Abstracts

POSTER 1

Sex Differences during Spore-Mediated Infections in a Mouse Model of Cryptococcosis

Sehrish Afsheen¹, Nicolas Pereira¹, and Christina M. Hull¹ ²

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

Cryptococcus neoformans is an opportunistic pathogen that causes fatal human infections. Spores are thought to be infectious agents responsible for cryptococcal meningitis in immunocompromised people. Although extensive research has been conducted on the virulence and pathogenic mechanisms of Cryptococcus, the effects of sex differences on infection outcomes remain unexplored for both the spore and yeast forms. This study aims to identify differences between spore- and yeast-mediated cryptococcal disease in male and female mice. We used a mouse inhalation model of cryptococcosis to determine outcomes in spore and yeast infections, including days of survival after infection and organ fungal burdens in both sexes. Our preliminary findings indicate that male (n=10) and female (n=10) C57/Bl6 mice exhibited different survival rates in response to Cryptococcus spores. Males showed increased susceptibility to spore-mediated infections relative to females with a significant decrease in time-to-disease (p = 0.0359). In contrast, yeast-infected mice did not show any significant differences in disease susceptibility between sexes by the endpoint (n=10/sex). Fungal burden in the brain, lung, kidney, and spleen did not show a significant difference in fungal colony counts between both sexes infected with spores at end point. We hypothesize that dissemination to organs outside the lung occurs earlier in males, measure by colony forming units in organs over the course of CNS disease. Additionally, the roles of sex hormones in modulating immune responses likely influences sex-based differences in time-to-disease. Future studies will determine the timeline of dissemination in males vs. females with differences in immune cell functions during infections. This study emphasizes the significance of considering sex as a biological variable in studying fungal infections and also highlights the potential need for sex-specific therapeutic considerations when treating cryptococcosis.

POSTER 2

Investigating the Role of Vitamin D3 in Modulation of IL-12 and Nitric Oxide During MycobacteriumInfection

Azka Ahmed¹, Maya E. Gough³, Elebeoba May^1,2,3*

¹Medical Microbiology & Immunology, University of Wisconsin – Madison, Wisconsin, USA, ²Wisconsin Institute for Discovery, University of Wisconsin – Madison, Wisconsin, USA, ³Biomedical Engineering, University of Houston – Houston, Texas, USA

Mycobacterium tuberculosis (Mtb) results in 1.5 million deaths annually. Macrophages are the first cells to respond to Mtb and produce interleukin-12 (IL-12) and nitric oxide (NO) which are key components of the immune response against the bacteria; IL-12 helps activate T cells to produce IFN-γ and under certain conditions stimulates the autocrine production of IFN-γ by macrophages. IFN-γ then binds to receptors on the macrophage to activate NO production which directly contributes to bacterial killing in the phagosome and macrophage cell death. Macrophages are known to synthesize vitamin D3, which plays an important immunomodulatory and protective role in Mtb infection. Previous in-vitro studies have demonstrated that vitamin D3 regulates cytokines and effector molecules important in host cell defense against M.smegmatis in an infection-level dependent manner which in turn impacts bacterial clearance and host cell death. However, there are no studies to date that determine the differences in vitamin D3’s regulation of macrophage immune response during high vs. low M. bovis BCG infection with respect to IL-12/ IFN-γ signaling which in turn leads to NO production and the potential impact of this infection-level dependent NO regulation on bacterial load and host cell death. To test the hypothesis that vitamin D3-treated macrophages produce increased levels of IL-12, IFN-γ and NO to kill bacteria during high infection, but not during low infection vitamin D3/ethanol-treated and untreated J774 cells were infected with BCG at a MOI of 1 and 10. We found that vitamin D3-treated macrophages respond differently to pathogenic M.bovis BCG.  IL-12 and NO levels of vitamin D3-treated cells were reduced at  both high and low infection possibly as a protective mechanism to minimize host cell death. IFN-γ in vitamin D3-treated cells was higher than infected control cells, but significantly lower than ethanol-treated control. Finally, in high infection vitamin D3-treated cells had a higher intracellular bacterial load compared to infected control cells, but no significant changes in the extracellular load of vitamin D3-treated cells was observed. Future studies will involve investigating the differences in the immune signaling pathways activated in vitamin D3-treated cells in repsonse to M.smegmatis and M.bovis BCG.

POSTER 3

Advancing Spatio-Temporal Gene Expression Analysis with a Dual Fluorescent Reporter System in E. coli

Daniel Ajuzie^1,2, Jayaraman Tharmalingam¹, Stefan Gavosto¹, Angela Day¹, Azka Ahmed^1,2, Elebeoba E. May^1,2,3

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

Fluorescent reporter systems have become invaluable for real-time monitoring of gene expression at the single-cell level, enabling high-temporal and spatial resolution analyses in systems biology. Here, we present the Dharmacon-May library—a dual fluorescent promoter expression system in Escherichia coli—that builds upon the traditional Dharmacon library by incorporating an IhfB–mCherry plasmid to monitor bacterial growth alongside GFP-based promoter activity. Our system was evaluated using four gene constructs (fimD, fur, ihfB, and lacZ), and regression analyses comparing GFP expression in single versus dual fluorescent strains revealed strong consistency in gene expression measurements. Comparison with PCR based gene expression data confirmed these observations. Notably, while the addition of the mCherry plasmid imposed a modest metabolic burden that reduced overall growth rates, GFP expression remained largely unaffected, particularly in the ihfB construct where expression consistency was highest. Furthermore, correlations among optical density, fluorescent signals, and colony-forming unit counts underscored the enhanced accuracy of multiplex microreader data in quantifying cell density under various growth conditions. The Dharmacon-May library, therefore, provides an alternative for precise and reliable gene expression platform for dissecting complex, multi-phase cellular processes such as quorum sensing, biofilm formation, and stress responses in a spatio-temporal manner. These advances offer significant potential for integrating dynamic gene expression data into comprehensive metabolic models, particularly under conditions of multi-stress.

POSTER 4

Xenorhabdus szentirmaii is Natural Resource of Antimicrobial Compounds

Matin Kamara Ali¹, Steven Forst¹, Shama Mirza², Troy Skwor³ and Madhusudan Dey¹

¹Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA, ²Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, USA, ³Department of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

Antimicrobial resistance (AMR) poses a significant global health threat. To address this issue, it is crucial to discover new antimicrobial compounds with new chemistry. A potential source is the bacterium Xenorhabdus szentirmaii, a symbiont of nematodes that invade the insect larvae. The nematodes regurgitate the bacterium, which kills the insect larvae by releasing toxins and antimicrobials.

Recently our lab demonstrated that antimicrobial production in X. szentirmaii is primarily driven by 17 operons (designated as ste1 to ste17) that encode non-ribosomal peptide synthetases (NRPSs). In this study, we show that disruption of ste17 operon – predicted to encode a putative fabclavine-like compound – significantly reduced the ability of X. szentirmaii to secrete antimicrobial compounds. This result underscore ste17 as a promising target for the engineering and discovery of novel antimicrobial compounds.

POSTER 5

Engineering Penicillium Expansum for Enhanced Discovery of Cryptic NRPS-Derived Cyclopeptides

Mira Syahfriena Amir Rawa¹, Justin L. Eagan¹, Ben Haefner², Roberto Regalado¹, Christina Hull³, and Nancy P. Keller^1,2

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

Genetic dereplication by targeted deletion of major secondary metabolite biosynthetic pathways is an effective strategy to uncover novel compounds and cryptic biosynthetic gene clusters (BGCs). In this study, we engineered a “flatline” strain of Penicillium expansum by knocking out key biosynthetic genes responsible for producing citrinin, patulin, roquefortine C, andrastin C, and communesins. Comparative metabolomic analyses of the flatline and wild-type strains revealed a marked increase in the production of five-residue cyclopeptides featuring the unusual residue pipecolic acid when cultivated on rice medium, as determined by MS/MS fragmentation patterns. Isolation and structural elucidation of two of these cyclopeptides led to the identification of MBJ-0110 (1) and a new derivative (2), the latter distinguished by hydroxylation at the β-carbon of isoleucine. Bioinformatic analysis using antiSMASH predicted two non-ribosomal peptide synthase (NRPS) BGCs, each containing five to six adenylation domains putatively involved in the biosynthesis of these cyclopeptides. Our ongoing work aims to characterize the structure–function relationships of compound 2, disrupt the candidate NRPS genes to validate their role in cyclopeptide production, and assess the conservation of this cyclopeptide BGC across Penicillium species.

POSTER 6

Cryptococcus Spores Can Germinate in the Near Absence of Oxygen

Barnes, M¹ Spieles, J^1,2 Hull, CM^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

Cryptococcus spores must germinate into yeast to cause disease in mammalian hosts. Because Cryptococcus is a non-fermentative, obligate aerobe in its yeast growth state, we anticipated that hypoxic conditions would prevent oxidative phosphorylation and block germination. However, we discovered that it is possible for a subset of Cryptococcus spores to germinate in the near-absence of oxygen, revealing an apparent conflict between the need for ETC activity during germination and the lack of need for oxygen at the same time. To determine the role of the ETC in germination, we tested inhibitors of ETC complexes and conducted a transcriptomic analysis of spores germinating hypoxically. Our data suggest that a second electron transport pathway that uses part of the canonical ETC in conjunction with an alternative oxidase (AOX1) acts during hypoxic germination. Future experiments will determine the mechanisms of AOX1 function in the ETC of germinating Cryptococcus spores.

