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-10, IL-12, and Nitric Oxide During Mycobacteria Infection

Azka Ahmed¹, Maya E. Gough³, Elebeoba May¹²³

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

Mycobacterium tuberculosis (Mtb) remains a leading cause of global mortality, responsible for ~1.5 million deaths annually. Macrophages are the first responders to infection and produce key immunoregulatory mediators, including interleukin-12 (IL-12), interleukin-10 (IL-10), and nitric oxide (NO). IL-12 promotes pro-inflammatory signaling and NO-mediated bacterial killing, whereas IL-10 suppresses these responses. Vitamin D₃, an immunomodulator synthesized by macrophages, has been implicated in host defense; however, its role in regulating IL-12, IL-10, NO production, bacterial burden, and host cell death across infection conditions remains unclear.

We hypothesized that vitamin D₃ enhances NO production through an IL-12–driven autocrine signaling cascade in a dose-dependent manner. To test this, J774 macrophages were infected with Mycobacterium bovis BCG at multiplicities of infection (MOI) of 1 and 10 and treated with vitamin D₃. Contrary to our hypothesis, vitamin D₃ suppressed IL-12, IL-10, and NO production across both infection levels. At high infection (MOI 10), vitamin D₃ increased intracellular bacterial burden while reducing host cell cytotoxicity, suggesting enhanced bacterial containment and improved macrophage survival. In contrast, at low infection (MOI 1), vitamin D₃ did not significantly alter bacterial load but increased host cell cytotoxicity.

To further investigate treatment timing and host–pathogen dynamics, we employed a dual-fluorescence live-cell imaging system using mCherry-labeled Mycobacterium smegmatis to track bacterial burden and GFP-labeled J774 macrophages to monitor host cell viability. Macrophages treated with vitamin D₃ at seeding maintained higher viability over time, whereas pre-conditioning prior to infection reduced cell viability. Across conditions, vitamin D₃-treated macrophages consistently exhibited increased bacterial-associated fluorescence, indicating greater intracellular bacterial accumulation.

Collectively, these findings demonstrate that vitamin D₃ exerts complex, infection dose– and timing–dependent immunomodulatory effects on macrophage function. Ongoing work will extend these studies to additional mycobacterial strains and primary macrophages to inform host-directed tuberculosis therapies.

POSTER 3

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

Daniel C. Ajuzie^1,2,3, Jayaraman Tharmalingam^1,2, Azka Ahmed^1,2, Elebeoba E. May^1,2,3

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

Fluorescent reporter systems have become invaluable for real-time monitoring of gene expression, enabling high-temporal-resolution analyses across the bacterial growth cycle. A key limitation of existing approaches is that standard optical density normalization fails outside balanced exponential growth, where OD increasingly misrepresents viable biomass. Here we present the Dharmacon-May library a dual fluorescent promoter expression system in Escherichia coli that incorporates a constitutive mCherry plasmid as an internal biomass reference alongside GFP-based promoter activity reporters. Evaluated across four promoters (fimD, fur, ihfB, and lacZ), ratiometric mCherry normalization captured up to 84% of qPCR-measured transcript variance compared to 60% for OD-based methods, with the largest advantage being its applicability to lag and stationary phases where OD is least reliable. While dual-plasmid carriage imposed a modest metabolic burden that delayed growth commitment in three of four strains, GFP expression tracked single-plasmid controls with high fidelity, confirming that the mCherry plasmid does not compete with GFP reporter output. Mechanistic analysis further shows that the GFP/mCherry fluorescence ratio provides a direct, phase-independent readout of promoter regulatory state, corrected only for a predictable fluorophore maturation offset without requiring cell lysis or OD measurement. The Dharmacon-May library therefore provides a robust, growth-phase-aware platform for dissecting dynamic transcriptional programs in complex multi-phase processes such as biofilm formation, stress responses, and quorum sensing, with direct utility for integrating live-cell reporter data into quantitative gene expression models.

POSTER 4

An Army Marches on its Stomach – Metabolic Reprogramming Fuels C.rodentium Expansion in an Inflamed Gut

Parijat Bandyopadhyay¹ and Vanessa Sperandio¹

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

Inflammatory bowel disease (IBD) is associated with an increased susceptibility to enteric infections which often result in worsened clinical outcomes including treatment failure and surgical intervention. This heightened susceptibility can be attributed to immune dysfunction, epithelial barrier disruption and microbiome dysbiosis, all of which collectively result in a significantly modified metabolic landscape. However, how pathogens exploit this inflammation derived novel niche during early colonization remains inadequately studied. To address this, we employed the dextran sodium sulfate (DSS) mouse model of colitis to interrogate the role of inflammation in altering the gut metabolome and influencing pathogen fitness. DSS treated mice exhibited significantly increased susceptibility to the murine enteric pathogen Citrobacter rodentium (C.rodentium) with greater pathogen load in both cecum and colon. Untargeted metabolomics of the cecal contents revealed a distinct inflammation associated metabolic signature with marked enrichment of diverse lipid species, amino acids, polyamines and nucleobases. To functionally assess how this altered metabolic environment influences adaptive bacterial responses we cultured C.rodentium in media containing cecal content extracts from healthy and DSS treated mice and performed transcriptional profiling to uncover specific pathways which directly contributed to its increased growth. These analyses revealed that pre-existing inflammation of the gut plays an important role in early colonization profile of C.rodentium, providing mechanistic evidence of its metabolic adaptability in altered niches.

POSTER 5

Cryptococcus Spores use the Electron Transport Chain to Germinate Hypoxically

Madison Barnes¹, Jackie Spieles^1,2, and Christina Hull^1,2

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

Cryptococcus spores must germinate into yeast to cause disease in mammalian hosts. Because Cryptococcus is an 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 and that the electron transport chain (ETC) is necessary for this process. This finding reveals 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 characterized the phenotype of spores from the alternative oxidase complex (AOX1) knockout strains. Our data suggest that the entire electron transport chain functions during hypoxic germination and that the ETC must be using an electron acceptor other than oxygen. Because fumarate reductase has been shown to function during hypoxic respiration in other eukaryotic organisms, we are currently testing the role of fumarate as an alternative electron acceptor during Cryptococcusgermination under hypoxic conditions.

