Abstracts

POSTER 1

Investigating The Role of Vitamin D3 in modulation of IL-12 and Nitric Oxide during Mtb infection

Azka Ahmed^1,2, Maya E. Gough³,Taha Salim² , and Elebeoba May^1,2,3

¹Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA, ²Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA, ³ Biomedical Engineering, University of Houston- Houston, TX, USA.

Tuberculosis (TB) is an infectious disease caused by the respiratory pathogen Mycobacterium tuberculosis (Mtb). In 2021, there were 10.6 million cases of tuberculosis (TB) with an estimated global total of 1.6 million deaths (World Health Organization[WHO], 2022). Deficiency of vitamin D3 (25(OH)D) has been reported as one of the many factors that influence the incidence and progression of active TB (Kim et al., 2014, Chocano-Bedoya et al., 2009). Although vitamin D3 is classically associated with musculoskeletal health, various studies have identified biological mechanisms by which vitamin D3 modulates innate and adaptive immune systems to enhance host defense against Mtb (Wei & Christakos, 2015). Macrophages, primary phagocytes of Mtb, are known to express the vitamin D receptor (VDR) and the enzyme CYP27B1 for conversion of inactive (25(OH)D) to active 1,25(OH)₂D. Host pathogen interactions via toll-like receptors (TLR2/1) upregulates VDR and CYP27B1 expression in macrophages allowing 1,25(OH)₂D to bind VDR and activate VDR-directed signaling that promotes bacterial clearance through production of antimicrobial proteins and oxidative species in the phagosome (Di Rosa et al., 2011). We previously investigated the role of vitamin D3 in differential modulation of bacterial load and effector molecules produced by infected macrophages based on infection level and found in high infection conditions, vitamin D3 sufficient cells release significantly higher levels of IL-12 and reactive oxygen and nitrogen species (H₂O₂ and NO) when compared to low infection (Gough et al., 2017). Follow up studies examining the combined effects of in vivo vitamin D3 deficiency and ex vivo supplementation showed that vitamin D3 sufficient/exogenously supplemented cells generated lower levels of IL-12, IFN-γ and nitric oxide(NO) (Gough et al., 2019). Specifically, IFN-γ production was positively correlated to IL-12 and the production of both pro-inflammatory cytokines was positively correlated to NO production (Gough et al., 2019). It was shown that macrophages release IFN-γ upon combined stimulation with IL-12 and M. bovis bacillus Calmette-Guerin (BCG) through nuclear translocation of STAT4 (Munder, M et al., 1998, Schindler, H et al., 2001).  IFN-γ activation of JAK/STAT pathway promotes inducible nitric oxide synthase enzyme (iNOS) gene expression (Salim et al., 2016).  What remains to be determined is the connection between IL-12 and NO, and whether vitamin D3 associated NO production is IFN-γ dependent. In-vivo/in-vitro studies provide some dynamic data necessary for predicting the macrophage immune response to Mtb over a period of several hours or days. Currently only a few in-silico models exist that capture the impact of vitamin D metabolism on intracellular immune mechanisms activated in macrophages during Mtb infection (Gough et al., 2016, Gough et al., 2018). There does not exist a detailed macrophage intracellular model that integrates the impact of vitamin D3 uptake on pro-inflammatory cytokine and effector molecule production. Using computational modeling we investigate the mechanistic pathways by which vitamin D3 modulates IL-12 which in turn regulates NO production.  Our model is used to determine whether NO production occurs via the endogenous release of IFN-γ or in an independent manner. Understanding the role of vitamin D3 as a regulator of effector molecules will provide insight into the ramifications of host vitamin D3 deficiency on the risk and progression of TB infection as well as provide a platform by which host immune responses can be manipulated.

POSTER 2

Modeling Bacterial Adaptation to Iron Homeostasis and Oxidative Stress in Escherichia coli

Daniel C. Ajuzie^1,2, Seyed A. Arshard¹, Komal Rasaputra¹, Elebeoba E. May^1,2,3

¹Department of Biomedical Engineering, University of Houston, TX, USA, ²Wisconsin Institute for Discovery, University of Wisconsin-Madison, WI, USA, ³Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, WI, USA

Iron metabolism in bacteria is impacted by the presence of other molecules in the microenvironment, particularly oxidants. To understand the cellular response to dynamic iron stress, we developed an in-silico model that integrates Escherichia coli K12 iron and peroxide stress response. Using experimental data showing proliferative, bactericidal, and bacteriostatic phenotypes when challenged with different stress combinations, including low and high peroxide stress in iron-rich and iron-deficient environments, we validated our model and compared multi-phenotype (MP) modeling approach to standard single phenotype (SP) approaches. The MP model accurately recapitulated 80% of responses measured by corresponding experimental model, while ensemble modeling reduced modeling error by up to 11% across different treatments for both the MP and SP models. Using the MP model, we identified siderophore production, growth, and peroxide-dependent transcriptional regulation as the most critical sub-components of the iron homeostatic machinery in E. coli. Additionally, our model explains bacterial siderophore response in the context of dynamic iron and peroxide stress, showing that enterobactin is causatively correlated with cellular growth parameters and likely confers cellular protection under oxic environments. Our simulations indicate that when bacteria experience low levels of peroxide stress in iron-rich environments, they face a distinctive challenge that requires a more complex response for adaptation compared to other stress combinations. Understanding variations in bacterial persistence resulting from differences in the maintenance of iron homeostatic processes could aid in the identification of potential therapeutic targets or novel therapeutic strategies for host-pathogen dynamics.

POSTER 3

Micromonosporaceae’s Biosynthetic Gene Cluster (BGC) Diversity Highlights Need for Broad Spectrum Investigation

Imraan Alas¹, Doug R. Braun¹, Spencer S. Ericksen², Tim S. Bugni¹

¹School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA, ²Small Molecule Screening Facility, University of Wisconsin-Madison, Madison, Wisconsin, USA

Investigations of the bacterial family Micromonosporaceae have enabled the development of secondary metabolites critical to human health. Historical investigation of bacterial families for natural product discovery has focused on terrestrial strains, where time-consuming isolation processes leads to the rediscovery of known compounds. To investigate the secondary metabolite potential of marine-derived Micromonosporaceae, 38 strains were sequenced, assembled, and analyzed using antiSMASH against BiG-SLiCE. BiG-SLiCE represents a near-comprehensive dataset of approximately 1.2 million publicly available BGCs from primarily terrestrial strains³. Our marine-derived Micromonosporaceae were directly compared to BiG-SLiCE’s preprocessed database, and genetic diversity within our strains was uncovered using BiG-SCAPE and multidimensional scaling analysis (MDS). Our analysis of marine-derived Micromonosporaceae emphasized the need for broader genomic investigation of marine strains to enhance natural product drug discovery efforts for unknown compounds.

