounds with anakinra, an IL-1R antagonist, partially rescued wound healing, suggesting that IL-1 signaling driven by Lm infection leads to wound healing defects. Ongoing studies are aimed at further investigating if IL-1 signaling can serve as a potential therapeutic target for treating infected wounds.
The Genetic Basis of Metronidazole Resistance in C. Difficile
Madeline Topf1, Madison Youngblom1, Abiola Olaitan2, Chetna Dureja2, Kelli Palmer3, Kevin Garey3, Caitlin Pepperell1, Julian G. Hurdle2
1Department of Medical Microbiology and Immunology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI, USA, 2 Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA, 3 Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, USA
Hospital-acquired C. difficile infection has become an epidemic, and treatments are decreasing in efficacy. Highly transmissible strains of C. difficile are resistant to the first-line antibiotic metronidazole, however the genetic basis of resistance is not known. We hypothesize that metronidazole resistance contributed to epidemic spread of C. difficile, and therefore resistance-associated loci are be under positive selection. We surveyed C. difficile clinical isolates using a genome-wide association study (GWAS). We identified a SNP in the promotor region of nimB which confers metronidazole resistance and is associated with rapid clonal transmission. Notably, the nimB SNP co-occurs with a SNP in gyrA associated with fluoroquinolone resistance, and both SNPs are under positive selection. Taken together, we conclude that metronidazole resistance conferred by the nimB SNP plays a role in the high transmissibility of epidemic, antibiotic-resistant C. difficile.
Systematically Phenotyping Acinetobacter Baumannii Essential Genes Using Antibiotics
Jennifer Tran1,2, Amy Banta1,3, Ryan Ward1,4, Jason Peters1,2,3,5,6
1Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2 Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA, 3Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA, 4Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA, 5Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 6Department of Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Considered an urgent threat to public health, Acinetobacter baumannii is a Gram-negative bacterium and opportunistic pathogen that can cause multidrug-resistant, hospital-acquired infections. Antibiotics used to treat these infections target genes and pathways that are essential for bacterial growth and survival; however, the functions of most A. baumannii essential genes have not been experimentally verified or remain unknown. By studying these genes, we can better understand antibiotic mechanisms and essential gene networks and potentially discover novel drug targets. We used CRISPR interference to knock down all predicted essential genes in A. baumannii and screened the knockdown library with a panel of antibiotics and other chemicals. From this, we identified novel drug-gene interactions and observed phenotypes for previously uncharacterized genes that provide clues about their function. These data provide the framework to establish an essential gene network for A. baumannii, allowing us to define new weaknesses that can be therapeutically exploited and providing greater understanding about A. baumannii biology in the context of antibiotics.
Transcriptomic Analysis Reveals Novel Regulatory Outputs for Vibrio Fischeri Biofilm Formation
Jacob Vander Griend1,2, Mark J. Mandel1,2
1Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA,2 Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA
Biofilms are critical components of the host colonization process for both pathogens and beneficial symbionts. The specific symbiosis between the Hawaiian bobtail squid (Euprymna scolopes) and the bioluminescent bacterium Vibrio fischeri provides an accessible model system to study regulators controlling biofilm formation during host colonization. Using a colony biofilm model that closely approximates the in vivo biofilm, we applied RNA sequencing to identify genes that are regulated in different biofilm-inducing conditions. We identified a core biofilm transcriptome that is shared across all biofilm induced mutants, capturing all characterized target genes. We additionally identified 92 target genes that are regulated independent of the known biofilm response regulator, SypG, with multiple new targets encoding putative roles in surface attachment. The most highly induced genes are found in a locus associated with the production of curli amyloid fibers. Our current work is focused on completing further bioinformatic analyses and examining the in vivo relevance of hypotheses generated from the transcriptome dataset.
Genetic Tradeoffs Underlying Synergetic Antibiotic Function in Acinetobacter baumannii
Ryan Ward1,2, Amy Banta1,3, Jennifer Tran1,4, Jason Peters1,2,4
1Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, USA, 3Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA, 4Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Acinetobacter baumannii is an opportunistic gram-negative bacterial pathogen that has become increasingly resistant to antibiotics of last resort, including colistin. Antibiotics inhibit essential gene function, but most of our understanding comes from studying isolated genes in model organisms. Furthermore, most antibiotic combination therapies are not understood mechanistically. A. baumannii is an unusual gram-negative bacterium due to its propensity to accumulate mutations in lipopolysaccharide synthesis genes – a structural part in the outer membrane and the target of colistin. Here, we systematically probe essential gene function in A. baumannii by screening a CRISPRi library against clinically relevant antibiotics. Based on these screens, we were able to describe the genetic mechanism of synergy between two antibiotics with seemingly distinct modes of action. This work aims to explain drug synergy by systematically probing genetic networks to reveal functional tradeoffs – in which the genes responsible for increased resistance to one drug also increase susceptibility to another. These tradeoffs can explain the genetic basis of known combination therapies, but more importantly they represent a new platform to predict effective drug synergies to overcome resistance mechanisms.