POSTER 6

Exploring Natural Antimicrobial Agents: Oregano-Peppermint Essential Oil Increases Antibiotic Production in Soil Bacteria

Aubrey Behling¹, Claire Adams¹, Jamie Garcia¹, Kayleigh Pigsley¹

¹Department of Plant Pathology, University of Wisconsin – Madison, Madison, WI, USA

A worldwide global crisis of antibiotic resistance is rapidly gaining recognition. As medicine rapidly evolves and pathogens continue to mutate, there is a significant need for research and discovery in antibiotics. This study investigated whether oregano-peppermint essential oil (Origanum vulgare and Mentha × piperita) could be used as a selective pressure to help isolate antibiotic-producing bacteria from soil. Bacteria isolated from control and oil-treated conditions were screened for antibiotic activity against six safe ESKAPE relatives (Staphylococcus epidermidis, Pectobacterium carotovorum, Enterobacter aerogenes, Pseudomonas putida, Acinetobacter baylyi, and Bacillus subtilis) by observing zones of inhibition. A Fisher’s Exact Test was used to compare the proportion of antibiotic-producing colonies between treatments. Following the conclusion of the experiment, the addition of oregano-peppermint essential oil was found to have produced a statistically significant increase in the number of antibiotic-producing colonies between the experimental and control groups. These results suggest that oregano-peppermint essential oil may be an effective method for isolating antibiotic-producing bacteria and could help improve approaches for discovering new antibiotics.

POSTER 7

The Impact of Garlic Exposure on Antibiotic Producing Properties of Soil Bacteria

Reese Bertram¹, Grace LeCaire¹, Kallen Hoffman¹, and Matas Korotkevic¹

¹Tiny Earth, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Antibiotic resistance is a growing global health problem contributing to rising healthcare costs and mortality rates. To address this, exploration of alternative antimicrobial agents that can influence bacterial communities is necessary for advancing antibiotic discovery. Bacteria from soil samples were cultured with and without sterilized garlic solution and screened against ESKAPE strain relatives. After analyzing experimental and control soil colonies, it was found that the colonies exposed to garlic had a higher percentage of antibiotic producers than the control colonies (two-tailed Fisher Exact Test, p = 0.000216). Further testing included PCR and MacConkey Agar tests to identify taxonomic groups of surviving colonies and their Gram staining reaction. Overall, the findings highlight the role of chemical stressors in promoting antibiotic production and demonstrate the potential of soil microbial diversity for discovering novel antimicrobial agents.

POSTER 8

Identification of Listeria monocytogenes Virulence Factors Associated with Adverse Outcomes during Perinatal Infection

Alyssa M Brokaw¹,John-Demian Sauer¹

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

Although foodborne listeriosis risk increases during pregnancy, mechanisms driving Listeria monocytogenes (Lm) perinatal infection and adverse pregnancy outcomes (APO) remain unknown. To identify host and Lm determinants that mediate severe perinatal infection, we used a perinatal listeriosis mouse model to compare the pathogenesis of the reference strain 10403S and LM2203, a human clinical isolate from an outbreak in which 90% of cases experienced severe APO. These strains caused similar spleen and liver burdens in pregnant dams infected at E14, but LM2203 burdens were 10-1000x higher than 10403S in gestational tissues and correlated with elevated risk of preterm birth and pup death. Internalin P is known to be important for 10403S placental infection but LM2203ΔinlP exhibited minimal virulence attenuation, suggesting LM2203 possesses additional factors that mediate perinatal virulence. Although systemic inflammation was similar in 10403S- and LM2203-infected dams, LM2203 increased uterine MCP-1 and prostaglandin-E₂ levels, supporting subsequent preterm labor. Finally, compared to 10403S LM2203 infection produced ~2x larger plaques in fibroblast monolayers, suggesting that increased intracellular replication and/or cell-to-cell spread may correlate with perinatal virulence. Ongoing experiments will determine whether LM2203 is hyper-responsive to environmental and intracellular stresses through the alternative sigma factor, SigB, and will test if modulation of SigB activity impacts plaque size. As an orthogonal approach, a genetic screen will identify mutations that revert 2203 plaques to 10403S-size, allowing us to uncover mechanisms that may explain LM2203 hypervirulence. Future murine experiments will define how MCP-1 and prostaglandin-E₂ contribute to Lm replication in gestational tissues and subsequent APO. Together, these approaches will define both host and bacterial mechanisms that mediate gestational tissue infection and APO caused by LM2203, allowing for future assessment across a broader panel of Lm isolates and perinatal pathogens.

POSTER 9

Characterizing LuxR-Type Receptors Through Homoserine Lactone-Based Small Molecule Probes

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

¹Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

Quorum sensing (QS) is a cell-to-cell communication used by many opportunistic pathogens, including Pseudomonas aeruginosa, to regulate group behaviors in a population density dependent manner. Because QS systems often control virulence phenotypes in bacteria, such as biofilm growth, swimming and swarming motility, and exotoxin production, understanding QS can provide strategies to target bacterial virulence. The most common and well characterized QS system in Gram-negative bacteria uses LuxI/LuxR-type synthase/receptor pairs. The LuxI-type synthase produces N-acyl-L-homoserine lactones (AHLs), with AHL concentration increasing with bacterial density. LuxR-type receptors, cytosolic homodimeric transcription factors, detect AHLs and alter QS gene expression accordingly. 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. Many of these AHLs were strong modulators of LuxRs, behaving as both agonists and antagonists. Interestingly, many of the antagonists displayed non classical partial agonism behavior. The SARs summarized here present a useful starting point for developing hypotheses about the role of AHLs in multi-microbial systems and characterize ideal acyl tail lengths for receptor agonism and antagonism across a range of LuxR-type receptors.