POSTER 4

Physiological Roles of an Acinetobacter-specific σ Factor

Emily Bacon^1,2, Kevin Myers³, Michael Place³, Jason Peters^1,3,4,5,6

¹Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, USA, ²Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA, ³Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, Madison, WI, USA, ⁴Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ⁵Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁶Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA

The Gram-negative pathogen, Acinetobacter baumannii, is classified as an urgent threat due to its resistance to nearly all clinically relevant antibiotics. Bacteria respond to antibiotic-induced stress on the cell envelope via signal transduction pathways that often utilize extracytoplasmic function (ECF) σ factors. Other Gram-negative pathogens, such as Escherichia coli and Pseudomonas aeruginosa, mitigate cell envelope stress via the well-characterized ECF σ factor σ^E. Although A. baumannii contains an ECF σ factor that we call σ^Ab, our in silico analysis of putative DNA-binding residues suggests that σ^Ab does not recognize the same promoter sequence as σ^E. Disruption of the sigAbgene results in sensitivities to many antibiotics, but its specific function and regulon are unknown. Here, we use RNA-sequencing in sigAb knockdown and overexpression strains to characterize the σ^Ab regulon. We show that σ^Abactivates many genes involved in transport and metabolism and find that several drug efflux pumps are members of its regulon. We also identify a σ^Ab-dependent promoter region upstream of the sigAb gene and show that sigAbexpression is necessary and sufficient for promoter activity. We aim to further characterize phenotypes associated with σ^Ab and determine its role in regulating antibiotic resistance genes.

POSTER 5

CRISPR Tools for Functional Genomics in Diverse Bacteria

Amy B. Banta^1,2, Ryan¹ D. Ward, Amy L. Enright^1,2, William J. Heelan¹, Jason M. Peters^1,2,3,4,5

¹Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, USA, ²Great Lakes Bioenergy Center, Wisconsin Energy Institute, ²Great Lakes Bioenergy Center, ³Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ⁴Department of Medical Microbiology,  and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁵Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA

Programmable, CRISPR-based approaches for gene perturbation have revolutionized bacterial functional genomics. Here, we develop mobilizable CRISPR tools for gene function discovery in diverse bacteria. We focus on CRISPR knockdown (CRISPRi) and CRISPR targeted transposition (CRISPRt) systems to alter gene expression or disrupt coding sequences. Building on our “Mobile-CRISPRi” platform, we demonstrate gene perturbation at scales from the individual gene to genome scale in medically and industrially relevant bacteria (e.g., Pseudomonas aeruginosa and Zymomonas mobilis). Given the robustness of CRISPR and the organism-agnostic nature of our delivery strategies, we anticipate that our CRISPR tools will be broadly applicable to the diverse research interests of the bacterial genetics community.

POSTER 6

Endogenous Oxylipins Promote Hyphal Tolerance of Echinocandin Antifungals in A. fumigatus

Dante G. Calise^1,2, Sung Chul Park¹, Jin Woo Bok¹, Nancy P. Keller^1,3

¹Department of Medical Microbiology and Immunology, University of Wisconsin – Madison, Madison, WI, USA, ²Microbiology Doctoral Training Program, University of Wisconsin – Madison, Madison, WI, USA, ³Department of Plant Pathology, University of Wisconsin – Madison, Madison, WI, USA

Aspergillus fumigatus is the leading etiological agent of invasive aspergillosis (IA) with a mortality rate upwards of 50 percent even with proper treatment. Azoles are the primary therapeutic option, but poor response demands the use of second line drugs including the cell wall targeting echinocandins like caspofungin. Echinocandins are a fungistatic drug against Aspergilli due to compensatory remodeling of hyphal architecture characterized by increases in branching, septation, and cell wall chitin. These morphological features are also known responses to the endogenous oxylipins 5,8-diHODE and 8-HODE produced by the linoleate diol synthase PpoA. The similarity in the morphological and transcriptomic responses to these compounds led us to hypothesize that 5,8-diHODE and 8-HODE are involved in activating echinocandin tolerant growth. In support of our hypothesis, we found that treatment of wild type A. fumigatus Af293 with caspofungin induced robust expression of ppoA as well as production of 5,8-diHODE and 8-HODE. Further, treatment with either oxylipin protected germlings against caspofungin mediated lysis. In addition to the commonly used lab isolates Af293 and CEA10, this protection was conserved in two clinical isolates of A. fumigatus showing high caspofungin susceptibility and tolerance respectively. Testing of deletion and overexpression mutants revealed the oxylipin responsive transcription factor ZfpA to be only a partial mediator of this effect suggesting the role of other transcription factors in this protection by oxylipins. Together these results demonstrate that 5,8-diHODE and 8-HODE act as signals to turn on an echinocandin tolerant growth program characterized by highly branched, chitin-rich hyphae.

POSTER 7

GRasp-ing new connections: uncovering genetic pathways in the mold, Aspergillus fumigatus, with our novel gene regulatory network resource

Cristobal Carrera Carriel¹, Saptarshi Pyne², Spencer A. Halberg-Spencer^2,6, Sung Chul Park⁵, Hye-won Seo⁵, Aidan Schmidt⁵, Dante Calise^5,7, Jean-Michel Ané^3,4, Nancy P. Keller^3,5, Sushmita Roy^2,6

¹Department of Genetics, University of Wisconsin-Madison, Madison, WI, USA, ² Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA, ³ Departments of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ⁴ Department of Agronomy, University of Wisconsin-Madison, Madison, WI USA, ⁵Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁶Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, USA, ⁷Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI USA

Aspergillus fumigatus is a pathogenic fungus responsible for harmful, occasionally lethal, respiratory conditions known as aspergillosis. Understanding the regulatory relationship between genes can not only shed insight into the fungus’ growth and development but also into the determinants of pathogenicity. Using publicly available expression datasets of Aspergillus fumigatus, we constructed a comprehensive gene regulatory network resource, GRAsp (Gene Regulation of Aspergillus fumigatus). We demonstrate that this resource can successfully recapitulate known regulatory pathways such as response to hypoxia, iron and zinc homeostasis, and secondary metabolite synthesis. We also experimentally validate one of GRAsp’s predictions: that the transcription factor AtfA is required for fungal responses to microbial signals known as lipo-chitooligosaccharides – a first and significant step to understanding this pathway. We further unveil an online, user-friendly version of GRAsp to allow the exploration of new and significant pathways in A. fumigatus.