POSTER 7

Adaptive Strategies of Zymomonas mobilis Under Isobutanol Solvent Stress Revealed by Population Sequencing

Sydney Bradley^1,2,3, Julio Rivera-Vazquez^1,2,4, Eashant Thusoo^1,2,4, Isabella Colon^1,2, Evrim Fer^2,4, Kevin Meyers¹, Daniel Amador-Noguez^1,2

¹DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ³Cellular and Molecular Biology Graduate Program University of Wisconsin-Madison, Madison, WI, USA, ⁴Microbiology Doctoral Training Program University of Wisconsin-Madison, Madison, WI, USA

Microbial biofuels offer a sustainable alternative to our current dependence on fossil fuels; however, solvent toxicity remains a significant challenge for industrial-scale production. Adaptive laboratory evolution (ALE) leverages natural selection to drive microbial adaptation under controlled stress conditions, enabling the development of strains with enhanced resistance to solvent stress. To investigate the resistance mechanisms acquired by Zymomonas mobilis under isobutanol stress, we conducted an ALE experiment involving ten independent experimental lines and four controls. We incrementally increased isobutanol concentrations from 0.05M to 0.175M to promote adaptation and select for more tolerant strains. Here, we analyze six of the evolved populations and two controls through high-coverage population sequencing at eight distinct time points spanning 180 days. Despite relatively few genic mutations (an average of seven per line), distinct mutational trajectories emerged across replicates, revealing common adaptive strategies. Frequent mutations in pyruvate kinase (pyk), LytS, and MucR suggest these genes play key roles in regulating the broad stress response and enhancing solvent tolerance. Some populations exhibited competing adaptations, with multiple alleles of the same gene transiently coexisting before one variant became dominant, highlighting the dynamic nature of selection. These findings indicate that Z. mobilis employs a limited yet convergent set of genetic solutions to withstand isobutanol stress, providing valuable insights for future strategies in engineering more resilient biofuel-producing strains.

POSTER 8

Comprehensive Investigations into the Effect of N-acyl L-homoserine Lactone Tail Length and Oxidation State on Agonism and Antagonism Activity Across Seven LuxR-type Quorum Sensing Receptors

Cannell, I.D.¹­, Manson, D.E.¹, Blackwell, H.E.¹

¹Department of Chemistry, University of Wisconsin–Madison

Many common Gram-negative opportunistic pathogens, including Pseudomonas aeruginosa, regulate group behaviors in a population density dependent manner through a cell-cell communication system called quorum sensing (QS). QS systems often control virulence phenotypes in bacteria, such as biofilm growth, swimming and swarming motility, and exotoxin production. The most common and well characterized QS system in these bacteria is based on LuxI/LuxR-type synthase/receptor pairs. The LuxI-type synthase produces N-acyl-L-homoserine lactones (AHLs), with AHL concentration increasing with bacterial density. AHLs are detected by the transcription factors LuxR-type receptors, which then alter QS gene expression at high densities. AHLs share a common L-homoserine lactone headgroup across species, so specificity of AHLs for different LuxR-type receptors is dependent on tail structure. In native AHL signals, the tail structure varies in the length of the carbon acyl tail and the oxidation state of the third carbon in the tail, which can contain a 3-oxo, 3-hydroxy, or a fully reduced “straight chain”. In this study, a comprehensive set of straight chain and 3-oxo AHLs with tail lengths ranging from 1-20 carbons was synthesized and tested in seven well-characterized LuxR type receptors, providing structure-activity relationships (SARs) between tail length and LuxR-type receptor activity. These SARs allowed us to form hypotheses about the role of AHLs in multi-microbial systems and to characterize ideal acyl tail lengths for receptor agonism and antagonism across a range of LuxR-type receptors.

POSTER 9

Role and Regulation of Dispersal in an Opportunistic Pathogen, Candida Albicans

Eli Cytrynbaum¹, Megan McClean²

¹Department of Cellular and Molecular Biology, University of Wisconsin – Madison, Madison, WI, USA, ²Department of Biomedical Engineering, University of Wisconsin – Madison, Madison, WI, USA

Candida albicans is a prevalent, generally commensal member of the human mycobiome carried by most people their entire lives. Simultaneously, C. albicans is a major opportunistic pathogen – in immunocompetent individuals this generally manifests in vulvovaginal (yeast infections) and cutaneous infections while in immunocompromised people, C. albicans can also cause superficial oral infections (thrush) or internal solid organ or bloodstream infections. C. albicans’ pathogenesis is associated with a cycle of biofilm formation and dispersal, and its ability to invade a variety of host tissues while evading the immune system and treatment is in part due to its ability to switch between cellular states with distinct metabolic signatures, adhesins, and morphologies. Candida cell state transition has also been linked to host immune and metabolic regulation. Despite the importance of these transitions, dispersal– the process by which hyphal cells give rise to yeast-form cells is poorly understood. Contrary to previous results, we have found that dispersal from biofilms occurs as a discrete event within biofilm development coinciding with the cessation of biofilm expansion regulated by intercellular signaling. By identifying the specific mechanisms of this regulation, we hope to uncover potential targets for treatment to encourage commensalism over pathogenesis.

POSTER 10

Tiny Earth Chemistry Hub (TECH): An Antimicrobial Discovery Pipeline

Martel DenHartog¹, Josephine Putnam¹, Tasha Miller^1,2, Neha Kashyap³, Marc Chevrette³, Sarah Miller¹, Jo Handelsman^1,2

¹Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, Wisconsin, USA, ²Department of Plant Pathology, University of Wisconsin–Madison, Madison, Wisconsin, USA, ³Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA,

Pathogenic microbes are poised to cause major human and plant health concerns as bacteria evolve resistance to current antibiotics and climate change expands pathogen habitats. Yet, industry has largely abandoned antimicrobial discovery efforts. Tiny Earth addresses this growing crisis with a global network of students and instructors discovering antimicrobial-producing bacteria from soil in a course-based undergraduate research experience (CURE). To learn more about the Tiny Earth network, visit tinyearth.wisc.edu. ​The Tiny Earth Chemistry Hub (TECH) was established in 2018 at the Wisconsin Institute for Discovery as part of the “Science and Discovery” core of Tiny Earth. TECH uses phylogenetics and genomics to identify candidates for chemical isolation and structural elucidation and develops screening methods to test Tiny Earth strains for suppression of plant diseases. Of particular interest to TECH are Tiny Earth strains that control oomycete plant pathogens such as Globisporangium ultimum, which causes damping off and root rot of important agricultural crops like wheat, soybean, and corn. Using in vitro and soybean plant assays, TECH identifies strains with disease-suppressive potential. TECH has also developed a Tiny Earth Summer Research Course (TESRC) that provides students at two-year colleges an authentic research experience at a university. Students who participated in TESRC increased their sense of belonging in science and research skills, and many students are now enrolled in four-year programs or working in STEM careers.

POSTER 11

Modulation of Th17 Activation by Beta-Hydroxybutyrate: A Double-Edged Role in Inflammatory Bowel Disease and Infection

Wenxuan Dong¹, Alejandra Ruiz¹, Gillian Hughes¹, Bethany Korwin-Mihavics¹, Kevin Schwartz¹, Kate Stack¹, Chi (Felix) Yan¹, Gustavo Caballero-Flores¹, Margaret Alexander¹

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

T helper 17 (Th17) cells are involved in the pathogenesis of autoimmune diseases but are also vital for preserving barrier integrity and defending against extracellular pathogens. Overactivation of Th17 cells exacerbates autoimmune conditions such as inflammatory bowel diseases (IBD). Paradoxically, clinical use of interleukin 17 (IL-17, the Th17 effector cytokine) inhibitors has been associated with exacerbation of IBD symptoms, suggesting that IL-17 levels need to be modulated within a homeostatic range, as deviations to either side may contribute to pathogenesis of IBD. Ketogenic diets (KD) and the KD-induced metabolite beta-hydroxybutyrate (BHB) can mitigate inflammation by reducing Th17 activation in mouse models of multiple sclerosis, a neuroinflammatory autoimmune disease. However, their impact on Th17 responses in IBD is unclear. In this study, we explored the effects of BHB on Th17 cell activation in the context of IBD. Analysis of public RNA-seq data revealed that proinflammatory Th17 gene markers are enriched in inflamed colon tissues from IBD patients, while ketogenesis-related genes are downregulated. Notably, IL17A expression, a key Th17 effector cytokine gene, was negatively correlated with HMGCS2, the rate-limiting enzyme in ketogenesis. To assess the direct immunomodulatory potential of BHB, we performed an in vitro Th17 skewing assay using splenic naïve CD4+ T cells skewed to Th17 fate. BHB inhibited IL-17a production in a dose- and pH- dependent manner. Next, we supplemented the mice with 1,3-butanediol (BD), a BHB precursor, to distinguish between the effects of KD and BHB on Th17 cells. Both KD- and BD-treated mice exhibited elevated circulating BHB levels compared to control mice. Flow cytometry analysis revealed that both KD and BD significantly reduced intestinal Th17 cell levels. Moreover, ileal Th17 cell levels were negatively correlated with circulating BHB concentrations, suggesting a potential role for BHB in limiting Th17 activation in vivo. In disease models, KD exacerbated dextran sulfate sodium (DSS)-induced colitis while reducing ileal Th17 cells. BD treatment worsened Citrobacter-induced systemic infection, likely due to impaired Th17-mediated mucosal immunity against this extracellular pathogen. These findings highlight the dual role of Th17 cells in gut immunity – while pathogenic in chronic inflammation, Th17 cells appear protective during acute infection and colitis. In conclusion, our findings provide preliminary evidence that KD and BHB suppress Th17 activation but may disrupt intestinal immune homeostasis under certain inflammatory conditions.