POSTER 10

Aspergillus fumigatus Metal-Chelating Secondary Metabolite Regulation in Co-culture with Pseudomonas aeruginosa

Ava Clark¹, Harrison Estes¹, Sung Chul Park¹, Marc Chevrette², Nancy Keller¹

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

Aspergillus fumigatus and Pseudomonas aeruginosa are environmental microbes and are the most common fungal and bacterial opportunistic pathogens in the cystic fibrosis lung environment. The pathogens are commonly found to co-occur and lead to persistent infections in cystic fibrosis patients and are associated with advancing disease progression. Metal-related competition is central to the relationship between A. fumigatus and P. aerguinosa. P. aerguinosa produces several secondary metabolites that enhance virulence and assist in competition against A. fumigatus, including phenazines, which increase reactive oxygen species (ROS) in the fungal cell, and siderophores, which starve A. fumigatus of iron and other metals, which are necessary for the detoxification of ROS. A. fumigatus is also known to produce the metal chelating secondary metabolites xanthocillin and hexadehydroastechrome (HAS), which are known to be co-regulated and serve antibacterial and virulence functions. However, it is not known how the regulation of the xanthocillin and HAS gene clusters is affected by co-culture with P. aeruginosa. We co-cultured xanthocillin and HAS overexpression and knockout mutants of A. fumigatus with P. aeruginosa. The strains were cultured in a variety of media conditions, including rich and minimal media, as well as metal-replete and starved conditions. By observing the phenotypes and metabolomic profiles of the co-cultures, we can determine the genetic and nutritional factors responsible for the differential expression of secondary metabolites produced by P. aerguinosa and A. fumigatus.

POSTER 11

No ESKAPE: Exploring the Effect of Microbial Growth Byproducts on Secondary Metabolite Production in Soil BacteriaIsolates

Jake Clavijo¹, Aubrey Keddington¹, Nola McGarry¹

¹Tiny Earth, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Today, multidrug resistant isolates, the most prominent being the ESKAPE pathogens, have become one of the top three threats to global public health. However, the search for and discovery of antibiotic producing bacteria has decreased significantly since the discovery of Penicillin. This study investigates whether exposure to sterilized Pseudomonas putida bacteria byproducts can increase antibiotic production in soil bacteria. Bacteria colonies from soil samples collected in and around the UW-Madison campus were isolated using a serial dilution process, exposed to filtered Pseudomonas putida cell filtrate, and screened against 6 ESKAPE relative pathogens and four agricultural fungi. Antibiotic activity was tested by presence or absence of zones of inhibition. Results showed that 11 out of 56 treated colonies and 7 out of 55 untreated colonies exhibited antibiotic activity, with no statistically significant difference between groups. However, treated colonies appeared to show multi-pathogen antagonism in contrast to untreated isolates. While sterilized bacteria may not have significantly increased antibiotic production, the addition of P. putida filtered supernatant as an additive showed promising increase of broad-spectrum frequency of isolated bacterial colonies, suggesting the need for further research on whether bacterial byproducts can enhance the production of secondary metabolites in soil bacteria.

POSTER 12

Design and Application of Small Molecules for the Interception of Quorum Sensing in Staphylococcus aureus

A.P. Clay¹, Thomas J. Polaske¹, Helen E. Blackwell¹

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

Quorum sensing (QS) is the process by which bacteria of the same species coordinate behavior, including virulence, at a high population density. The accessory gene regulator (agr) QS system found in the Gram-positive pathogen Staphylococcus aureus uses agr to regulate production of toxins and biofilm formation. Small molecules that inhibit agr QS have the potential to be used as therapeutics to combat S. aureus infection or as probes to study agr QS. Previous small molecules that have been reported to inhibit agr QS suffer from low potencies, poorly characterized mechanisms of action, and/or associated toxicities that render them impractical for use as potential therapeutics or probes. We have developed a new class of small molecule agr inhibitors, and are studying their biochemical mechanisms of agr inhibition. We have designed a new synthetic route to the core structure of a best-in-class small molecule inhibitor (CP-20), which is the most potent small molecule inhibitor of S. aureus agr QS to date. This synthesis enables facile functional group variation for the delineation of structure activity relationship analyses to improve compound potency, membrane permeability, solubility, and stability. Characterization of the mechanism of agr inhibition by CP-20 is currently underway. Almost all known agr inhibitors target autoinducer (AIP) binding to the transmembrane histidine kinase AgrC or intracellular response regulator AgrA:DNA binding. We have demonstrated that CP-20 is a competitive inhibitor of AIP:AgrC binding, although the exact mechanism of inhibition remains unknown. We have shown that CP-20 inhibits hemolysis, a key virulence phenotype associated with agr QS. We are also investigating other small molecule inhibitors of S. aureus quorum sensing identified through a high-throughput screen based on vesicle lysis.

POSTER 13

Glycolytic Nucleotide Preference Ties Carbon Intake to Nucleotide Metabolism

Matthew B. Cooke¹, Yanxiu Li¹, Jue D. Wang¹

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

Glycolysis is the universal hub for energy source intake and processing. Carbon, in the form of sugars like glucose is taken up and broken down into smaller chains amenable to further metabolism. Flux through this pathway is thought to be driven by the final step, conversion of phosphoenolpyruvate to pyruvate through the activity of the enzyme pyruvate kinase. This conversion also yields glycolysis’ first ATP generation. Previously, the Wang lab discovered that pyruvate kinase frequently charges nucleotides other than ADP. Here, I performed a pan-bacterial assessment of pyruvate kinase nucleotide selectivity and find that bacteria frequently host multiple pyruvate kinase subtypes which are promiscuous in their nucleotide selection. This choice between strict nucleotide selection (ADP) or broad nucleotide selection (NDP) appears to govern the efficiency of carbon uptake and the flexibility of bacterial nucleotide metabolic networks. This work suggests that glycolytic ATP generation is much less universal than previously thought and highlights a role for charging non-ADP nucleotides in central metabolism and growth.