POSTER 8

Discovery of Natural Antimicrobials from the Cheese Ripening Microbiota

Yu-Xing Chen¹, Aaron R. Gall¹, Tu-Anh Huynh¹

¹Department of Food Science, University of Wisconsin-Madison, Madison, WI, USA

Fermented foods harbor diverse and complex microbial communities that have been shown to exhibit antimicrobial activities. Cheese is a fermented food that is exceptionally well studied for microbiota composition and diversity. We found that Listeria monocytogenes, a human and animal bacterial pathogen, was greatly inhibited on the surface of wooden boards used in cheese ripening. Thus, we hypothesize that the wooden cheese board microbiota produces antimicrobials against L. monocytogenes. We systematically isolated bacteria from wooden boards used for different cheese types, and identified two species that inhibits L. monocytogenes: Serratia marcescens and Bacillus safensis. In response to these antagonistic bacteria, L. monocytogenes globally reprograms its gene expression related to acid stress response, efflux pump, carbohydrate utilization, and transcriptional and translational machineries, among other cellular pathways. Work is in progress to identify those antimicrobials and their mechanisms of action.

POSTER 9

Directed evolution to alter substrate selection in a model NRPS

Erin Conley^1,2, Ivy Lucier¹, Michael G. Thomas¹

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, ²Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA

Nonribosomal peptide synthetases (NRPS) are responsible for producing numerous natural products with impressive bioactivities, many of which have industrial, therapeutic, and agricultural applications. Engineering these enzymes for altered substrate selection is a long-standing goal because it would allow for formation of designer peptides. Despite decades of research in this area, a reliable method of altering substrate selection in NRPS enzymology has remained elusive. Here, a directed evolution pipeline involving random mutagenesis and an in vivo selection is used to identify functional enzyme variants that have undergone a change in substrate selection in a model NRPS. Variants exhibit altered substrate selection in vivo and in vitro, and DNA sequencing identifies the associated amino acid substitutions. Experiments are underway to assess substitution tolerance at the identified residue positions, and to determine transferability of the identified substitutions to additional NRPS systems. Our findings identify residue substitutions that would not be targeted in a rational reprogramming campaign, expanding our understanding of the full determinants of substrate selection in NRPS enzymology, and highlighting the utility of directed evolution to further the goal of generating designer peptides.

POSTER 10

Inhibiting the Intramembrane Aspartic Acid Protease PilD: Targeting a lynchpin of virulence in Pseudomonas aeruginosa

Christopher Dade^1,2, Jessica Cabading¹, Kate McKay^1,2, and Katrina T. Forest¹  

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA

The World Health Organization’s prognoses that the world is on the precipice of a “post-antibiotic” era underscores the critical need for antibiotic drug innovation; this includes novel drug targets in pathogens of concern and a reconceptualization of approaches to treating infections from anti-biotic to anti-infective strategies. One new target is the GxGD-type intramembrane cleaving aspartic acid protease PilD, which is essential for the assembly and function of both the Type 4 Pilus and Type 2 Secretion System in the opportunistic human pathogen Pseudomonas aeruginosa. PilD, however, is not essential for survival. While this enzyme has been biochemically characterized, PilD has never been systematically investigated as a drug target despite the apparent advantages of affecting multiple virulence phenotypes simultaneously. We are developing a high-throughput FRET-based PilD peptidase assay to screen potential inhibitors. Initial screening will be performed with an FDA-approved drug library, selected inhibitors of other aspartic acid proteases, and rationally designed peptides and peptidomimetics derived from the native PilD substrate. An orthogonal post-peptidase PilD methyltransferase inhibition assay is being optimized to validate initial inhibitor candidates. Work is ongoing to optimize the FRET-assay for high-throughput application and improve the orthogonal assay robustness. Inhibitors will be validated as anti-infective leads through bacteriostatic and bactericidal counter screens, and in vivo efficacy will be confirmed in a C. elegans infection model. This inhibitor screening and validation will identify the first specific inhibitors of PilD, provide insights into inhibiting intramembrane cleaving aspartic acid protease, and may produce new anti-infective drug candidates for the treatment of P. aeruginosa infections.

POSTER 11

Chemical tools to secure a niche: Penicillium expansum utilizes secondary metabolites to modulate microbial communities

Justin L. Eagan¹, 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, WI, USA, ²School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA,  ³ Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA, ⁴ Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Penicillium expansum is a postharvest pathogen of apples that contaminates products with two toxic secondary metabolites, patulin and citrinin. Mutant strains incapable of producing patulin or citrinin are still pathogenic, which prompts us to ask what benefit these compounds have to P. expansum. We hypothesize that secondary metabolites provide P. expansum an ecological fitness advantage by inhibiting other microorganisms in its environment. To test this hypothesis, we assessed growth of 138 yeast and bacterial apple isolates from three orchards in Wisconsin against extracts from wildtype, a patulin deletion mutant, a citrinin mutant or a double patulin/citrinin mutant of P. expansum. Extracts containing patulin exhibit antimicrobial activity against the majority of our apple microbiota, with the majority of bacterial isolates showing higher sensitivity than yeast isolates. Citrinin does not play a significant inhibitory role. A subset of bacterial isolates are sensitive to extracts from the double mutant, suggesting other P. expansum secondary metabolites have antimicrobial activity. Additionally, we are employing a model microbiome community called THOR to coculture with our P. expansum mutants to determine if fungal secondary metabolites can alter the composition of

POSTER 12

A potential Toxoplasma gondii lipoxygenase is necessary for virulence and changes localization associated with the host immune response.