POSTER 13

Functional Determinants of Bacillus subtilis Type IV Pili

Rachel Erickson^1,2, Kasia A Gromek², Briana M Burton²

¹CALS Honors in Research, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

Bacteria can acquire genetic information through the process of horizontal gene transfer including via natural transformation, in which bacteria actively take up DNA from the environment and incorporate it into their genome. In Bacillus subtilis, this process requires a type IV pilus (T4P) primarily composed of ComGC proteins. Each ComGC monomer contains two cysteine residues that form an intramolecular disulfide bond. There is evidence of this disulfide isomerizing and forming intermolecular disulfide bonds between the ComGC proteins. Evidence from Westerns run in reducing and nonreducing conditions suggests that the disulfide bond might isomerize to form intermolecular bonds between the ComGC proteins. This intermolecular disulfide bond would make the ComGC system unique considering other pilus systems are held together through noncovalent protein interactions. However, this evidence not conclusive. We asked whether an inter-subunit interaction is necessary for the activity of the T4P during B. Subtilis transformation. Using predicted R-group distances from PyMol models, we replaced the cysteine residues with salt-bridge forming residues and created strains with only the salt bridge-containing comGC. We tested these salt bridge mutants for their ability to facilitate natural transformation. Transformation efficiency assays revealed these mutants were un-transformable. Western blot analysis showed the mutant proteins were expressed at considerably lower levels compared to WT. To assess possible reasons for the lack of transformability, we purified protein variants and ran gel filtration profiles. The salt bridge variant was soluble and had a different elution profile compared to WT. The results from gel filtration suggested two possible scenarios, either the salt bridge variant was running as a dimer or has adopted a different conformation than the wild type ComGC . After rerunning the salt bridge variant under high-salt conditions where it migrated at the same size, we favor the idea that the salt bridge variant has an altered conformation.  Next we will conduct in vivo imaging of the salt bridge variant to see whether it can still assemble into pili.

POSTER 14

An Aspergillus fumigatus Homeobox Transcription Factor Regulates Multiple Developmental Responses to Copper Starvation

Harrison Estes¹, Jacob Gutierrez², Sung Chul Park³, Dante Calise³, Grant Nickles³, Jin Woo Bok³, and Nancy Keller^3,4

¹Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA, ³Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁴Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Copper homeostasis is a fundamental property in all organisms, yet the developmental response to copper starvation remains poorly characterized in the opportunistic filamentous fungal pathogen Aspergillus fumigatus. Machine learning analysis of RNAseq datasets identified Afu4g04320, one of seven putative homeobox transcription factors in A. fumigatus, as a candidate transcription factor relevant to copper response pathways. Through deletion and overexpression of this transcription factor, we investigate its influence upon physiology and development under both standard and copper-depleted conditions. Phenotypic assessments revealed that Afu4g04320 modulates multiple developmental processes across both environmental contexts. Notably, overexpression of this transcription factor under copper starvation substantially enhanced biomass accumulation and germination efficiency, while its deletion impaired germination under identical conditions. There were also notable phenotypic differences present under copper replete conditions, including altered hyphal morphology and increased germination in the overexpression strain. These findings suggest Afu4g04320 to be important in A. fumigatus developmental transitions that are most pronounced under copper starvation.

POSTER 15

A Broad-Spectrum Antimicrobial from the GRAS Fungus Aspergillus Oryzae to Combat Human Pathogens

Xingrui Fan^1,2,3, Dasol Choi^1,2, Lucy M. Wersinger^1,2, Alanah S. Kaufmann^1,2, Jae-Hyuk Yu^1,2,3 

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ²Food Research Institute, University of Wisconsin-Madison, Madison, WI, USA, ³Department of Food Science, University of Wisconsin-Madison, Madison, WI, USA

Antimicrobial resistance (AMR) represents a critical global health challenge, as multidrug-resistant bacterial and fungal pathogens render conventional treatments increasingly ineffective. To address this global threat, we have developed Natural Protectant (NP), an antimicrobial product derived from the Generally Recognized As Safe (GRAS) fungus Aspergillus oryzae. NP is produced via the fermentation of food substrates, showcasing broad-spectrum antimicrobial activity against both bacterial and fungal pathogens, including those identified as serious AMR threats by the CDC and WHO. In broth culture, NP effectively eradicated methicillin-resistant Staphylococcus aureus (MRSA) and enteropathogenic Escherichia coli O157:H7, achieving greater than 1,000-fold reductions within 24–48 hours. Additionally, NP demonstrated strong inhibitory effects against other ESKAPE pathogens, a group of antibiotic-resistant bacteria highly associated with healthcare-associated infections. Beyond its antibacterial properties, NP exhibited potent antifungal activity, effectively killing human pathogenic fungi Candida auris, Candida albicans, and Aspergillus fumigatus,including azole-resistant strains. Encouraged by these results, we aim to further develop NP as a therapeutic for bacterial and fungal infections. Preliminary toxicity testing has shown that NP was not toxic to human MCF-7 cells, suggesting its potential for clinical application. Future efforts will focus on isolating and identifying the specific antimicrobial molecules in NP to develop a novel, broad-spectrum antibiotic that could play an important role in combating AMR and improving public health.

POSTER 17

Determining the Surface Composition of Cryptococcus Neoformans Spores

Seth Greengo¹ and Christina 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

The makeup of the exterior of a cell is a crucial determinant of how it will interact with its environment, such as a host organism during infection. The basidiomycete yeast Cryptococcus neoformans is a meningitis-causing pathogen of humans, especially in immunocompromised people. The surface composition of Cryptococcus yeast is generally understood – it has a cell wall composed of chitin, chitosan, and alpha- and beta-glucans, as well as a polysaccharide capsule that is primarily made up of glucuronoxylomannan (GXM). Though the yeast surface has been the subject of many studies, far less is known about the surface of Cryptococcus spores. Spores are the product of the Cryptococcus sexual reproduction cycle and are thought to be infectious particles in cryptococcal disease. Previous research has demonstrated that the exterior of spores, called the spore coat, contains some of the same components as the yeast exterior, including GXM, but also some spore-specific polysaccharides or arrangements of polysaccharides. Additionally, it is known that Cryptococcus spores and yeasts interact differently with host phagocytes, which in turn can produce different disease outcomes. By using techniques like fluorescence microscopy and flow cytometry, we are investigating the surface composition of the Cryptococcus spore and comparing it to that of the yeast. We will also examine the way the cell surface changes during spore germination. These early studies will lead to greater explorations into the biosynthetic pathways that produce these polysaccharides and assemble the spore coat.

POSTER 18

The Effect of Melatonin in the Regulation of EHEC virulence

Ebru Guver¹, Vanessa Sperandio¹

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

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne human pathogen colonizing the colon, causing global outbreaks of bloody diarrhea and potentially leading to life-threatening hemolytic uremic syndrome. The pathogen’s low infectious dose and severe complications make it particularly dangerous. Identifying the molecular mechanism of EHEC virulence is a challenging task to elucidate how colonization is modulated within the gut environment.

EHEC employs a type III secretion system (T3SS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to produce attaching and effacing (AE) lesions. EHEC modulates virulence gene expression in response to environmental cues within the gastrointestinal (GI) tract, including neurotransmitters. Serotonin, a tryptophan derivative neurotransmitter produced predominantly by GI enterochromaffin cells, has been shown to decrease EHEC virulence. Building on this, we explored the effects of melatonin, another tryptophan-derived molecule, which is found in the GI tract at concentrations up to 400 times higher than in the pineal gland. While melatonin is known to play a role in immune defense against certain bacterial infections, its impact on EHEC pathogenesis remains unexplored.

Here, we specifically asked if melatonin is shaping host-microbial interactions that can be utilized for the management of EHEC. Optimizing in vitro conditions, certain concentrations of melatonin lead to increased LEE expression in transcription studies. Transcriptomic analysis confirmed up-regulation of LEE genes, T3SS secreted effector and structure proteins, while tryptophanase and flagellin genes were down-regulated with melatonin treatment. Colonizing mice with Citrobacter rodentium as a murine model of EHEC infection, we investigated the effects of melatonin administered via drinking water or intraperitoneal (IP) injection. The in vivo studies shows that mice receiving melatonin in drinking water exhibited higher CFUs and reduced survival, suggesting that gut-mediated delivery impacts colonization and host health differently than systemic IP administration. Together, this work highlights the impact of melatonin on EHEC virulence and how environmental signals in the gut can shape the progression of enteric infections.

POSTER 19

Community-Regulated Biosynthetic Gene Cluster Alters Gene Expression Networks in Bacillus cereus

Austin Hall^1,3, Natalia Rosario-Meléndez^2,3, Rilee Schmit¹, Jo Handelsman^1,3;

¹Plant Pathology, University of Wisconsin- Madison, Madison, WI, USA, ² Microbiology Doctorate Training Program, University of Wisconsin- Madison, Madison, WI, USA, ³Wisconsin Institute for Discovery, Madison, WI, USA

Secondary metabolites are non-essential compounds produced by plants, bacteria, and fungi to increase fitness in an environment-dependent manner. Many secondary metabolites function as antibiotics, and others act as molecular signals, enhancing host growth or regulating gene expression in other community members. To explore the effect of community life on regulation of secondary metabolite production, we performed RNASeq on a model rhizosphere community containing three members—plant symbiont, Bacillus cereus UW85, Pseudomonas koreensis, and Flavobacterium johnsoniae. Notably, expression of several biosynthetic gene clusters (BGCs) in B. cereus was altered by the presence of the other two community members. For example, the presence of P. koreensis downregulated one BGC, a hybrid trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS-NRPS) cluster, that is normally highly expressed in monoculture. Comparative metabolomics of the wild type and a mutant containing a deletion of the entire BGC and revealed several candidate molecules produced by products of this cluster. Furthermore, comparative transcriptomics and correlation analysis demonstrated that deletion of the gene cluster alters production of other secondary metabolites in B. cereus, leading to differences in regulation of P. koreensis biofilm formation and antagonism of the fungal plant pathogen Sclerotinia sclerotiorum. Our data suggest that the product of the PKS-NRPS cluster regulates expression of other secondary metabolites in B. cereus.