POSTER 14

Innate Pathway Selection Modulates Antibody and T Cell Responses to Mosaic Influenza Nucleoprotein in Cattle

Clara Cole¹, Thomas Cleven¹, Marlee Henige², Keith Poulsen⁵, Mike Maroney⁴, Lautaro Rostoll-Cangiano³, Doerte Doepfer², and M. Suresh¹

¹Department of Pathobiological Sciences, University of Wisconsin – Madison, Madison, Wisconsin, USA,²Department of Medical Sciences, University of Wisconsin – Madison, Madison, Wisconsin, USA, ³Animal and Dairy Sciences, University of Wisconsin – Madison, Madison, Wisconsin, USA, ⁴Research Animal Resources and Compliance, University of Wisconsin – Madison, Madison, Wisconsin, USA, ⁵Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin – Madison, Madison, Wisconsin, USA

Highly pathogenic avian influenza (HPAI) is a lethal disease of poultry that has recently spilled over into mammals, including dairy cattle and humans, heightening concerns for livestock health, food security, and pandemic emergence. While vaccines that induce neutralizing antibodies against hemagglutinin and neuraminidase provide strain-specific protection, durable cross-subtype immunity requires T cell responses targeting conserved internal antigens such as nucleoprotein (NP). To leverage these conserved targets, we utilized a previously engineered mosaic nucleoprotein (MNP) incorporating T cell epitopes from thousands of influenza A virus (IAV) strains, conferring broad protection against epidemic (H3N2) and pandemic (H1N1) IAV in mice. Here, we tested whether precision adjuvancy could differentially imprint adaptive immunity to MNP in cattle. Combination formulations paired the carbomer-based nano-emulsion Adjuplex (ADJ) with either a STING agonist (cyclic dinucleotides; CdN) or a TLR4 agonist (glucopyranosyl lipid A; GLA) to program distinct inflammatory milieus. Both formulations elicited circulating IFN-γ–producing T cell responses and NP-specific antibodies in serum and milk. However, STING activation via CdN generated more potent and consistent cellular and humoral immunity than TLR4 engagement. These data demonstrate that selective activation of innate sensing pathways functionally imprints adaptive immune magnitude and quality in a large animal host. By advancing a broadly protective, T cell–focused vaccine strategy in cattle, this work supports a One Health framework to mitigate H5N1 transmission risk at the human–animal interface.

POSTER 15

Role and Regulation of Dispersal in Candida albicans Commensalism and Pathogenicity

Eli G. Cytrynbaum¹ and Megan N. McClean^2,3

¹Department of Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA, ³University of Wisconsin Carbone Cancer Center, 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. Using innovative underoil microfluidic technology, we have found that dispersal from biofilms occurs as a discrete event within biofilm development regulated by environmental cues and extracellular signaling. We observe that dispersed cells are morphologically and functionally heterogenous, genetically diverse, and primed for the formation of new biofilm – exhibiting increased immune evasion, stress tolerance, fitness in competition with other microbes, and rapid adhesion and filamentation. By modeling the regulatory pathways controlling dispersal, we hope to uncover potential targets for treatment to encourage commensalism over pathogenesis.

POSTER 16

Effects of Apple Cider Vinegar on Antibiotic Production in Soil Bacteria

Olivia Davis¹

¹Tiny Earth Lab, Wisconsin Institute of Discovery, University of Wisconsin – Madison, Madison, WI, USA

Antibiotic resistance as a global concern supports the need for further research on factors that can influence bacterial competition and antibiotic production, like acidity. This study examined whether exposure to apple cider vinegar (ACV), a source of acetic acid, affects antibiotic production in soil bacteria. Soil samples collected from multiple locations on the University of Wisconsin–Madison campus were cultured, isolated, and grown on Luria Bertani (LB) agar plates with and without ACV treatment. Antibiotic activity was assessed using zones of inhibition against safe relatives of ESKAPE pathogens. Of 55 ACV-treated colonies, 18.2% exhibited antibiotic production compared to 13.8% of 58 untreated colonies. Statistical analysis (Fisher’s exact test, p = 0.61) indicated no significant difference between groups not due to random chance. These findings suggest that mild acid stress from ACV does not significantly influence antibiotic production in the soil bacteria tested.

POSTER 17

Antimicrobial Discovery from Soil-Derived Bacteria Using a Genome-Level Approach

Martel L. DenHartog¹, Murrel Saldanha^1,2, Tasha Miller¹, Delaney Miller¹, Kaetlyn T. Ryan³, Sarah Miller¹, Marc G. Chevrette^1,4, Jo Handelsman^1,4

¹Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA, ²Department of Biophysics, University of Wisconsin-Madison, Madison, Wisconsin, USA, ³Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA, ⁴Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA

Resistance to pesticides and drugs is increasing plant and animal diseases, threatening production of agriculture and medicine. Soil bacteria are a rich source of many natural products used as antimicrobials. The challenge is to minimize rediscovery of known compounds to detect new ones. In classrooms at 486 institutions across the United States, college students enrolled in a research course (Tiny Earth) isolate antimicrobial-producing bacteria from the soil and send them to a collection of 4,400+ isolates at the University of Wisconsin–Madison where scientists extract DNA for PacBio sequencing at the Joint Genome Institute. To find novel antimicrobial compounds, we take a comparative genomics approach using antiSMASH to mine the genome sequences for biosynthetic gene clusters (BGCs) that encode the machinery for production of secondary metabolites with antimicrobial potential. Taxonomic classification conducted viaGTDB-tk and OrthoFinder is used to perform a multi-locus sequence analysis and phylogenetic tree construction. An annotated phylogeny with mapped BGC data provides researchers with genetic insight into the biosynthetic potential and evolutionary patterns of BGCs for soil bacteria across geographical samples. This analysis of 417 genomes reveals the antimicrobial-producing potential of soil-dwelling bacteria and provides a basis for prioritizing isolates for chemical analysis of bioactive compounds. We have identified strains that protect soybeans from oomycete infection and solved structures of 20 bioactive molecules, including a nematicidal indole compound from abacterial strain active against several pathogens that cause plant diseases. Genomic analysis provides a new basis for sorting through the chemical cornucopia of the soil microbiome.