Carlos J. Ramírez-Flores, Andrés M. Tibabuzo Perdomo, Billy J. Erazo, Katie L. Barnes, Sarah K. Wilson, Carolina Mendoza Cavazos, Laura J. Knoll

Department of Medical Microbial & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA

While the asexual cycle of Toxoplasma gondii can occur in any warm-blooded animal, the sexual cycle is restricted to the feline intestine. We previously determined that because cats lack delta-6-desaturase activity in their intestines, they build up excess linoleic acid, which signals T. gondii to undergo sexual development. We hypothesized that T. gondii oxygenates linoleic acid to signal sexual development, so we examined the T. gondii genome for potential lipoxygenases (TgLOX) enzymes. We identified seven potential TgLOXs that were at least 100-fold more abundant in the cat intestinal versus the tissue culture tachyzoite stage. Parasites deleted in TgLOX1 (TgDLOX1) had no significant growth differences in tissue culture fibroblast cells. Because the sexual development assay begins with brain cysts, we infected mice with TgDLOX1 and were surprised to find that TgDLOX1 had reduced virulence. The TgDLOX1 parasitemia was reduced by 3 days postinfection and largely cleared by 7 days postinfection. At 3 days postinfection, the cytokines IFN-γ, IL-6, MCP-1, and TNF-a were significantly reduced in TgDLOX1-infected mice, which prompted us to examine TgDLOX1 in IFN-γ KO mice. We found that IFN-γ KO mice infected with TgDLOX1 succumbed to acute infection with the same kinetics as the parental and complemented strains, suggesting the role of TgLOX1 in mice was IFN-γ dependent. In tissue culture fibroblasts, TgLOX1 was localized within the parasite, but in leukocytes from infected mice and activated macrophages, TgLOX1 was localized within the host cytoplasm. These results suggest that TgLOX1 changes localization in response to host immune activation.

POSTER 13

Roles of Essential Genes in Pseudomonas aeruginosa Biofilm Formation

William Heelan¹, Amy Banta^1,3, Warren Rose⁶, Jason Peters^1,2,3,4,5

¹Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, WI, USA, ²Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA, ³Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA, ⁴Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ⁵Department of Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁶Pharmacy Practice Division, University of Wisconsin-Madison, Madison, WI, 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.

POSTER 14

PASTA kinase-dependent control of peptidoglycan synthesis is required for adaptation of Listeria monocytogenes to cell wall stress in the cytosol

Jessica L. Kelliher¹, Caroline M. Grunenwald¹, McKenzie E. Daanen¹, John-Demian Sauer¹

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA

The mammalian cytosol is restrictive to microorganisms. Professional cytosolic pathogens must therefore possess adaptations that allow them to survive and replicate in this niche. Previously, we demonstrated that the PASTA kinase PrkA in the intracellular pathogen Listeria monocytogenes is required for cytosolic survival and ultimately virulence. PASTA kinases sense cell wall stress and phosphorylate multiple targets to mediate global physiological changes in response. Loss of PrkA function, either through genetic deletion or pharmacological inhibition, potentiates the effects of β-lactam antibiotics and other cell wall stressors. Here, to understand the mechanisms through which PrkA mediates resistance to cell wall stress, we used phosphoproteomics to identify PrkA targets, finding that during β-lactam exposure PrkA phosphorylates 23 proteins with functions in cell wall homeostasis, central metabolism, stress responses, and more. We show that proper phosphoregulation of the PrkA substrates ReoM and GpsB, both of which are involved in peptidoglycan metabolism, is required for the response to cell wall stress in vitro and survival in the cytosol. Cumulatively, our phosphoproteomics analysis indicates that PrkA is a global regulator of physiology, and our findings further suggest that a critical role of PrkA in vivo is modulating peptidoglycan synthesis to facilitate cytosolic survival and virulence.

POSTER 15

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence

Hans B. Smith¹, Kijeong Lee¹, Matthew J. Freeman¹, David M. Stevenson², Daniel Amador-Noguez², John-Demian Sauer¹

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI,USA, ²Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD⁺ while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD⁺/NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

POSTER 16

Analyzing Altered Substrate Selection in NRPS Enzymology

Ivy Lucier¹, Erin Conley¹, Michael Thomas¹

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

Nonribosomal peptide synthetases (NRPS) are multi-domain enzymes responsible for the production of specialized peptide metabolites, also known as natural products. The modular nature of the biosynthesis of these natural products, many of which have medicinal, industrial, or agricultural applications, makes them an attractive target for molecular engineering. However, to be able to introduce alternative precursors into natural products, we must first understand how to change the substrate specificity of NRPS enzymes. This project uses the NRPS EntF, responsible for production of the siderophore enterobactin, to find the amino acid changes necessary to alter substrate selection in this model system. Using a directed evolution pipeline, we have identified a functional variant of an EntF fusion protein that exhibits altered substrate selection. Here, we analyze this variant to determine which of the amino acid substitution(s) revealed via DNA sequencing is responsible for altered substrate selection. Using direct mutagenesis, we introduce the substitution hypothesized to alter substrate selection and assess in vivo fitness, as well as in vitro substrate selection of the purified enzyme. Identifying the amino acid substitutions necessary to alter substrate selection in this model system will inform future efforts to rationally reprogram NRPSs, with the ultimate goal of expanding our approach to additional NRPS systems.

POSTER 17

Post-Biogenesis Maturation of Pathogenic Fungal Spores Expands Germination Competence

Megan McKeon¹ and Christina M. Hull^1,2 

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA

Under growth-limiting conditions, the human fungal pathogen Cryptococcus initiates sexual development and produces spores to aid in the colonization of new, more suitable environments. To survive, these stress-resistant cells must remain dormant until they encounter favorable conditions for germination, an essential differentiation process in which dormant spores transition into vegetatively growing yeast. While spores and yeast are both able to cause disease, the identity of infecting particles has a profound effect on disease progression and outcome in a murine model of infection, with spores causing higher fungal burdens in the brain. Despite the known of impact of spore-mediated infection, the molecular networks governing essential spore processes remain poorly understood. Based on observations that spore responses vary within a population, we hypothesized that spores undergo changes after biogenesis (maturation) that enable successful germination in diverse conditions. To determine the nature ofCryptococcus spore maturation, we carried out a series of quantitative germination assays in various environmental conditions and inhibitors of specific biological processes. We discovered that immature spores are less efficient at germinating in complex carbon sources and that chromatin accessibility and translational capacity are differ between immature and mature spores. Transcriptomic analyses revealed distinct changes at the transcript level over the course of maturation. Taken together, our data suggest that spore maturation includes establishing a suitable chromatin state, modulating stored mRNA transcripts, and increasing ribosome biogenesis to poise spores to both sustain dormancy and germinate in diverse environments. These data also provide the first of evidence for a maturation process in spores of a basidiomycete fungus. We are continuing to define the intrinsic link between spore maturation and germination using multi-omics approaches and high-resolution, quantitative germination assays. Defining the molecular mechanisms governing these spore-specific processes will further our understanding of spore-mediated disease and aid in the development of new, more effective antifungal disease treatments.