POSTER 20

Gut Microbiota-Derived Metabolite Trimethylamine N-oxide Alters the Host Epigenome through Inhibition of S-adenosylhomocysteine Hydrolase

Jessica H. Han^{1, 2, 3}, Federico E. Rey², John M. Denu^{1, 3}

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA, ²Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, ³Wisconsin Institute for Discovery, Madison, Wisconsin, USA

Trimethylamine N-oxide (TMAO), a metabolite derived from gut microbiota, is produced from dietary choline, carnitine, and phosphatidylcholine. It has been implicated in the progression of inflammatory and metabolic diseases. Prior studies in gnotobiotic mice revealed that colonization with choline-consuming bacteria leads to elevated systemic TMAO levels when fed a high-choline diet.  These mice exhibited reduced DNA methylation across various tissues compared to genetically modified counterparts incapable of producing TMAO. These findings highlight the profound impact of gut microbial TMAO on host metabolic and transcriptional processes.

Building on these observations, our research investigates the metabolic and epigenomic alterations induced by TMAO in mammalian hosts. We aim to delineate TMAO’s effects on chromatin remodeling, metabolic pathways, and its inhibition of the critical enzyme S-adenosylhomocysteine hydrolase (SAHH).

In conventionally raised mice, elevated TMAO levels resulting from a high-choline diet or direct TMAO supplementation induced significant chromatin remodeling and altered circulatory levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Exposing cultured cells to pathophysiological levels of TMAO led to reductions in homocysteine and adenosine levels and increased SAH, suggesting inhibition of SAHH activity. Using recombinant SAHH in an LC-MS-based enzymatic assay, we found that TMAO functions as a noncompetitive inhibitor, reducing enzymatic efficiency and impairing adenosine production. These findings were further supported by chromatin analyses, which revealed hypomethylation of histones at loci similar to those observed with SAHH inhibitor, 3-deazaadenosine.

To investigate TMAO’s maximal inhibitory effects, we are treating cells with higher TMAO concentrations at 500µM and conducting chromatin analysis to identify additional hypomethylation sites. As a potential therapeutic strategy, we propose overexpressing MAT2A, a SAM-producing enzyme, to restore SAM levels and counteract SAH accumulation. This approach may recover methyltransferase activity and normalize histone methylation disrupted by TMAO.

This study highlights TMAO’s role as a modulation of chromatin remodeling and metabolic regulation. By elucidating the interplay between diet-derived metabolites and epigenetic modifications, our findings provide a foundation for developing therapeutic strategies targeting TMAO-related pathologies.

POSTER 21

Mapping Origins of DNA Replication in Cryptococcus

Amelia Hansen¹, Timothy Hoggard², Catherine A. Fox², and Christina M. Hull²

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

Eukaryotic DNA replication is a vital and necessary step to transfer genetic information to progeny and thereby perpetuate the species. Eukaryotic DNA replication is tightly regulated temporally and spatially with DNA replication strictly occurring during the S-phase of the cell cycle and initiating at discrete genomic loci called origins of DNA replication, henceforth origins. Disrupting the distribution of origins in a genome can sensitize a cell to replication pathologies, copy number changes, and breaks in chromosomes. While much work has led to our understanding of origin licensing in model eukaryotic genomes ranging from the budding yeast Saccharomyces cerevisiae to humans, far less is known in other fungi, especially pathogenic fungi. Cryptococcus is a pathogenic budding yeast and model organism for spore-forming pathogenic fungi. Previous work suggests that Cryptococcus lacks several of the “conserved” and essential components that form the pre-replicative complex (pre-RC). This observation raises the question of how and where Cryptococcus forms origins in its genome. To determine where origins can form in Cryptococcus, we are using SortSeq.  SortSeq uses flow cytometry-based sorting coupled with high-resolution next-generation sequencing (NGS) to compare copy number changes between S-phase and G1-phase populations. Inflections in copy number ratio (S:G1) reflect DNA replication initiation events and thus origins of replication. Importantly, we are using SortSeq to map origins in both Cryptococcus yeast and germinating spores to compare replication for these two cell types to better understand both genome organization and timing of gene expression during escape from dormancy.

POSTER 23

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, Wisconsin, USA, 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.

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 24

Bacteriophage Infection Drives Loss of β-lactam resistance in Methicillin Resistant Staphylococcus Aureus

Angel J. Hernandez Viera¹, My H Tran¹, Patricia Q. Tran¹, Charlie Y. Mo¹

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

Staphylococcus aureus is a bacterium that causes a wide range of diseases, from endocarditis to pneumonia. Methicillin-resistant S. aureus (MRSA) displays resistance towards β-lactam antibiotics, one of the most commonly prescribed drug classes. The usage of bacteriophages (phages for short) has been proposed and used as an alternative to combat antibiotic resistant bacterial infections. However, similar to antibiotics, phage predation exerts a strong selective pressure on bacteria, making the rise of phage resistance inevitable. Crucially, evolution of phage resistance can cause genetic trade-offs that resensitize bacteria to antimicrobials. Here we report that staphylococcal phages drive three MRSA model clinical strains to evolve sensitivity to β-lactam antibiotics, resulting up to a 1000-fold reduction in minimal inhibitory concentration (MIC) against select β-lactams. Sequencing analysis showed that each MRSA strain evolved distinct mutational profiles, suggesting that different strains of the same bacterial species can take different evolutionary trajectories to evolve resistance to phage predation. Transcriptomic analysis further revealed that phage-resistant MRSA strongly modulated their gene expression profiles, notably down-regulating genes involved in virulence. Indeed, we found that phage predation reduced hemolytic and clumping ability – two key virulence determinants – in the MRSA strains. Finally, we show that the reduction in β-lactam resistance caused by phage predation is conserved in eight MRSA clinical strains that were isolated between 2008 and 2011. These findings open up new possibilities to reduce drug resistance and virulence in MRSA infections.

POSTER 25

Regulation of EHEC Virulence by Pyruvate and BtsS/BtsR 

Jacob Hotvedt¹, Vanessa Sperandio¹

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

Enterohemorrhagic E.coli (EHEC) is a foodborne pathogen affecting an estimated 63,000 Americans per year. An EHEC infection is usually acquired through ingesting contaminated water or food, with the first outbreaks involving consumption of undercooked meat. Most cases involve vomiting and bloody diarrhea. A key pathogenicity island that leads to gastrointestinal disease is named the Locus of Enterocyte Effacement (LEE), which encodes the type three secretion system, effectors, chaperones, and other virulence factors. The LEE genes are activated by the master regulator, Ler, which is highly regulated and under the control of many signals and other transcription factors.  In addition to the LEE, EHEC carries the phage-encoded toxin, Shiga toxin, which is toxic to the kidneys and leads to Hemolytic Uremic Syndrome (HUS) in 3-7% of cases. Other non-LEE virulence factors include the Non-Lee Encoded effectors NleA-H. Among the molecules regulating EHEC virulence gene expression is pyruvate.

The pyruvate regulator system, BtsS/BtsR, formerly known as YehU/YehT, is a two-component system which senses pyruvate to upregulate expression of the pyruvate importer BtsT. In a screen of E.coli K12 engineered to contain the LEE, BtsS was identified as a possible regulator of the LEE. We have identified BtsS/BtsR and pyruvate as possible repressors of EHEC virulence gene expression and virulence factor secretion in vitro. A ∆btsS mutant exhibited increased virulence in a cell culture model of infection. Preliminary data indicates that this regulation is direct through binding of BtsR to the regulatory region of the master regulator Ler. Further work is in progress to determine the sequence of the binding site, along with any other possible binding sites. Additionally, based on earlier studies suggesting BtsS functions as a peptide sensor, and our recent findings that, when co-cultured with EHEC, Enterococcus faecalis produces bioactive fractions containing small peptides, we are also investigating if BtsS senses autoinducing peptides.

POSTER 26

Defining Evolutionary Pathways to Drug Resistance in Influenza Virus

Rodrigo Ibarra¹, Katherine Amato¹, Andrew Mehle¹

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

Influenza A viruses (IAV) constant evolution results in recurring epidemics with the chance to cause pandemics. This evolutionary process occurs on a global scale where positive selection drives fixation of fitter variants. Conversely, on the local scale within an infected individual, evolution seems more random and potentially fit variants rarely rise to high frequency. This results in a conundrum between global and local evolution, where evolutionarily advanced variants detected on the global scale are rarely seen in acute infections on the local scale. How and where influenza virus evolution changes from a process driven mostly by randomness to one that is mostly deterministic remains unclear. Here we use the emergence of drug resistance to dissect selection and emergence of evolutionarily advanced viruses within a host. Drug resistance could emerge once and sweep the population, or it might need to arise independently on multiple lineages before it dominates. Both pathways have distinct implications on the barrier to drug resistance and population-level fitness. We used barcoded virus libraries to uniquely track and quantify over 10⁵ distinct lineages. These barcoded viruses were challenged with the polymerase inhibitor Baloxavir Marboxil (BXA; Xofluza). Serial passaging in the presence of BXA produced drug-resistance at a population level. Barcode sequencing revealed reduced barcode diversity on BXA treated viruses after each passage with a single lineage dominating the population suggesting the selection of drug resistance variants. Viral populations were sequenced to identify BXA-resistance mutations. Genetic linkage between escape mutations and barcodes revealed the specific evolutionary pathways taken as influenza virus acquired drug resistance. Together, these data provide a fine-grained understanding of how BXA resistance emerges and the evolutionary constraints this type of selection has at a population level.

POSTER 27

Euprymna Berryi as a Comparative Model Host for Vibrio Fischeri Light Organ Symbiosis

Avery M. Imes^1,2, Morgan N. Pavelsky³, Klodia Badal³, Derrick L. Kamp⁴, John L. Briseño⁴, Taylor Sakmar⁵, Miranda A. Vogt⁵, Spencer V. Nyholm⁴, Elizabeth A. C. Heath-Heckman⁶, Bret Grasse⁵, Alecia N. Septer³, Mark J. Mandel^1,2

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA, ²Genetics Training Program, University of Wisconsin-Madison, Madison, WI ,USA, ³Department of Earth, Marine & Environmental Sciences, University of North Carolina, Chapel Hill, NC, USA, ⁴Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA, ⁵Eugene Bell Center, Marine Biological Laboratory, Woods Hole, MA, USA, ⁶Department of Integrative Biology, Michigan State University, East Lansing, MI, USA

Functional studies of host-microbe interactions benefit from natural model systems that enable exploration of molecular mechanisms at the host-microbe interface. Bioluminescent Vibrio fischeri colonize the light organ of the Hawaiian bobtail squid, Euprymna scolopes, and this binary model has enabled advances in understanding host-microbe communication, colonization specificity, in vivo biofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid, Euprymna berryi, can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which the E. berryi light organ symbiosis parallels known processes in E. scolopes. However, the nature of the E. berryi light organ, including its microbial constituency and specificity for microbial partners, have not been examined. We isolated bacteria from E. berryi animals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes in E. berryi and E. scolopes hatchlings. This study reveals E. berryi to be a valuable comparative model to complement studies in E. scolopes.