POSTER 18

Less is More: Surprising Activation of Bacterial Biofilm with a Truncated Regulator

Anna M. Dickfoss^1,2, Jacob A. Vander Griend^1,3, Mark J. Mandel^1,2,3

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA,²Cellular and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA, ³Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA

The symbiotic relationship between bioluminescent Vibrio fischeri bacteria and their squid hosts, Euprymna scolopes, is a well-characterized model for studying beneficial colonization. Biofilm formation is required for successful colonization and depends on the symbiosis polysaccharide (Syp) pathway. Expression of the syp locus is controlled by a phosphorelay centered around the hybrid histidine kinase SypF. The SypF histidine phosphotransferase (HPt) domain transmits phosphoryl groups to transcriptional activator SypG, and the HPt domain of SypF is the only domain required for function. We found that a construct containing only the 3’ end of sypF with no added promoter was sufficient for function: partial function if the construct contains only the HPt-encoding segment, and full function if an additional 81 bp upstream of the domain is included. These data demonstrate that a minimum region of sypF(“sypF-C”) under the control of an internal promoter within the gene is sufficient for biofilm formation. We propose a model where sypF-C is produced independent from the rest of the syp locus from a promoter in the 81 bp-region, and that production of this fragment is critical for activation of the biofilm locus. Combined with additional transcriptomic data from the lab, we speculate that SypF-C may be constitutively produced in the cell, awaiting host signal(s) to rapidly switch on the broader biofilm pathway.

POSTER 19

Investigating How Strain-specific Sequence Variation Impacts Activity of the influenza A Virus Endoribonuclease PA-X

Cynthia Feng^1,2, Marta Maria Gaglia²

¹Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA,
²Institute for Molecular Virology and Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA

Influenza A virus strains that cause recurring seasonal and newly emerging zoonotic disease display varying severity, highlighting the need to understand how strain-specific differences determine infection severity. A key determinant of severity during influenza infection is lung damage induced by inflammatory immune responses. The influenza A virus ribonuclease PA-X globally reduces cellular gene expression during infection through RNA degradation and modulates immune responses and inflammation, thus altering disease severity. Though found in all influenza A virus strains, PA-X exhibits a high degree of sequence variability. Preliminary evidence suggests there are also differences in its RNA degradation and immunomodulation across strains. However, how changes in PA-X sequence impact its activity remains unknown. To link PA-X sequence variation to activity, we are comparing the RNA cleavage activity of PA-Xs from different strains using a novel quantitative in-cell RNA cleavage assay. We have discovered that PA-X activity is modulated by strain-specific sequence variation and are working on defining links between key residues in PA-X sequence and its RNA cleavage activity. Our goal is to investigate what impact differences in PA-X ribonuclease activity have on immunomodulation during infection to determine how restriction of host gene expression contributes to differences in influenza infection across strains.

POSTER 20

Impact of Pseudomonas aeruginosa on Phage and Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus

Doran A. Goldman¹, Charlie Y. Mo¹

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

Understanding how bacteria evolve resistance to bacteriophage (phage) infection is a major goal for the design of phage-based therapies. Many studies of phage resistance focus on a single phage and host pair, but the fitness costs of resistance can drive divergent evolutionary and ecological outcomes in the presence of competing bacterial species. We investigated the evolution of phage resistance by methicillin-resistant Staphylococcus aureus (MRSA) in the presence of Pseudomonas aeruginosa, a pathogen that co-infects a variety of body sites and competes with S. aureus. MRSA strain MRSA252 grown in rich medium typically acquires high levels of phage resistance following exposure to the lytic phage Evo2, while simultaneously suffering a strong reduction in β-lactam resistance. These phenotypes are likely mediated by changes in peptidoglycan cross-linking caused by mutations in the femA gene. In contrast, passaging MRSA252 in sterile P. aeruginosa spent medium for ~50 generations prior to phage infection results in a variety of genetic and phenotypic outcomes, including the emergence of S. aureus populations with distinct mutations in wall teichoic acid (WTA) biosynthesis genes alongside other mutations in genes that may play roles in resisting stress from toxic P. aeruginosa exoproducts. WTA mutant strains exhibit striking differences in phage and β-lactam resistance depending on growth condition, remaining phage susceptible and antibiotic resistant in rich medium but exhibiting phenotypes like those of femA mutants in spent medium. These findings underscore the diverse impacts that a single non-host species can have on the evolution of phage and antibiotic resistance.

POSTER 21

Determining Cryptococcus Spore Surface Composition and Rearrangement Mechanisms

Greengo, SD¹ & Hull, CM^1,2

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

The yeast Cryptococcus is a significant cause of mortality in immunocompromised populations and is initiated when Cryptococcus spores or desiccated yeasts enter the lungs. The composition of the cryptococcal surface is a key mediator of interactions with the surrounding environment, including, in the context of mammalian infection, host phagocytes. The Cryptococcus cell wall contains chitin, chitosan, glucans, and various proteins. In addition, Cryptococcus produces a polysaccharide capsule in times of stress. However, the surface composition of spores is relatively unknown. Additionally, how the Cryptococcus spore exterior changes during germination is unclear. Using fluorescence microscopy, we have identified spore-specific binding patterns by an array of cell surface probes, indicating unique components and/or component exposure in spores compared to yeast. Additionally, we have tracked the locations of these components over the course of germination and found that they do not migrate with the germinating cell surface, suggesting that new surface material is synthesized during germination. As the cell wall and spore germination are both spore-specific biology, they make ideal potential drug targets, both separately and in tandem. These experiments also allow further investigation of the interactions between spores and immune cells, including potentially identifying the host receptor(s) for Cryptococcus spores.