POSTER 18

Thermal adaptation of Cryptococcus deneoformans to gradual increases in temperature

Nasya Miller¹, Jackie Spieles^2,3, Christina M. Hull^2,3

¹Department of ^\Genetics, University of Wisconsin-Madison, Madison, WI, USA, ² Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA, ³ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA

Climate change poses many threats, including the potential evolution of certain environmental microbes to adapt to living at human body temperature. One of these microbes, Cryptococcus deneoformans, is an invasive human fungal pathogen that produces germinating spores. C. deneoformans has been developed as a model for pathogenic fungal biology. Once spores are produced and inhaled, they germinate into yeast that spread throughout the body to other tissues, such as the brain. Because germination is required for disease, our lab has focused on the conditions that initiate germination and developed a quantitative germination assay (QGA) to measure it. Using microscopy, we determine the size and aspect ratios (w/l) of spores as they germinate to calculate the proportion of the sample with spore versus yeast characteristics. To assess the ability of non-pathogenic strains of C. deneoformans to adapt to growth at higher temperatures, we will slowly expose them to incrementally higher temperatures from 30°C to 37°C over time. We will then produce spores from this population. Using our germination assay, we will test how these spores germinate at high and low temperatures compared to non-adapted spores. We predict that spores from adapted strains will germinate more effectively at high temperatures relative to non-adapted spores. We will also test additional characteristics of the adapted strains using phenotype plates and other methods. Our data may help provide information on how global temperature increases due to climate change will impact environmental fungal pathogens.

POSTER 19

Engineering of Streptomyces for the Export of Fatty Acid Bioproducts

Neil T Miller¹, Michael G Thomas¹

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

Fatty acids can be utilized as fuel additives, lubricants, and plastics depending on their mixture, lengths, and branching. The microbial synthesis of such fatty-acid-based bioproducts is a possible way to add value to a biofuel synthesis pipeline. Because purification of bioproducts can be costly, it would be beneficial to produce materials that accumulate in spent media to avoid costs associated with obtaining the desired materials from cellular components. The actinobacterium Streptomyces sp. NP10 has been shown to accumulate fatty acids in spent media. An uncharacterized biosynthetic gene cluster in Streptomyces sp. NP10 was later shown to be responsible for the high production of free fatty acids in this strain. Although homologs to this gene cluster are rare, we have identified homologous gene clusters (termed fasA-K) in several other Streptomyces species, including Streptomyces griseussubsp. griseus ATCC 12648 (S.gg). In addition to possessing the fasA-K cluster, S.gg can produce detectable fatty acids in spent media. To characterize the fasA-K cluster, it was cloned into Streptomyces lividans, a strain that does not have homologs to the fasA-K cluster and has no detectable extracellular fatty acids. Initial characterization of this strain suggests that expression of the fasA-K cluster confers the ability to accumulate fatty acids in the spent media.

POSTER 20

Copper regulation of the antimicrobial isocyanide, brassicicolin A in Alternaria brassicicola

Nischala Nadig ¹, Sung Chal Park², Jin Woo Bok² , Nancy Keller, ^2,3

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, ²Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, ³Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI

Phytopathogenic Alternaria species are renown to produce toxins that contribute to virulence on target plants. Typically, these toxins belong to well known secondary metabolite chemical classes including polyketides, non-ribosomal peptides, and terpenes. However, chemical analysis of the purported host toxin brassicicolin A produced by A. brassicicola showed it to be an isocyanide, a chemical class whose genetics and encoding gene structure is largely unknown. The chemical structure of brassicicolin A shows it to have similarity to the recently characterized Aspergillus fumigatus fumicicolins derived from the isocyanide synthase CrmA. Examination of the A. brassicicola genome identified AbCrmA, a putative homolog with 64% identity to A. fumigatus CrmA. Deletion of AbcrmA resulted in loss of production of brassicicolin A. Contrary to reports that brassicicolin A is a host-specific toxin, the ΔAbcrmA mutants were equally virulent as the wild type on Brassica hosts. However, similar to A. fumigatus CrmA generated metabolites, we find that brassicicolin A increased 30-fold under copper starvation conditions. Also, like A. fumigatus CrmA metabolites, we find brassicicolin A to be a broad-spectrum antimicrobial. We speculate that these copper regulated isocyanide synthases provide the microorganisms they are present in a fitness advantage under copper deplete environments.

POSTER 21

Mining for a New Class of Fungal Natural Products: The Evolution, Diversity, and Distribution of Isocyanide Synthase Biosynthetic Gene Clusters

Grant R. Nickles¹, Brandon Oestereicher, Nancy P. Keller^1,2*, Milton T. Drott^3*

¹ Department of Medical Microbiology and Immunology, University of Wisconsin—Madison, Madison, WI, USA, ²Cereal Disease Laboratory, Agricultural Research Service, United States Department of Agriculture, Saint Paul, MN, USA, ³ USDA-ARS Cereal Disease Lab (CDL), St. Paul, MN, USA

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) have strong bioactivities that mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these genes across the Fungal Kingdom. We developed the first genome-mining pipeline to identify ICS BGCs, locating 3,800 ICS BGCs in 3,300 genomes. Genes in these clusters share promoter motifs and are maintained in contiguous groupings by natural selection. ICS BGCs are not evenly distributed across Fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the dit1/dit2 gene cluster family (GCF), which was thought to only exist in yeast, is present in ~30% of all Ascomycetes, including many filamentous fungi. The evolutionary history of the dit GCF is marked by deep divergences and phylogenetic incompatibilities that raise questions about convergent evolution and suggest selection or horizontal gene transfers have shaped the evolution of this cluster in some yeast and dimorphic fungi. Our results create a roadmap for future research into ICS BGCs. We developed a website (www.isocyanides.fungi.wisc.edu) that facilitates the exploration, filtering, and downloading of all identified fungal ICS BGCs and GCFs.