POSTER 28

This poster has been withdrawn from the session.

POSTER 29

Identification and Characterization of Regulators of Germination Initiation in the Human Fungal Pathogen Cryptococcus neoformans

Nasya Miller¹, Megan McKeon¹, Christina M. Hull^1,2

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

Cryptococcus neoformans is an invasive human fungal pathogen that causes severe meningitis in immunocompromised people. Spores are infectious propagules that germinate into yeast when inhaled into the lung and can spread to other tissues of the body. Spores must germinate to cause disease; therefore, we are especially interested in understanding the molecular mechanisms underlying germination to identify targets for inhibition. To identify key pathways and molecular processes important for germination, we evaluated a collection of single gene deletion mutants for phenotypes in germination. We discovered that hgr2∆ and isp2∆ spores show an unexpected “early” germination phenotype in which mutant spores initiate and complete germination 2 hours earlier than wild type spores. Hgr2 is a sequence-specific High Mobility Group Domain transcription factor. Isp2 is a protein found to be enriched in spores relative to yeast that has no conserved domains. Because hgr2∆ and isp2∆ spores showed the same germination phenotype, we hypothesized that Hgr2 and Isp2 regulate similar molecular pathways in germination. To test this hypothesis, we carried out RNA-seq analysis on hgr2∆ and isp2∆ mutant spores during germination and discovered that they have strikingly different transcriptomes. These preliminary data suggest that they function in independent regulatory pathways. To further test and refine our hypothesis, we will create hgr2∆ isp2∆ double mutant spores and evaluate their cellular and metabolic phenotypes under diverse germination conditions and use transcriptomics to determine their molecular phenotypes. By comparing wild type, hgr2∆, isp2∆, hgr2∆ isp2∆ spores, we will determine the similarities and differences between Hgr2 and Isp2 regulation and gain insights into the fundamental pathways that function to regulate initiation of germination.

POSTER 30

Unearthing Nature’s Hidden Arsenal: Mining Fungal Genomes for a New Class of Natural Products

¹Grant Nickles, ¹Dr. Sung Chul Park, ¹Dr. Natália Sayuri Wassano, ¹Dr. Jin Woo Bok, ²Dr. Mickey T. Drott, and ¹Dr. Nancy P. Keller

¹Department of Medical Microbiology and Immunology Madison, WI, USA, ²U.S. Department of Agriculture-Agricultural Research Service, Cereal Disease Laboratory, St. Paul, MN, U.S.A.

Isocyanides (also called isonitriles) are a chemical class of SM produced by bacteria and fungi produced by isocyanide synthases (ICS). These compounds are created from non-canonical biosynthetic gene clusters (BGCS) that prior to our research were not detected by existing genome-mining software. They are characterized by the presence of the highly reactive isocyanide functional group, which is formed by the conversion of the amino group on select amino acids. Possessing potent bioactivities, isocyanides can engage in unique chemical reactions due to their affinity for chelation to metals, with some isocyanides targeting specific transition metals and metalloproteins. 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. Here, we present the first genome-mining pipeline, assembled from preexisting tools, to identify fungal ICS BGCs. We discovered 3,800 ICS BGCs in 3,300 fungal genomes, establishing ICSs as the fifth-largest class of SMS when compared to canonical BGC classes found by antiSMASH. Our results create a roadmap for future research into ICS BGCs. This work lays the foundation for further exploration of ICS BGCs, bolstered by our dedicated website which offers comprehensive access to all identified fungal ICS BGC files. In addition, I will present updates on our targeted investigations into a family of highly bioactive fungal isocyanides, the cyclopentyl isocyanides, that are known to cause an agricultural disease in ruminating animals.

POSTER 31

Discovery of New Cycloamanides from Amanita Phalloides Using a MSDIN Sequence Based Bioinformatic Pipeline

Sung Chul Park¹, Milton T. Drott², Anne Pringle³, and Nancy P. Keller^1,4

¹Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, WI, USA, ²Cereal Disease Laboratory, USDA-ARS, St. Paul, MN, USA, ³Department of Botany, University of Wisconsin–Madison, Madison, WI, USA, ⁴Department of Plant Pathology, University of Wisconsin–Madison, Madison, WI, USA

Amanita phalloides, also known as ‘death cap’ mushroom, is well known for producing the highly toxic amatoxins and phallotoxins. Through research on the biosynthesis of these cyclic peptides, it has been revealed that both types of toxic peptides are originated from the MSDIN sequence. Further studies on death cap derived compounds have unveiled cyclopeptides named cycloamanides A–D and antamanide, which are also the MSDIN sequence derived peptides like amatoxins and phallotoxins. According to the previous sequence analysis has shown that Amanita phalloides harbor more than 40 MSDIN sequences, but only four of these sequences are directly involved in the synthesis of toxic substances. While some researchers have identified the presence of other cycloamanides through MS/MS fragmentation patterns, to the best of our knowledge, none of natural products has been isolated and elucidated using MSDIN-based approaches. Traditional extraction methods with a buffer containing methanol:water:0.01 M hydrochloric acid (5:4:1) are optimized for the isolation of toxic peptides; thus, we optimized the extraction condition focused on extracting other cycloamanides. Here, we have isolated and elucidated two new cycloamaides, cycloamanides E (1) and F (2), based on the MSDIN sequence approach. Additionally, two new non-MSDIN derived peptides (3, 4) were also isolated and determined. The absolute configurations of these compounds were assigned based on the Marfey’s analysis. We anticipate that the newly obtained cycloamanides (1 – 4) will play a significant role in figuring out the physiological activity and internal mechanisms of non-toxic substances, which are primarily dominated by the MSDIN sequences.

POSTER 32

Sensing, Germinating, and Disseminating: Determining the Triggers of Cryptococcus Spore-Mediated Infection

Nicolas Pereira^1,2,3, Megan McKeon^1,2, Christina Hull^1,2

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA, ²Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA, ³Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA

Cryptococcus is an environmental yeast capable of causing fatal meningitis in humans. Cryptococcus causes disease when it is inhaled and then disseminates from the lungs to the brain via poorly understood mechanisms. To cause disease Cryptococcus must germinate from a spore into a replicating yeast. In cryptococcal infections germination appears to occur very early, but the kinetics of this process and how they affect dissemination and disease are not known. We aim to understand how both spore characteristics and germination environment impact germination kinetics and dissemination in the context of mammalian disease. To understand Cryptococcus germination in vitro we developed a high throughput, quantitative germination assay (QGA) that has facilitated the characterization of germination under diverse conditions. Previous work showed that spores germinate synchronously in response to glucose over ~12 hours in vitro. In vivo, little is known about how germination is triggered or what kinetics occur. To begin to address germination in a lung environment, we are developing a germination medium for QGAs produced from mouse lung homogenates. Preliminary data show that spores germinate in lung homogenates, but with differing kinetics and frequency of replication compared to rich growth media. At the same time, we are using a mouse model of intranasal infection to determine germination timing in vivo and rate of spread to other organs. Both approaches will facilitate studies of the germination kinetics and early dissemination capacity of a range of Cryptococcus strains and mutants. Studying the characteristics of early infection in physiologically relevant environments promises to inform our understanding of early events in Cryptococcus infection and will provide insights into how spores mediate fatal cryptococcal disease.

POSTER 33

Managing Enterohemorrhagic E. Coli Infection with Virulence Inhibitors  

Quentin Perraud¹, Vanessa Sperandio¹

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

Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that can cause hemolytic uremic syndrome (HUS), a life-threatening condition. The use of antibiotics for the treatment of EHEC infections is controversial since it often causes an increase in production of Shiga toxin, the main contributor to HUS.

A different approach for the handling of EHEC infection would be to target virulence by hijacking bacterial signaling systems controlling the attachment of bacteria to the intestinal epithelium. One such signaling system involves the QseC histidine kinase, a sensor whose inhibition has been shown to drastically diminish EHEC virulence and for which a small molecule inhibitor, LED209, have been previously characterized^1,2.

The bioactive moiety of LED209 consists of a sulfonamide scaffold substituted by two aromatic rings, one of those bearing a thiocyanate function able to cause covalent inhibition of QseC². Considering the challenges in developing novel antimicrobial drugs for niche use, we have turned toward re-purposing already FDA-approved molecules as potential virulence modulators by looking for structural analogues of LED209 in the sulfa drugs family.

In this study, we explore the use of sulfasalazine as a virulence modulator for E. coli O157:H7 infection. Using an approach combining in vitro tools such as immunoblotting, fluorescent actin staining and RNA sequencing, we were able to show a decrease in transcription and production of proteins encoded on the locus of enterocyte effacement (LEE) pathogenicity island, as well as a diminution of attachment to epithelial cells.

This decrease of virulence production can also be observed in Citrobacter rodentium, a LEE-bearing murine pathogen. We were able to demonstrate that our anti-virulence strategy significantly increases odds of recovery of C3H/HeJ mice challenged with a Shiga toxigenic C. rodentium strain.