A Host-Derived Peptide Enhances Sensitivity of Non-Typhoidal Salmonella to Antibiotics
Trina L. Westerman1, Johanna R. Elfenbein1,2
1Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2 Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA
Non-typhoidal salmonellae are a leading cause of bacterial food-borne gastroenteritis worldwide. The increased incidence of antibiotic resistant non-typhoidal Salmonella infections presents a significant challenge for effective patient treatment, making it essential to develop new therapeutic strategies. Antimicrobial peptide therapy in combination with traditional antibiotics offers one method to improve treatment. Here, we evaluate the effect of a peptide derived from the mammalian protein, Myristoylated Alanine Rich C-Kinase Substrate, on Salmonella. We show that our peptide, Anti-Salmonella Peptide (ASP), decreases the in vitro survival of Salmonella in various media, regardless of bacterial growth phase or atmospheric conditions. We utilized fluorescent chemical uptake assays to demonstrate that ASP permeabilizes both outer and cytoplasmic membranes. In addition, ATP concentrations decrease in Salmonella treated with ASP, suggesting dissipation of proton motive force across the permeabilized cytoplasmic membrane. Next, we hypothesized the membrane damage caused by ASP might enhance the bactericidal effects of antibiotics. We found that combinatory treatment of ASP with sub-inhibitory concentrations of chloramphenicol, gentamicin, azithromycin, ceftiofur or carbenicillin caused enhanced killing of antibiotic-sensitive Salmonella. Furthermore, we found that combinatory treatment of ASP with chloramphenicol sensitizes chloramphenicol-resistant strains of Salmonella Infantis to chloramphenicol. Overall, these results highlight that ASP has the potential to enhance effectiveness of multiple classes of antibiotics against Salmonella. Furthermore, ASP also has the potential to enhance sensitivity of multi-drug resistant Salmonella strains to antibiotic killing, thereby restoring antibiotic efficacy to drug resistant bacteria.
Biosynthesis of Quorum Sensing Inhibitors of Staphylococcus Aureus
Danielle L. Widner1, Helen E. Blackwell1
1Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Many pathogenic bacteria coordinate virulence through the release of quorum sensing (QS) signals. Several Gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, and Clostridioides difficile, utilize accessory gene regulator (agr) type QS systems. The signaling molecules of agr-type QS systems are autoinducing peptides (AIPs). Minor changes to the native AIP sequence can switch the molecules from activators to strong inhibitors of QS. In this work, I provide the first evidence that AIP analogs can be biosynthesized in a surrogate organism. I demonstrate that two of the most potent S. aureus QS inhibitors, AIP-I D5A and AIP-III D4A, can be biosynthesized at levels sufficient to inhibit QS in all four S. aureus agr groups.
Human Oral Streptococcus spp. Defend Against Pathogen Colonization
Susan Zelasko1, Mary-Hannah Swaney2, Reed Stubbendieck1, Shelby Sandstrom2, Caitlin Carlson1, Julian Cagnazzo3, Athena Golfinos4, Nasia Safdar5,6, David Andes5, Lindsay Kalan2,5, Cameron Currie1
1Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA, 2Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, USA, 3Department of Engineering, Computing and Mathematical Sciences, Lewis University, Romeoville, Illinois, USA, 4Department of Oncology, University of Wisconsin, Madison, Wisconsin, USA, 5Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA, 6William S. Middleton Veterans Affairs Medical Center, Madison, Wisconsin, USA
Understanding how changes in the microbiome render it vulnerable to pathogen colonization is essential, as carriage of multidrug-resistant organisms (MDROs) is a major risk factor for developing serious infections. One mechanism by which our microbiome provides colonization resistance is through antimicrobial specialized metabolites (ASMs) to inhibit the growth of competing microbes. To advance our understanding of ASM production by human-associated microbes, we characterized salivary and fecal microbiome samples previously collected through the Winning the War on Antibiotic Resistance in Wisconsin study. High-throughput bioactivity screening revealed oral bacteria isolated from carriers of one or more MDRO (n=133) demonstrate decreased inhibition of Gram-negative pathogens of high clinical priority, compared to isolates from non-carriers (n=160). Subsequent shotgun metagenomic analysis of oral swabs from study participants (n=63) revealed enrichment of Streptococcus and Prevotella spp. among MDRO non-carriers, compared to carriers. To explore whether pathogen inhibition may be mediated by these taxa, we performed bioactivity-guided fractionation of microbial extracts from isolates with evidence of strong inhibition of Gram-negative pathogens. This screen identified a lead fraction from a Type III polyketide synthetase–containing Streptococcus salivarius strain isolated from a MDRO non-carrier. Subsequent analysis using an in vivo murine model of infection revealed this extract fraction significantly reduced the infectious burden of Acinetobacter baumannii and Escherichia coli without evidence of toxicity. Together, our data provides evidence that oral bacteria shape this dynamic microbial community and may serve as an untapped source for much-needed antimicrobial drug leads.
Inhibition Mechanism of Small Molecules and Peptidomimetics in the Quorum Sensing System of Staphylococcus Aureus
Ke Zhao1, Thomas J. Polaske1, and Helen E. Blackwell1
1Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
The quorum sensing (QS) system in Staphylococcus aureus controls the production of various virulence factors. It contains four main components, AgrB, D, C, and A, and is regulated by the binding of the native QS signal AIP to the histidine kinase receptor AgrC. Small molecules and peptides have been previously developed to attenuate QS in S. aureus. However, the QS system targets of these compounds remains unknown. Herein, we have developed in vitroassays to determine the inhibition mechanism of each type of inhibitor. In this work, we performed competition assays between the native AIP and our inhibitors to look for reduction in auto-phosphorylation of AgrC using nanodiscs, and we saw this reduction in some inhibitors, indicating they are targeting the AgrC receptor. We also developed assays to look for both the phosphate transfer from AgrC to AgrA and the binding of response regulator AgrA to the promoters controlling virulence regulation genes. Using these tools, we were able to identify the target of each of our compounds, which can lead to more in-depth study of protein-ligand interactions and rational design of further QS inhibitors.