POSTER 22

Novel bioactive prenylated phenols derived from heterologous expression of a Pseudogymnoascus destructans squalene synthase gene in Aspergillus nidulans

Sung Chul Park¹, Jin Woo Bok¹, Rosa Ye², Raveena Gupta³, Matthew T Robey³, Chengcang C. Wu², Neil L. Kelleher³, Nancy P. Keller^1,4,*

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ²Intact Genomics, St. Louis, MO, USA, ³Department of Chemistry, Northwestern University, IL, USA,⁴Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Filamentous fungi produce a wealth of uncharacterized natural products (NPs) that are often challenging to characterize due to cryptic expression in laboratory conditions. Previously we have had success in isolating novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here we present a twist to FAC utility where we demonstrate that heterologous expression of a Pseudogymnoascus destructans FAC induced silent A. nidulans terpenes. Transformation of PdFAC1 into A. nidulans resulted in significant production of the host aspernidine-type metabolites compared to PdFAC1 free host extract. Of the total 10 prenylated phenolic compounds (1–10) isolated, four new aspernidine derivatives (1–4) were identified. Each of the 10 metabolites contained a farnesyl pyrophosphate (FPP) tail with different aromatic head, which could result in a significant difference in bioactivity. Nidulene A (1) and nidulene B (2) showed antibacterial activity against gram positive Staphyloccocus aureus and gram negative Escherichia coli. PdFAC1 contains a squalene synthase that when deleted resulted in loss of production of these terpenes. We hypothesize that expression of the P. destructans native squalene synthase in A. nidulans increases the pools of FPP, the precursor to aspernidine synthesis in A. nidulans.

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POSTER 23

High-throughput exploration of evolutionary trends within gene-clusters

Rauf Salamzade ¹, Patricia Tran ², Cody Martin ², Karthik Anantharaman ², Lindsay R. Kalan ^1,3,4

¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ² Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA, ³ Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA, ⁴ M.G. DeGroote Institute for Infectious Disease Research, David Braley Centre for Antibiotic Discovery, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

Recently, several software have been developed to visualize sequence-based relationships between homologous gene-clusters; however, these are largely limited in the information they provide and scale at which they can be applied. Here we introduce fai and zol, new bioinformatics tools that enable the rapid detection of homologous gene-clusters across thousands of genomes and subsequent evolutionary exploration of the genes within them using a table-based report. We applied these programs to search metagenomes for instances of a virus as well as to understand evolutionary trends of biosynthetic gene clusters encoding for toxins and anti-insecticidal secondary metabolites in fungi. To further demonstrate the scale at which the programs can be applied, we searched for a gene-cluster encoding a cell-wall associated polysaccharide across >5,000 genomes from the diverse bacterial genus of Enterococcus. Subsequent evolutionary exploration of orthologous instances of the gene-cluster uncovered that a key enzyme, previously shown to decorate the polysaccharide and aid mammalian gut colonization, exhibited substantial sequence diversity, with certain allelic clades potentially predating the emergence of common nosocomial pathogens.

POSTER 24

New Regulators of Gliotoxin Synthesis, HsfA and RogA, Identified through the Systems Biology Network GRAsp

Hye-won Seo¹, Cristobal Carrera Carriel², Saptarshi Pyne³, Spencer A. Halberg-Spencer^3,4, Sung Chul Park¹, Aidan Schmidt¹, Dante Calise¹, Jean-Michel Ané^5,6, Sushmita Roy^3,4, Nancy P. Keller^1,7*

¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI USA, ² Department of Genetics, University of Wisconsin-Madison, Madison, WI USA, ³ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI USA, ⁴ Department of Biostatistics and Medical Informatics, ⁵Departments of Bacteriology, University of Wisconsin-Madison, Madison, WI USA, ⁶ Department of Agronomy, University of Wisconsin-Madison, Madison, WI USA, ⁷ Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI USA

Aspergillus fumigatus, a world health organization priority pathogen, causes the often-fatal disease invasive aspergillosis in immunologically weakened patients. One of the well characterized virulence factors of this opportunistic fungus is the production of several toxic secondary metabolites such as gliotoxin and fumagillin. Although the biosynthetic pathways for A. fumigatus toxins are well characterized, there are still many unanswered questions on upstream regulators of these pathways. We have recently constructed a comprehensive gene regulatory network resource called GRAsp (Gene Regulation of Aspergillus fumigatus) to analyze A. fumigatus regulatory pathways (see the poster Carriel CC, 1). Using GRAsp, we have identified 13 genes predicted to regulate several A. fumigatus secondary metabolites. To start our study, we focused on two genes, AFUA_5G01900 and AFUA_3G11990, linked to gliotoxin regulation. AFUA_5G01900 encodes the heat shock protein HsfA (2) and AFUA_3G11990 encodes a C6 transcription factor we term RogA (Regulator of Gliotoxin). Gene expression data suggest RogA and HsfA regulate gliotoxin synthesis in opposite ways. RogA negatively regulates gliZ, the gliotoxin gene cluster transcription factor whereas HsfA is a positive regulator of gliZ. Interestingly, RogA and HsfA also appear to regulate endogenous gliotoxin self-protection pathways determined by gliT (encoding an oxidase) and gtmA (encoding a S-methyltransferase) expression. In synthesizing gliotoxin, dithiol gliotoxin, which is a toxic intermediate, is converted into gliotoxin or bis(methyl)gliotoxin by GliT and GtmA, respectively (3). Chemical analysis supports RogA as a negative regulator of gliotoxin synthesis with ongoing analysis of HsfA mutants.

POSTER 25

Infection induced inflammation impairs wound healing through IL-1b signaling

Simone Shen¹, Veronika Miskolci¹, John-Demian Sauer¹, and Anna Huttenlocher^1,2

¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Wisconsin, USA, ² Department of Pediatrics, University of Wisconsin-Madison, Wisconsin, USA

Wound healing is impaired by infection. How infection limits tissue repair and the role of inflammation remains unclear. We took advantage of the optical transparency of zebrafish and a genetically tractable microbe, Listeria monocytogenes, to probe the role of inflammation in healing of infected wounds. We found there is a critical window of antibiotic treatment needed to limit inflammation and enable repair. A strain engineered to activate the inflammasome, Lm-Pyro, induced robust inflammation and impaired healing, despite low bacterial burden. By contrast an apoptosis inducing strain, Lm-Apo, had similar burden but was associated with rapid repair. Inflammatory infection induced il-1bexpression and blocking IL-1b partly rescued wound healing despite persistent infection. We conclude that the inflammation associated with infection impairs wound healing and that the type of cell death influences outcome. These findings suggest that the dynamics of inflammation determines repair in complex tissue damage.