POSTER 34

Investigation of the Interspecies Interactions of Micromonosporaceae for Natural Product Drug Discovery

Christopher Roberts¹, Doug Braun¹, Adriana B. Perea¹, Tim S. Bugni¹

^​1Pharmaceutical Sciences Division, University of Wisconsin–Madison, Madison, WI, USA

Bacterial natural products are an invaluable source of clinically relevant small molecules. Historically, these small molecules were isolated from Streptomyces spp. However, this intense focus on a single genus has created a challenge in discovering new structurally diverse compounds. Shifting the focus to understudied genera, such as those within the family Micromonosporaceae, presents an opportunity to discover structurally and mechanistically novel small molecules. Although many of these understudied genera display slow-growth in laboratory conditions, which reduces their attractiveness, we’ve isolated a Mycobacterium sp. that shows the ability to modulate the growth and metabololite production of Micromonosporaceae members. This cross-species interaction was characterized with a focus on how this interaction changes the growth phenotypes and metabolite production of bacteria from the family Micromonosporaceae. The discovery of Mycobacterium sp. WMMD1722 will aid in the effective exploration of understudied bacterial families to find clinically relevant small molecules.

POSTER 35

Bacillus Cereus Enhances Pseudomonas Koreensis Biofilm Formation

Natalia Rosario-Meléndez^1,2, Julia Nepper¹, Xingjian Yang^1,2, Kavya Illath Kandy¹, Sarah Uhm¹, and Jo Handelsman^1,3

¹Wisconsin Institutes for Discovery, University of Wisconsin–Madison, Madison Wisconsin, USA, ²Department of Bacteriology, University of Wisconsin–Madison, Madison Wisconsin, USA, ³Department of Plant Pathology, University of Wisconsin–Madison, Madison Wisconsin, USA

The rhizosphere microbiome is essential for plant and soil health. Elucidating the molecular crosstalk that governs microbial interactions within communities is essential for understanding their impact on their environment and for developing strategies to sustainably enhance agricultural production. Microbes respond to environmental cues like secondary metabolites, yet the mechanisms behind their production and function in natural habitats remain poorly understood. Using the three-member model rhizosphere community The Hitchhikers Of the Rhizosphere (THOR)—composed of Pseudomonas koreensis, Bacillus cereus, and Flavobacterium johnsoniae—I aim to understand how secondary metabolites mediate interspecies signaling in a community context. Previous work in the Handelsman lab has shown that coculturing P. koreensis with its rhizosphere neighbor B. cereus elicits augmented biofilm formation by P. koreensis. Here, we show that B. cereus induces biofilm in P. koreensis through the production of zwittermicin, a unique aminopolyol antibiotic produced by B. cereus. We found that 5µg/mL of zwittermicin enhanced the levels of extracellular proteins and viable cells in the biofilm matrix. Additionally, P. koreensis contains two exopolysaccharide biosynthesis pathways: poly-beta-1,6-N-acetyl-D-glucosamine (PGA) and alginate. Transcriptomic analysis (RNAseq) of zwittermicin-treated biofilms revealed a significant increase in the expression of the sigma factors rpoN and rpoH, and alginate, but not PGA. This suggests that subinhibitory concentrations of other translation-inhibiting antimicrobials also increase biofilm formation in P. koreensis. Exposing Chromobacterium violaceum to B. cereus secreted metabolites and zwittermicin induced the production of the purple antibiotic violacein, suggesting zwittermicin inhibits translation. Our work suggests that B. cereus enhances biofilm formation by inhibiting translation in P. koreensis. Future work will seek to understand why translation inhibitors enhance biofilm formation in P. koreensis. The results of this study will contribute to the growing interest in dissecting and manipulating microbial communities for plant and soil health.

POSTER 36

Defining Structure-Function Relationships in Immune Response to Mycobacteria – A Model Driven Approach

Marina Slawinski^1,3, Thomas Potts³, Dr. Jayaraman Tharmalingam³, Dr. Elebeoba May^1,2,3

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

Tuberculosis (TB) is the infectious disease which causes the greatest number of deaths worldwide, and an estimated quarter of the world’s population is currently infected. Upon inhalation of Mycobacterium tuberculosis bacilli, alveolar macrophages are the first to respond to infection, phagocytosing bacteria and attracting other populations of immune cells. Immune response results in a granuloma which attempts to physically contain and eliminate the bacteria, either successfully clearing the infection or containing bacteria in a latently infected state without clinical symptoms for decades with ~10% risk of bacterial dissemination and progression to active infection. Understanding the interactions between host immune cells and bacteria is important for predicting which patients are most at risk of developing active infection. Here, we developed fluorescently labeled murine macrophage cell lines and infect them with fluorescently labeled M. smegmatis bacteria to characterize structure and function in TB-associated mycobacterium infection using confocal microscopy. Using this platform, we examine the spatiotemporal response of three different murine macrophage cell lines (RAW, J774, and MH-S) to infection under different environmental conditions with the goal of extracting movement and cell phenotype features to inform a multiscale agent-based model of granuloma formation. Imaris software is utilized to process confocal microscopy data, implementing cell tracking to extract parameters related to macrophage structural response, including intracellular bacteria load (quantified via RFP), volume, velocity, acceleration, displacement, and directedness, and additional tools will be used to track phenotypic changes, including multinucleated giant cells and macrophage activation or death. An agent-based model of granuloma formation will be developed in Python and MATLAB, using previously developed models and extracted features to inform movement rules and phenotypic changes of macrophages. The final model should be able to represent aspects of structural organization, including emergence of macrophage-derived cell populations, compactness, number of cell clusters, and resulting bacterial counts pre-granuloma formation. Given that immune structural response contributes to disease outcome and effectiveness of drugs, modeling early granuloma events predictive of disease outcome will help inform improved treatment regimens and drug development.

POSTER 37

Role of Lactobacillus Tryptophan Metabolism in Th17 Activation and Multiple Sclerosis

Kate Stack¹, Wenxuan Dong¹, Gillian Hughes¹, Bethany Korwin-Mihavics¹, Kevin Schwartz¹, Felix Yan¹, Margaret Alexander¹

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

The relationship between diet, the gut microbiota and immune cells has an impact on autoimmune diseases including multiple sclerosis. The mouse model of multiple sclerosis, Experimental Autoimmune Encephalomyelitis (EAE), can be utilized to study these interactions in-vivo. In prior studies, mice following a ketogenic diet have reduced EAE disease compared to those on a high-fat diet in a microbiota-dependent manner. The Lactobacillus strain, L. murinus, is increased in abundance in murine microbiota stable in-vitro communities derived from ketogenic diet fed mouse fecal samples. L. murinus produces indole lactic acid (ILA), a tryptophan metabolite that has been previously seen to inhibit Th17 cells – major immune drivers of multiple sclerosis. However, whether ILA production by Lactobacillus is necessary for Th17 inhibition and downstream disease phenotypes is unknown. To determine the necessity of the ILA metabolite for EAE disease protection and impact on Th17 cells, tryptophan pathway genetic deletions were generated in L. reuteri, aLactobacillus species that produces ILA. The deletion targeted the aromatic aminotransferase gene (ArAT) which is responsible for the first enzymatic step of the conversion of tryptophan into ILA. Both wild type L. murinus and  L. reuteri cell free supernatants were sufficient to inhibit Th17 cells in vitro compared to the ILA knockout strain (L. reuteri ArAT KO). To determine the effect of eliminating ILA production in vivo, mice on a high fat diet were orally gavaged every other day with wild type L. reuteri, L. reuteri ArAT KO, or PBS prior to EAE induction and throughout the duration of disease. Mice treated with L. reuteri ArAT KO bacteria exhibited increased disease scores compared to the ILA-producing wild type L. reuteri group, with no statistical difference when compared to the control group. These preliminary results indicate that ArAT and ILA have a potential role in Lactobacillus-mediated Th17 inhibition and protection during EAE.

POSTER 38

Investigations into the Structural Determinants of Associative and Dissociative Mechanisms in LuxR-type Quorum Sensing Receptors 

Irene M. Stoutland¹, Suzy A. Walker¹, Helen E. Blackwell¹

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

LuxR/I-type quorum sensing (QS) regulates a variety of cell density-dependent phenotypes, including biofilm formation, virulence, and symbiosis, in many common species of Gram-negative bacteria. Small molecules that target QS are of interest as chemical probes to better understand QS systems and for potential applications in antivirulence, antibiofouling, and synthetic biology. To this end, the Blackwell research lab has developed a variety of small molecule agonists and antagonists targeting LuxR-type QS receptors. These intracellular receptors are transcription factors that are activated by binding to small molecule autoinducer ligands. The general lack of information about LuxR receptor structure and the precise mechanisms of action of small molecule LuxR modulators, antagonists in particular, is a significant barrier to the design of more potent, specific, and stable probes. The current study aims to determine the structural features that differentiate LuxR receptors that are most active in the presence of ligand (associative) and those that are most active in the absence of ligand (dissociative). Through the design and generation of “chimeric” LuxRs combining domains from the associative LasR receptor of Pseudomonas aeruginosa, the dissociative EsaR receptor of Pantoea stewartii, and/or the dissociative ExpR2 receptor of Pectobacterium versatile, we have found that the ligand-binding domain, rather than the DNA-binding domain, determines whether a LuxR-type receptor is more active in the presence of ligand or in its absence. Select synthetic LasR antagonists were found to maintain their activity in chimeras with interchanged, dissociative-type DNA-binding domains. In addition, a complementary mutagenesis approach revealed that LasR, EsaR, and ExpR2 have divergent responses to changes in the length of the linker region between the ligand-binding and DNA-binding domains, which has broader implications for our understanding of signal transduction in general in this class of receptors. Collectively, these results provide a deeper understanding of the modes by which small molecules control the activity of mechanistically distinct LuxR-type receptors and suggests new routes for the manipulation of LuxR/I-type QS network.

Financial support for this work was provided by the NIH (R35 GM131817). I.M.S. was supported in part by an NSF Graduate Research Fellowship and is an affiliate of the UW−Madison NIH Chemistry−Biology Interface Training (CBIT) Program (T32 GM008505).