POSTER 22

Melatonin as a Modulator of Enteric Pathogen Virulence

Ebru Guver¹, Vanessa Sperandio¹

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

Environmental cues within the gut are key modulators of host–pathogen interactions, yet specific signals that shape enteric pathogenesis remain incompletely defined. Melatonin, produced in the gut approximately 400-fold higher than in the pineal gland, represents a critical gut-derived signal whose role on enteric infections is poorly understood. Enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen that colonizes the colon, senses local cues through two component systems (TCSs) to precisely regulate virulence. Considering its low infectious dose, severe clinical complications and reliance on environmental sensing for successful colonization, we investigated the role of melatonin as a regulatory signal during enteric infection.

EHEC and its surrogate murine model, Citrobacter rodentium, employ type III secretion system (T3SS), encoded by the locus of enterocyte effacement (LEE), to form attaching and effacing (A/E) lesions. We previously shown that enteric pathogens sense tryptophan-derived metabolites through a TCS and down-regulate virulence. Building on this, our post-transcriptional, transcriptional, and transcriptomic studies confirm that another tryptophan-derived gut signal, melatonin, enhances expression of LEE genes, and T3SS effector and structural proteins while repressing chemotaxis and flagella genes when sensed through multiple TCSs. These findings suggest that melatonin promotes a non-motile, adherent state to enhance epithelial attachment and A/E lesions. Mammalian infection further demontrates that melatonin-treated mice exhibit higher bacterial colonization and reduced survival, indicating that melatonin worsens disease severity.

Given the complexity of the gut environment, this work provides new insights into potential therapeutic strategies by characterizing the crosstalk between enteric pathogens and a remarkable gut modulator, melatonin.

POSTER 23

Disrupting Microbial Communities: Investigating Patulin as a Driving Force of Microbiome Community Dynamics

Haefner, B.J.¹, Park, S.C., ² Keller, Nancy^1,2

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

Microbial communities are shaped by a continuous exchange of chemical signals between fungi and bacteria, orchestrating interactions that range from cooperation to competition. This work investigates how fungal metabolites, specifically patulin, influence these processes. Patulin, a lactone mycotoxin produced by Penicillium expansum, exhibits important functions at low, subinhibitory concentrations that do not impact microbial growth. Rather than acting solely as a toxin, patulin functions as a communication-modifying molecule that disrupts bacterial quorum sensing (QS) and alters microbial community dynamics. At subinhibitory concentrations, patulin broadly interferes with QS-associated behaviors in Gram-negative bacteria, significantly reducing biofilm formation in Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Chromobacterium subtsugae, disrupting swarming motility in P. aeruginosa and Pseudomonas koreensis, and suppressing violacein production in the QS biosensor strain C. subtsugae. A LasR-GFP reporter assay in Escherichia coli further demonstrates that patulin disrupts AHL binding to the LasR receptor, directly interfering with a key QS regulatory pathway. The ecological consequences of this QS disruption were evaluated using the THOR (The Hitchhikers Of the Rhizosphere) synthetic microbiome, composed of P. koreensis, Flavobacterium johnsoniae, and Bacillus cereus. In this system, P. koreensis produces koreenceine antibiotics that inhibit F. johnsoniae, while B. cereus protects and stabilizes F. johnsoniae populations when present in the community. Fungal extracts from wild-type, but not patulin-deficient, P. expansum strains enhanced F. johnsoniae fitness and significantly reduced P. koreensis production of koreenceine A and C, the most bioactive forms against F. johnsoniae. These findings suggest that patulin disrupts competitive dynamics within the THOR community. Collectively, this work identifies patulin as a QS-interfering metabolite, highlighting communication disruption as a mechanism shaping microbiome community composition.

POSTER 24

Knockdown of Fatty Acid Biosynthesis Genes Increases Biofilm Formation in Pseudomonas aeruginosa

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

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

A biofilm is a collection of surface attached microorganisms existing in an extracellular matrix that serves as a protective barrier against antibiotics and other environmental stressors. Several studies identified non-essential genes that are important for biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa, but these studies lacked the ability to assess the roles of essential genes. Here, we use a CRISPRi-based essential gene knockdown library in P. aeruginosa to systematically uncover biofilm phenotypes associated with essential cellular processes. Our work reveals that perturbation of genes in the fatty acid biosynthetic pathway (FAS) enhances biofilm formation as evidenced by an increase in the overall biofilm volume under conditions of continuous media flow as well as substantially improved ability to attach to plastic surfaces under static conditions. We found that biofilm enhancement by FAS perturbation is dependent on the glycolipid class called rhamnolipids, and we have also shown that the distinct colony biofilm and attachment phenotypes require the production of the cationic biofilm exopolysaccharide, Pel. Essential gene products are the target of FDA approved antibiotics, but our work reveals that these essential gene perturbations may also counterintuitively enhance biofilm formation, a major driver of antibiotic resistance. By leveraging our pooled CRISPRi essential gene knockdown library, we discovered that FAS disruption in P. aeruginosa significantly increases its biofilm forming capacity and attachment properties. These results highlight the importance of continued investigation of essential gene perturbations and their potential to drive biofilm development in clinically relevant bacterial pathogens.