POSTER 26

Using TN-Seq to Identify Molecular Targets of Fungal Spore Germination Inhibitors

Jacqueline Spieles^1,2, Sébastien C. Ortiz¹, R. Blake Billmyre³, and Christina M. Hull^1,2

Departments of ¹ Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA, ² Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ³ Stowers Institute for Medical Research, Kansas City, MO, USA

Cryptococcus neoformans is an environmental fungus that produces spores as a fundamental survival mechanism. Germination of these spores is required for subsequent vegetative growth in new environments, including mammalian hosts. Spore germination has therefore been identified as a potential reservoir for new molecular targets for antifungal drug development. Previously, our lab identified 191 small molecule inhibitors of C. neoformans spore germination. Within this pool, eight prevalent substructure groups were identified, many of which also inhibit yeast growth and exhibit low cytotoxicity to mammalian fibroblasts. As an initial approach to identify targets of these novel antifungal molecules, we are employing genome-wide transposon mutagenesis with high-throughput sequencing (TN-seq). With this approach, we use a library of transposon insertion mutants in which each cell has a single transposon insertion, and the population is saturated to include mutations of every gene in the genome (an average one insertion per 13 bp genome-wide). When exposed to selective conditions (i.e., germination and growth inhibitors), cells with transposon-mediated mutations in genes required for survival become under-represented in the population. By sequencing just outside the transposon sequence in the selected population, we will identify genes differentially influenced by inhibitors during vegetative growth and spore germination. This method has been previously employed in C. neoformans to verify targets of the well-known antifungal drug fluconazole. Thus, with TN-seq we anticipate identifying affected pathways that will implicate molecular targets of these novel inhibitors of C. neoformans spore germination.

POSTER 27

The Combined Effect of Vitamin D and Ethanol on Murine PBMC Response to Mycobacterium bovis (BCG) Infection

Jayaraman Tharmalingam^1,2,3, Maya E Gough³, Elebeoba E May^{1,2,3, 4}

¹Wisconsin Institute for Discovery, University of Wisconsin – Madison, Madison, WI, USA, ²Department of Medical Microbiology and Immunology, University of Wisconsin – Madison, Madison, WI, USA, ³Department of Biomedical Engineering, University of Houston, Houston, TX, USA, ⁴Department of Medical Microbiology and Immunology, University of Wisconsin – Madison, Madison, WI, USA

Vitamin D, known to play a role in antimicrobial peptide synthesis in innate immune cells, plays a major role in adaptive immune response against bacterial infections. Globally millions of people are exposed or infected with Mtb, resulting in nearly 1 out of 4 people having latent Mtb infection (LTBI) (Ding C et al., 2022). The likelihood of active disease or LTBI outcomes increases in individuals with compromised or impaired immune systems (Kiazyk S and Ball TB., 2017). Host immunity can be impaired due to various factors, including nutritional status such as vitamin D deficiency (Sassi F et al., 2018) and alcohol use, which has many deleterious health effects including altered cellular immune response. Our previous studies explored the role of vitamin D in mycobacterium infection given in vivo conditioning and, in the presence, or absence of exogenous ethanol (Gough, et al. 2019). We expand our prior study to consider the compounding effect of concurrent vitamin D deficiency and alcohol use in immune cells. Mice were given vitamin D deficient/sufficient feed mixed with/without ethanol. Following the in vivo conditioning, we collected spleen and blood by cardiac puncture. Peripheral blood mononuclear cells (PBMC) from mice were infected with Mycobacterium bovis BCG. Culture supernatant was collected every 24hr from 0 to 96hr and used to quantify extracellular bacteria (ECB) and cell death. Our results indicate that ethanol-treated mice had reduced levels of circulatory immune cells and splenic cells regardless of vitamin D condition. We observed vitamin D sufficient cells oscillating between higher and lower ECB loads, indicating a modulatory role of in vivo vitamin D conditioning. Addition of ethanol negatively influenced the vitamin D dependent responses. In conclusion, ethanol alters the immune cell profile and increased cell death irrespective of vitamin D condition and extracellular bacterial counts. Our outcomes suggest that addition of vitamin D may enhance the bactericidal activity of antibiotics.

POSTER 28

Mapping an Acinetobacter baumannii essential gene network

Jennifer Tran^1,2, Ryan Ward^1,3, Amy Banta^1,4, Jason Peters^1,2,3,5,6

¹Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, WI, USA, ² Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA, ³Genetics Training Program, University of Wisconsin-Madison, Madison, WI, USA, ⁴Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA, ⁵Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ⁶Department of Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a significant threat to human health as a cause of antibiotic-resistant nosocomial infections. Despite its clinical relevance, our understanding of its core biology in the context of antibiotics is limited. Most A. baumannii essential genes have not been experimentally verified, and many remain uncharacterized. To better understand these essential genes, we used CRISPR interference to knock down all predicted essential genes and screened the resulting library with a panel of subinhibitory concentrations of antibiotics and other chemicals. By analyzing patterns of gene knockdown sensitivity and resistance, we created an essential gene network for A. baumannii. From this network, we identified several poorly characterized essential genes associated with cell division, raising the possibility of a unique A. baumannii divisome. We also showed that oxidative phosphorylation complexes (e.g., Complex I, Cytochrome bo3, ATP synthase) have distinct antibiotic sensitivity and resistance phenotypes, suggesting important roles for these complexes outside of electron transport. Furthermore, we identified chemical-gene interactions for small molecule inhibitors that offer insights into their modes of action. This study has provided a comprehensive understanding of essential genes and their response to antibiotics, thereby paving the way for new drug targets and strategies to combat A. baumannii infections.

POSTER 29

Determining transcriptional control of Cryptococcus spore germination using CRISPR/Cas9 and quantitative, high throughput microscopy

Olivia Valentine¹, Megan McKeon¹, Christina M. Hull^1,2

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA,  ²Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA

Cryptococcus is an environmental human fungal pathogen that grows as a budding yeast and produces spores that can be inhaled. To cause disease, Cryptococcus spores need to germinate into yeast and reproduce in the host. Therefore, understanding how spores germinate can help us develop approaches to prevent disease before yeast are formed and can grow to overwhelm the host. Based on previous RNA-sequencing data from germinating spores, we have identified four zinc finger transcription factors whose transcripts are highly dynamic during germination. We hypothesize that these factors play important roles in the regulation of Cryptococcus germination. To test this hypothesis, we are using CRISPR-Cas9 gene editing technology to knock out the genes encoding each of the transcription factors to generate deletion strains. Phenotype analyses and quantitative germination assays will be performed to assess the behaviors of the deletion strains and determine roles that deleted genes play in germination. These data will be used to better understand how germination in spores is regulated at the molecular level and inform further studies to develop germination as a drug target for prevention of fatal meningoencephalitis.

POSTER 30

Staphylococcal autoinducing peptide mimics capable of potent pan-group quorum sensing activation and inhibition in planktonic and biofilm communities.