POSTER 39

The Effect of Malate and Succinate on Salmonella enterica Typhimurium SPI-1 Heterogeneity

Crimson Stuckert¹, Matthew Warren¹, Alyson Hockenberry¹

¹ Department of Microbiology and Immunology, Loyola University of Chicago, Maywood, IL, USA

Salmonella Pathogenicity Island-1 (SPI-1) genes contribute to the virulence of Salmonella enterica Typhimurium (S. Tm) by allowing for host cell invasion. In a given S. Tm population, a portion of cells express SPI-1 (SPI-1+) and others do not express SPI-1 (SPI-1-), giving rise to phenotypic heterogeneity. It is known that heterogeneity drives persistence of S. Tm in the mammalian gut. According to previous studies, it appears that the tricarboxylic acid (TCA) cycle metabolite succinate may be important for S. Tm virulence gene expression. The C4-dicarboxylate antiporter, DcuABC, exports succinate as it imports malate and is required for S. Tm colonization and virulence in vivo. Thus, the TCA metabolite malate is also of interest. Our preliminary data indicates malate and succinate may play a role in SPI-1 heterogeneity. Metabolomics data shows that SPI-1+ cells produce malate, whereas SPI-1- cells consume malate and export succinate. Mathematical modeling of population dynamics supports the hypothesis that malate produced by SPI-1+ cells is imported and metabolized by SPI-1- cells to produce succinate. Perhaps this succinate is used either as a carbon source or a signal to induce SPI-1 expression, contributing to the cooperation between SPI-1+ and SPI-1- subpopulations.

POSTER 40

Staphylococcus epidermidis RP62a Modulates the Movement of Mobile Genetic Elements via Large Genomic Deletion

My Tran¹, Jessica Rosu¹, and Charlie Mo¹

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

Mobile Genetic Elements (MGE), such as phage and plasmids, play a dual role in bacterial evolution. On the one hand, phages and plasmids carry virulence genes and antibiotic resistance markers that can confer fitness benefits to the bacterial host. Conversely, phages infect and lyse bacterial cells, while parasitic plasmids impose a metabolic burden on the host cell. To counteract these threats, bacteria have evolved a diverse array of defense systems that target exogenous genetic materials. These defense systems are often clustered close to one another in what is known as a defense island, providing synergistic defense against a wide variety of MGEs. However, little is known about how these defense islands evolve, particularly how defense islands balance protection against harmful MGEs while allowing the acquisition of beneficial ones.

We study the methicillin-resistant Staphylococcus epidermidis RP62a which harbors a well-characterized anti-phage defense island consisting of four defense systems:  type III-A CRISPR-Cas locus (CRISPR), a type II restriction-modification system (RM), a serine-threonine kinase defense gene (stk2), and a defense system with nuclease-helicase activities (nhi). To understand how defense islands evolve, we passaged RP62a in rich media in the absence of phage and antibiotic selection. After 12 passages (~80 generations), RP62a exhibits a ~300 kb deletion, constituting more than 10% of RP62a’s total genome. The deletion consists of the antiphage defense island and virulence genes, such as theSCCmec cassette (which confers methicillin resistance) and the ica locus (responsible for biofilm formation). In phenotypic analyses, we found that the evolved populations had a diminished ability to restrict plasmid conjugation with CRISPR, phage susceptibility, attenuated resistance towards methicillin/oxacillin, and impaired biofilm production – consistent with the genomic deletion observed. The evolved populations demonstrate an enhanced ability to acquire beneficial plasmids in comparison to the unevolved wild-type strain. Intriguingly, defense against MGEs can be reinstated into the evolved RP62a population by applying a single selective pressure (phage, antibiotics, or biofilm selection). These findings suggest that the genomic heterogeneity and selection in S. epidermidis is a novel way of modulating horizontal gene transfer, with implications for bacterial adaptability and evolution.

POSTER 41

An RNA-Binding Protein Regulates Vibrio Fischeri  Symbiosis Behaviors

Jacob A. Vander Griend¹, Hunter C. Nottage¹, Andrew Mehle¹, Mark J. Mandel¹

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

Symbioses between animal hosts and microbial symbionts play important roles in host health, development, immune function, and nutrient acquisition. The symbiosis between the Hawaiian bobtail squid (Euprymna scolopes) and the bacterium Vibrio fischeri offers a naturally simplified model system to study the mechanisms by which beneficial colonization is established. In this interaction, host colonization follows a specific program at a dedicated symbiotic organ, with symbiont biofilm formation acting at a critical early stage. Host-associated biofilm formation is dependent on the symbiosis exopolysaccharide (SYP), the production and export of which is controlled by a phosphorelay network involving at least three hybrid histidine sensor kinases and two response regulators. While biofilm formation due to SYP production typically occurs only in vivo, manipulation of the regulators, including overexpression of sensor kinase RscS (rscS*), induces biofilm formation in vitro. The rscS* model is temperature sensitive, with growth at 28 ˚C resulting in significantly reduced biofilm formation compared to 25 ˚C. We hypothesized that this conditional phenotype could permit the isolation of novel SYP regulators and conducted a transposon screen for mutants capable of restoring biofilm formation at the restrictive 28 ˚C. The screen yielded hits in genes encoding a known biofilm inhibitor, BinK, as well as unstudied VF_2432. VF_2432 is a 160 amino acid protein with two predicted N-terminal transmembrane helices and a C-terminal RNA Recognition Motif 1 (RRM1) domain. The ability of ∆VF_2432 mutants to induce biofilm formation at 28 ˚C appears to be due to increased expression of the 18-gene syp locus, suggesting that VF_2432 may directly or indirectly target this locus. VF_2432 requires conserved RRM1 domain motifs for its biofilm regulatory phenotype. Overexpression of VF_2432 additionally inhibits cell growth and flagellar motility. Through UV-crosslinking followed by immunoprecipitation of a chromosomal 3X-FLAG tagged VF_2432 allele, complexes of VF_2432 bound to RNA have been detected. RNA ligands from the complexes were isolated and are being analyzed through a CLIP-seq approach. As VF_2432 has detectable orthologs throughout the Vibrionaceae including V. cholerae, this work will shed further light on a previously cryptic regulator present in many animal symbionts and pathogens.

POSTER 42

Nutrient Conditions Impact F. johnsoniae Growth in the Presence of the Antimicrobial Koreenceine A

Rachel Vogel^1,4,5, Natalia Rosario-Melendez^1,2, and Jo Handelsman^1,2,3

¹Wisconsin Institutes for Discovery, University of Wisconsin–Madison, Madison, WI, USA, ²Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA, ³Department of Plant Pathology, University of Wisconsin–Madison, Madison, WI, USA, ⁴Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA, ⁵Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

Microbial communities produce secondary metabolites that mediate inter and intra-species interactions, including antagonism and signaling. Complex interactions within in vivo systems make them difficult to understand. To dissect these complicated microbial interactions, we used a simplified model microbial system called The Hitchhikers of the Rhizosphere (THOR), which is composed of Pseudomonas koreensis, Flavobacterium johnsoniae, and Bacillus cereus. In THOR, P. koreensis produces the secondary metabolite Koreenceine A, which has antimicrobial activity. Koreenceine A regulates gene expression within the THOR community and inhibits the growth of F. johnsoniae, while B. cereus protects F. johnsoniae from this inhibition. First, we showed how nutrient availability modulates the growth of THOR community members in the presence of Koreenceine A. Then, we look at how increasing concentrations of Koreenceine A impacts F. johnsoniae growth under constant nutrient conditions. We found that F. johnsoniae has higher growth ratein high nutrient conditions despite a constant concentration of Koreenceine A. Conversely, increasing Koreenceine A concentration decreases growth rate of F. johnsoniae. Future studies will characterize F. johnsoniae modification of Koreenceine A into other secondary metabolites under favorable growth conditions. Understanding how bacteria respond to antibiotics across a nutrient gradient will enhance our understanding of the effect of antimicrobials on growth and persistence of microbes in the environment.

POSTER 43

Modulation of AGR Quorum Sensing Alters Mixed Staphylococcal Biofilm Formation and Community Composition

Troy D. Vulpis¹, Emma L. Eisenbraun¹, Jordan T. York¹, Helen E. Blackwell

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

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, primarily due to its ability to form robust biofilms on abiotic surfaces such as indwelling medical devices. S. epidermidis uses the accessory gene regulator (agr) quorum sensing (QS) system to regulate biofilm adherence and dispersal in a density-dependent manner. Here, we demonstrate that a universal agr agonist can appreciably inhibit biofilm formation on medical catheters by two common strains of S. epidermidis. Despite activating the agr system, this universal agonist is unable to inhibit biofilm formation by mock communities containing multiple strains of S. epidermidis. Instead, we find that other, non-universally active QS modulators can significantly shift the composition of mixed-group and mixed-species biofilms over time. To our knowledge, this is the first instance in which agr QS has been leveraged to modulate the composition of a mixed-species biofilm community. Overall, this study further elucidates the role of agr system in mixed-microbial biofilm development and suggests a possible strategy for perturbing bacterial communities that exploits subtle differences in QS systems across Staphylococcus species.

POSTER 44

Light-Controlled Synthetic Zinc Finger Transcription Factors for Saccharomyces Cerevisiae

Timothy Wakiyama¹, Megan McClean¹

¹Department of Biomedical Engineering, University of Wisconsin- Madison, Madison, WI, USA,

Synthetic zinc finger transcription factors (TFs) are powerful tools that have been used to create multigene control, logic gates, and cooperativity. However, previous studies of these TFs have not fully explored optogenetics which enables rapid and precise control of nuclear localization dynamics. Based on computational modeling results of said dynamics, we constructed optogenetically controlled synthetic zinc finger TFs with differing nuclear import/export rates and nuclear TF saturation levels by balancing nuclear localization signals, nuclear export signals, and anchor proteins that sequester the TFs to the plasma membrane. In accordance with our models, a) TFs with faster import and slower export resulted in pulsatile light input yielding higher reporter expression than continuous light, and b) TFs with slower import and faster export resulted in the opposite expression pattern, showing the power of our model guided design strategy. Furthermore, we altered the number of transcription factor binding sites (TFBSs) in the cognate synthetic promoter and observed a non-linear relationship between basal level, induced expression level, and the number of TFBSs. This study lays the groundwork for designing more advanced gene expression systems with broad implications for synthetic biology and bioengineering by incorporating synthetic zinc finger TFs and cognate promoters into optogenetics.