POSTER 25

Defining Antibody-driven Mechanisms of Pathogen Clearance in a Murine Model of Infectious Diarrhea

Marienela Y. Heredia¹, Margaret L. Sleeth¹, Aidan J. Schmidt¹, and Gustavo Caballero-Flores^1*

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

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are major causes of infectious diarrheal disease worldwide, particularly in young children. These pathogens induce attaching-and-effacing lesions in the intestinal epithelium and can lead to severe morbidity. The murine pathogen Citrobacter rodentium serves as a well-established model to study host immune responses to enteric infection by EPEC/EHEC in vivo. While antibodies are known to contribute to protection against bacterial infections, how distinct antibody isotypes cooperate to eliminate C. rodentium remains incompletely understood. Therefore, our lab seeks to define the specific and coordinated contributions of IgM, IgG, and IgA to pathogen control and disease resolution in the gut.

To address this, we tracked C. rodentium infection kinetics in wild-type mice, single antibody knockout mice, and newly generated antibody double knockout mice. We found that mice lacking IgG, IgA, or IgM individually are able to efficiently eradicate C. rodentium, indicating a degree of functional compensation among isotypes. Consistent with this, mice lacking both IgG and IgA also clear infection efficiently. In contrast, mice lacking both IgG and IgM fail to effectively control infection during the primary response, demonstrating that IgM is required for pathogen control in the absence of IgG and highlighting a previously underappreciated role for IgM in host defense. Taken together, these findings reveal key aspects of coordinated antibody-mediated immunity during enteric infection and establish IgM as a critical contributor to pathogen clearance, providing a framework for antibody cooperation and informing the development of novel, immunotherapeutic strategies for vulnerable populations.

POSTER 26

The Microbiota Metabolite-Dependent Protective Effect of a Ketogenic Diet in a Mouse Model of Multiple Sclerosis

Gillian Hughes¹, Kate Stack¹, Margaret Alexander¹

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

Ketogenic diets improve multiple sclerosis in both human and animal studies, yet the mechanisms by which these diets impact disease are not fully understood, limiting our ability to explain variation in patient responses and to guide patient-specific dietary and therapeutic strategies. Ketogenic diets elevate β-hydroxybutyrate (βHB) and induce microbiota-dependent changes that shape host immune responses, including reductions in pathogenic Th17 cells that drive multiple sclerosis. Here, we delineate an aryl hydrocarbon receptor (AhR)-dependent mechanism linking diet-induced microbiota changes to suppression of Th17-driven autoimmunity. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, a ketogenic diet ameliorates disease severity in amicrobiota-dependent manner and alters the gut microbiota, including enrichment of indole-lactic acid (ILA)-producing bacteria such as Lactobacillus murinus and Lactobacillus reuteri. ILA is a tryptophan metabolite that is reduced in individuals with multiplesclerosis and increased following ketogenic-associated microbial shifts in mouse models, suggesting a role in diet-induced immunoregulation, consistent with prior evidence that ILA suppresses pathogenic Th17 responses. Administration of ILA alonereplicates the anti-inflammatory effect of ketogenic diet and BHB-ester-treatment in EAE. Because ILA is a known ligand for the arylhydrocarbon receptor (AhR), which is expressed by Th17 cells that drive EAE and multiple sclerosis, we hypothesized that ILA suppresses Th17 responses through AhR signaling. In contrast to wild-type cells, ILA fails to suppress Th17 activation in AhR-deficient cells. Moreover, AhR inhibition eliminates ILA-mediated protection in the EAE mouse model of MS. Additionally, ILA-mediated protection varies with AhR genotype, with low- and high-affinity AhR alleles exhibiting differential responses. Together, these findings support a model in which microbiota-derived ILA suppresses pathogenic Th17 responses through AhR signaling, and contributes to the protective effects of ketogenic diet in multiple sclerosis.

POSTER 27

Uncovering Biosynthetic Gene Clusters in Phage and Giant Virus Genomes

Neha Kashyap¹, Cody Martin^1,2, James Kosmopoulos^1,2, Karthik Anantharaman^1,3

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ²Microbiology Doctoral Training Program, University of Wisconsin–Madison, Madison, WI, USA, ³Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA

Secondary metabolites play a role in defense, communication, and adaptation during microbial interactions. Within the bacterial genome, colocalized genes called biosynthetic gene clusters (BGCs) encode the pathways needed to synthesize, modify, transport, and regulate expression of secondary metabolites. BGCs can be transferred between bacteria, giving recipient bacteria the ability to harness new biosynthetic pathways and create specialized metabolites like antibiotics. Evidence suggests that bacteriophages may also play a small but ecologically significant role in carrying and transferring BGCs between bacteria. If bacteriophages can encode BGCs in their genomes, it means phage infection could be beneficial to the host in such cases. To test this hypothesis on a dataset representative of the global virus ecosystem, five databases – IMG/VR, GOV 2.0, GSV Atlas, Prophage-DB, and VIRE (Terrestrial) – were compiled and filtered to only include complete and high-quality genomes. After dereplication of the viral proteomes, a broad screen to find BGCs was conducted using both deep-learning and rule-based approaches. Preliminary findings show that BGCs are found in phage and giant virus genomes, with over 15,000 BGCs detected from about 1.4 million genomes. Many of the detected BGCs fell under the terpene, RiPP, and other categories, and environment and taxonomy influenced the distribution and types of BGCs observed. Verifying the presence and patterns of phage-encoded BGCs will give us deeper insight into the ability of phages to transfer and spread secondary metabolism pathways. These results also bring us closer to realizing biotechnological applications like phage-antibiotic cocktails where the antibiotic is encoded on the phage genome.