Vulpis, T. D.¹; Eisenbraun, E. L.¹; Prosser, B. N.¹; Horswill, A. R.²; Blackwell, H. E.¹

¹Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA; ²Department of Immunology and Microbiology, University of Colorado, Aurora, CO, USA

We report the development of synthetic agonists and antagonists of the Staphylococcus epidermidis agr quorum sensing (QS) system capable of potent super-activation and complete inhibition, respectively. These non-native peptides maintain full efficacy across multiple agr specificity groups, offering exciting new utility for studying S. epidermidis in physiologically relevant mixed microbial communities. S. epidermidis is a leading cause of hospital-acquired and indwelling device infections due to its ability to form robust biofilms, a phenotype negatively regulated by the agr system. In cases of atopic dermatitis, however, S. epidermidis can exhibit virulence by producing the protease, EcpA, which degrades the skin barrier in an agr-dependent manner. Given this diverse behavior, these pan-group agr modulators are powerful tools for probing QS-controlled behavior in either setting and regardless of the specificity groups present. In the current study, we demonstrate that our pan-group agonists can appreciably decrease biofilm formation in vitro, underscoring the potential for their use as antivirulence agents. Furthermore, both the agonists and antagonists show strong, pan-group QS inhibition in Staphylococcus aureus, another opportunistic pathogen that produces a number of toxins under positive control of the agr system. Finally, we describe unexpected strong inhibitory behavior of certain of our pan-group agonists at sub-activating concentrations, which we hypothesize to be a result of divergent peptide secondary structure. Together, these tools will offer important insight into the role of QS in S. epidermidis infections and provide promising chemical strategies for combating broader Staphylococcal pathogenicity.

POSTER 31

Virulence reduction in enterohemorrhagic Escherichia coli caused by intestinal glutathione

Huiwen Wang¹, Vanessa Sperandio¹

¹Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that causes bloody diarrhea and even hemolytic-uremic syndrome (HUS) in humans. EHEC virulence includes a type III secretion system (T3SS) that is encoded by the locus of enterocyte effacement (LEE) pathogenicity island and results in attaching and effacing (A/E) lesions on intestinal epithelium, as well as the potent Shiga toxin responsible for HUS. Upon entry into the intestine, EHEC senses and responds to a complex milieu of metabolites derived from the host and intestinal microbiota, leading to precise modulation of its virulence program. Glutathione is a tripeptide that is highly abundant (1–10 mM) in the cytosol of most eukaryotes and some prokaryotes. Its major physiological role involves detoxification of reactive oxygen species. In addition to cytoplasm, glutathione is also abundant (about 10 mM) in human intestinal lumen, a reducing and anaerobic environment where glutathione remains the reduced form. Recent studies have shown that glutathione can be exploited by some intracellular bacterial pathogens, such as Listeria Monocytogenes and Burkholderia pseudomallei, as a signal to turn on the virulence programs. In this study, we examined the virulence-modulating effect of glutathione on EHEC using multifaceted approaches such as RT-qPCR and immunoblotting. When glutathione was supplemented in the in vitro anaerobic growth of EHEC, the bacterium showed a strong attenuation in the virulence, evidenced by the significantly decreased transcription and production of T3SS proteins encoded by the LEE pathogenicity island. Further studies are needed to investigate the molecular mechanism for virulence reduction caused by glutathione and in vivo role of glutathione on EHEC infection.

POSTER 33

Development of a Heterologous System for the Biosynthesis of Autoinducing Peptide Analogs that Inhibit Staphylococcus aureus Quorum Sensing

Danielle L. Widner¹, Hannah Vates¹, and Helen E. Blackwell¹

¹Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA

The Gram-positive pathogen Staphylococcus aureus requires an accessory gene regulator (agr) type quorum sensing (QS) system for coordinating virulence. This system is also required for S. aureus to colonize a human host, making quorum sensing inhibitors versatile tools for combating both S. aureus colonization and infection. There are four variants of S. aureus agr type QS systems, which are differentiated by the autoinducing peptide (AIP) signals. Several quorum sensing inhibitors have been discovered that attenuate signaling in all four agr types, including AIP analogs that differ from the native sequence by only a single amino acid. Previously, these AIP analogs and other related peptides have only been produced through solid-phase peptide synthesis. In this study, we show that AIP analogs can be robustly biosynthesized by modifying and transferring the AIP biosynthesis pathway into a non-pathogenic host. Specifically, strains of Bacillus subtilis were developed that constitutively express non-native AIP analogs at levels that fully inactivate S. aureus QS in a mixed microbial environment. This poster outlines the design and generation of the B. subtilis system and experiments toward characterizing the AIP products generated by these strains.

POSTER 34

Secondary Metabolite-Mediated Interkingdom Competition among Human Upper Respiratory Tract Microbiota

Susan Zelasko¹, Shelby Sandstrom², Mary-Hannah Swaney², James Gern³, Christine Seroogy³, David Andes⁴, Lindsay Kalan², Cameron Currie¹

¹Department of Bacteriology, University of Wisconsin, Madison, WI, USA, ²Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA, ³Department of Pediatrics, University of Wisconsin, Madison, WI, USA, ⁴Department of Medicine, University of Wisconsin, Madison, WI, USA

Microbial interactions mediating colonization resistance within the human respiratory tract remain understudied, particularly bacteria-fungal interactions that underly susceptibility to a range of clinically-significant infections. Herein, we characterized the antifungal potential of microbiota inhabiting the oral and nasal microbiomes of 212 participants from the Wisconsin Infant Cohort Study. Shotgun metagenomic sequencing of paired naso-oral samples and taxonomic annotation of quality-filtered reads via Kraken2/Bracken revealed significantly fewer fungal reads in nasal versus oral microbiomes. Alignment of reads to a curated in-house database of respiratory tract biosynthetic gene clusters (BGC) revealed significant differences in abundances of BGC classes across taxa inhabiting each body site. To elucidate patterns of antimicrobial inhibition, we performed high-throughput bioactivity screening of commensal bacteria from each body site, revealing nasal bacteria (n=766 isolates) demonstrate higher fractional inhibition of fungal pathogens, relative to oral bacteria (n=301 isolates). Subsequent bioactivity-guided fractionation of microbial extracts generated from nasal isolate SID4515 identified fractions with strong antifungal activity, including against Candida auris, via disc diffusion assay and an in vivo murine model of infection. Together, these data suggest nasal bacteria may utilize antimicrobial metabolites that mediate fungal inhibition in the human respiratory tract and such small-molecules serve as promising antifungal drug leads.