POSTER 45

Intestinal Glutathione Modulates the Virulence of Enterohemorrhagic Escherichia coli

Huiwen Wang¹, Vanessa Sperandio¹

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

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing bloody diarrhea and hemolytic-uremic syndrome in humans. EHEC’s major virulence factor is a type III secretion system that is encoded by the locus of enterocyte effacement (LEE) pathogenicity island and causes the characteristic attaching and effacing lesions on enterocytes. In the intestine, EHEC senses and responds to a complex milieu of metabolites derived from the host and intestinal microbiota, leading to the precise regulation of its virulence program. Of the thousands of metabolites, glutathione (GSH), a tripeptide comprised of glutamate, cysteine, and glycine, is highly abundant (at the millimolar concentration) in human intestine. In this study, we explored the effect of GSH on EHEC virulence modulation. When GSH (2 mM) was supplemented to EHEC culture, the EHEC displayed a strong increase in the LEE gene transcription and protein expression. The boost of EHEC virulence by GSH is likely due to the amino acid glutamate, because upon its import into EHEC cells, GSH is efficiently degraded to release glutamate, and glutamate was shown to promote the LEE gene expression similarly as GSH did. We further found that the leucine-responsive regulatory protein (Lrp) was responsible for the EHEC virulence upregulation resulting from GSH and glutamate. Deletion of the lrp gene fully abolished the increased LEE gene expression, while complementation to the Lrp mutant rescued the phenotype. The Lrp-mediated virulence promotion is indirectly through three prophage-encoded homologue regulators PchA/B/C. The pchA/B/C genes are distributed dispersedly on the EHEC genome, each of which contains an Lrp-binding consensus sequence on the promoter. Previous chromatin immunoprecipitation sequencing analysis shows that PchA/B/C bound to multiple promoter regions on the LEE pathogenicity island. Further, deletion of all the pchA/B/C genes led to a total abrogation of increased LEE gene expression caused by GSH and glutamate, while the presence of each individual PchA/B/C only partially restored the phenotype, indicating PchA/B/C work synergistically to augment the LEE gene expression. Together, this study elucidated the molecular mechanisms by which intestinal GSH promotes the virulence of EHEC.

POSTER 46

Machine Learning Reveals Key Structures Influencing Substrate Preference in Capsule Transporters of Streptococcus pneumoniae

Matthew Warren¹, Wan-Zhen Chua², Lok-To (Chris) Sham², Alyson Hockenberry¹

¹Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA, ²Yoo Loo Lin School of Medicine, National University of Singapore, Singapore, SG.

Streptococcus pneumoniae (pneumococcus) are surrounded by a layer of capsular polysaccharide (CPS). This CPS layer provides many evolutionary benefits.  It prevents phagocytosis, blocks complement deposition, enhances survival under starvation, and facilitates transmission. Pneumococci produce over a hundred structurally distinct CPSs. Each strain can synthesize and polymerize a single CPS structural variant, which determines its serotype.

CPS monomers are synthesized in the cytoplasm, transported across the membrane, and polymerized extracellularly.  During transport, CPS monomers are attached to a carrier lipid and a MOP (Multidrug/Oligosaccharide-lipid/Polysaccharide) family transporter, aka flippase, and “flip” the carrier lipid:CPS monomer complex from the cytoplasmic to extracellular face of the membrane.

These flippases are under strong co-evolutionary pressure with CPS monomers, as shown by a high-throughput screen of >6000 measurements revealing that non-cognate flippases often fail to flip foreign CPS monomers, leading to cell death.  The structural features influencing flippase-CPS recognition remain unclear.  To address this, we applied machine learning to analyze flippase sequences, predicted structures, and CPS monomers, in order to identify features that drive flippase specificity.  Our preliminary findings indicate that several methods of sequence encoding can provide insights into this specificity. Various interpretability methods allow these encodings to be better understood in terms of the underlying properties of the sequences.  This approach provides a more efficient method to understand CPS synthesis and may inform vaccine development and strategies to disrupt CPS production.

POSTER 47

A Platform for Design and Biosynthesis of Staphylococcus Aureus Non-Native Autoinducing Peptide Analogs by Engineered Bacillus Subtilis

Danielle L. Widner¹, Troy Vulpis¹, Hannah Vates¹, and Helen E. Blackwell¹

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

Both colonization and infection by Staphylococcus aureus are reliant on its accessory gene regulator (agr) type quorum sensing (QS) system. Inhibition of the S. aureus agr system can be achieved through a variety of probiotic and chemical strategies. This work aims to combine probiotic and chemical strategies by creating a heterologous platform for designing and biosynthesizing inhibitors of the S. aureus agr system that closely mimic the native signal. Previously, these analogs have only been produced through solid-phase peptide synthesis. In this study, we show that they can be robustly biosynthesized by modifying and transferring the 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. Surprisingly, these engineered strains outperform B. subtilis NCBI3610, a species known to naturally produce high levels of QS inhibitors, in most conditions.

POSTER 48

Microbiome Modulation: Investigating Lacticaseibacillus Pracasei in Vaginal Dysbiosis

Jingwen Zhao¹, Alyson Hockenberry

¹Department of Microbiology and Immunology, Loyola University Chicago, Chicago, IL, USA

Emerging evidence suggests that the vaginal microbiota plays a protective role against HPV-induced cervical carcinomas. A healthy vaginal microbiome is typically dominated by one of four major Lactobacillus species, which confer protection by producing lactic acid, lowering vaginal pH, and generating hydrogen peroxide. These mechanisms help suppress pathogen growth, prevent dysbiosis, and modulate inflammatory pathways, potentially limiting chronic HPV infections and their progression to cancer.

Recent studies indicate that Lacticaseibacillus paracasei, closely related to Lactobacillus spp., may enhance immunity, inhibit HPV oncogenes (E6, E7), reduce inflammation, and restore microbiome balance. However, its precise role remains understudied. Key questions include its capacity to produce hydrogen peroxide (H₂O₂), its ability to outcompete BV-associated pathogens, and how its protective effects compare to other dominant Lactobacillus species.

We hypothesize that interbacterial interactions influence the production of lactic acid, hydrogen peroxide, and other metabolites, thereby shaping the vaginal microbiome. L. paracasei may outcompete other Lactobacillus spp. as well as anaerobic and facultative bacteria. To test this, we will investigate the physiology of Lactobacillus spp., L. paracasei, in both monoculture and co-culture with other these microorganisms.

This study aims to elucidate how L. paracasei contribute to restoring a healthy vaginal microbiome. We will measure pH, lactic acid, hydrogen peroxide, and other metabolites to assess their impact on microbiome composition and function. Our findings will provide deeper insights into vaginal dysbiosis and inform the development of novel strategies for cervical cancer prevention, including vaccination and therapeutic interventions.

POSTER 49

Modeling The Impact of Low and High Dose Radiation on Macrophage Cell

¹Tochukwu Olie, ^2,3Daniel Ajuzie, ³Azka Ahmed, ³Jayaraman Tharmalingam, ¹Mark Harvey, ^2,3Elebeoba May

¹Physics Department, Texas Southern University, Houston TX, USA, ²Biomedical Engineering, University of Houston, TX, USA, ³Wisconsin Institute of Discovery, University of Wisconsin- Madison, Madison, WI, USA

Advances in medical imaging have led to increased exposure to low-dose ionizing radiation (LDR). On average, 80% of patients are exposed to 6.2 mSv of LDR annually, with diagnostic imaging accounting for half of this exposure. LDR induces DNA damage and cell death via direct and indirect ionization. This study focuses on the indirect effects of ionizing radiation through the process of water radiolysis, which involves the decomposition of water molecules due to ionization. We have developed a computational model to simulate the chemical cascade resulting from the excitation of water by ionizing radiation. The model, solved using MATLAB’s ode45 solver, successfully captured the creation of chemical species resulting from radiation impact. It provided insights into the dynamics of chemical species formation over time, elucidating the effects of radiation exposure on cellular processes. Hydrogen peroxide (H₂O₂), a key reactive oxygen species (ROS), was incorporated to evaluate macrophage cell responses. Experimental data provided valuable information on cell count and cell viability in response to H₂O₂ exposure over 1- and 72-hour periods. Notably, macrophage response to LDR-associated radical species revealed an immune reaction to H₂O₂. Despite exposure, a significant number of macrophage cells remained viable, suggesting a complex regulatory mechanism influencing immune response and cell lysis. These findings highlight the need for further investigation into the pathways governing macrophage survival, the cytotoxic effects of ROS, and the inflammatory cytokines that mediate immune response to ionizing radiation.

POSTER 50

Tiny Earth Chemistry: Promising antibiotic-producing isolates from Beloit College

Cassidy Felix¹, Sencio Eckland¹, Lauryn Volza¹, Kristin J. Labby¹

¹Department of Chemistry, Beloit College, Beloit, Wisconsin, USA

The Beloit College Spring 2025 CHEM 360: Microbes to Molecules: Antibiotic Discovery course is continuing the antibiotic discovery pipeline in collaboration with the Tiny Earth network. Tiny Earth Chemistry is a course-based undergraduate research experience (CURE) in which students engage in natural products chemistry. Collaborative efforts since 2019 have culminated in seven high-priority antibiotic-producing isolates. Students have worked to validate antibiotic activity, perform chemical extractions, and screen chemical extracts for antibiotic activity. Preliminary work has been done to separate and identify components of the extract using TLC, bioautography, and LC/MS.