POSTER 28

Malnutrition-Associated Microbiota Inhibit Immune Responses to Cryptosporidium Infection

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

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

Malnutrition is a major contributor of susceptibility to infection with the intestinal protozoan parasite Cryptosporidium, especially in young children. Despite a clear association between malnutrition and microbiota composition, the impact of malnutrition-associated microbiota on susceptibility to Cryptosporidium infection has not been explored. NLRP6 has been identified as a key regulator of immune responses to Cryptosporidium infection since its absence, but not the absence of other NLRs, results in a stark increase in parasite shedding compared to control mice. NLRP6 is known to respond to microbial cues, such as microbial metabolites, so I hypothesize that the microbiota modulate the immune response to C. parvum via the NLRP6 inflammasome. I employed a protein malnutrition model where healthy controls received a 20% protein diet and undernourished mice received an isocaloric 7% protein diet. Parasite fecal shedding varied with respect to malnutrition, host sex, and the facility in which mice were raised. Parasite shedding was significantly increased in malnourished mice raised in our facility, but not those raised in the breeding core facility. Increased parasite shedding in malnourished mice was observed in both sexes, but it was significantly more pronounced in males. IL-18 secretion by infected intestinal epithelial cells is expected when the NLRP6 inflammasome is activated. Despite an increase in parasite burden in malnourished groups, IL-18 secretion from infected ileal explants did not increase in the presence of microbiota. However, when the microbiota were knocked down with broad-spectrum antibiotics, IL-18 secretion was directly proportional to parasite burden. IL-18 is known to stimulate Type 1 immune responses in immune cells resulting in IFNγ production, which is key in coordinating immune responses to Cryptosporidium. We observed a decrease or no change in IFNγ production in lamina propria immune cells in malnourished mice despite higher parasite burden. Future studies will assess the metabolite pools and composition of malnutrition-associated microbiota to identify metabolites and microbes that may modulate NLRP6 activity or inhibit Cryptosporidium infection.

POSTER 29

Impact of Inorganic Fertilizer on the Frequency of Antibiotic-producing Soil Isolates in Garden and Prairie Soil

Aniella Kovacic¹, Olivia Titel¹, & Kianna Young¹

¹Tiny Earth Lab, Department of Plant Pathology, University of Wisconsin – Madison, Madison, WI, USA

Antibiotic resistance is a pressing concern for public health and safety in recent times, making finding new antibiotics increasingly important. One place to look for new antibiotics is in the soil microbiome, which contains a large diversity of bacteria. Wisconsin contains many fertilized soils, so does the fertilized or unfertilized soil have more antibiotic production in its bacteria? By culturing soil bacteria from both a previously fertilized and unfertilized sample with a dilute fertilizer solution, differences in bacterial growth and antibiotic production can be observed. It was ultimately found that there was no statistically significant difference between fertilized and unfertilized soil. However, the producers found in this study were all Gram-positive Bacillus species. This has broader implications for future farming practices, antibiotic resistance genes, and use of soil as a resource for novel antibiotics.

POSTER 30

Discovery of Antibacterial and Antifungal Fatty Amides using LC-MS-based Metabolomics

Tae Hyun Lee¹, Bailey A. Bell¹, Christopher D. Roberts¹, Doug R. Braun¹, and Tim S. Bugni¹

¹Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA

The rapid emergence of antimicrobial resistance in methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicansnecessitates the discovery of new bioactive small molecules from underexplored microbial sources. In our efforts to identify structurally intriguing secondary metabolites from marine-derived bacteria, LC–MS-based hierarchical clustering analysis combined with principal component analysis (hcapca) was performed on a library of 92 marine-derived bacterial strains, leading to the prioritization of 16 metabolically distinctive candidates. Subsequent PCA analysis identified WMMD794, a marine-derived Verrucosispora sp., as a chemically distinct candidate. Further metabolomics investigation using GNPS molecular networking revealed the presence of underexplored metabolite classes within this strain.

Chemical investigation of WMMD794 resulted in the isolation and purification of three new fatty amides, verrucamides A–C (1–3), along with two known analogs (4 and 5). Their structures were elucidated through comprehensive spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry. Detailed structural comparison revealed that compounds 1–3 possess branched isobutyl terminal groups, distinguishing them from their linear counterparts and suggesting potential functional relevance of terminal branching.

All compounds (1–5) were evaluated for their antimicrobial activities against MRSA and C. albicans. Compounds 1 and 2 exhibited significant antibacterial activity against MRSA (MIC 16 μg/mL), whereas the corresponding linear analog showed reduced potency. Notably, verrucamide A (1) displayed moderate antifungal activity against C. albicans (MIC 32 μg/mL), while compounds with extended alkyl chains or linear termini showed diminished or no activity. Comparative analysis across these structural analogs revealed that both terminal branching and optimal alkyl chain length (C12–C13) play critical roles in determining antimicrobial efficacy.

POSTER 31

Exploring the Relationship Between Winter Ice-Melting Practices and Antibiotic-Producing Microbes

Alexis Legge¹, Lindsay Peters¹, Ellen Chen¹, and Grace Behm¹

¹Tiny Earth, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Antibiotic resistance poses a severe threat to global health and the overall well-being of society. As more pathogens develop resistance to the current supply of antibiotics, the discovery of new antibiotics becomes crucial to solving this crisis. The question that this experiment is based on is, do current ice-melting practices in Madison, WI have an effect on the number of antibiotic-producing bacteria found in soil? This experiment began with a soil sample and was followed by plating the soil solution, patching the bacterial colonies in the presence of sand, salt, and a control, and plating the bacterial colonies that grew under each condition on plates with bacterial indicators to screen for zones of inhibition. It was found that there is no significant difference between the number of antibiotic-producing bacteria grown in the presence of sand, salt, and a control. This implies that the current practices related to road snow and ice control may not harm the number of antibiotic producers present; however, certain confounding factors, like pre-exposure, may complicate the true findings. Thus, the many producers identified will be sent for further analysis of their antibiotic-producing properties.

POSTER 32

Plant Derived Signals as a Potential Mediators of Interactions Between Soil Microbes

Marielle Martin¹, Marc G Chevrette^1,2

¹Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA,²Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin USA

Streptomyces are known for producing a large number and variety of natural products (NPs), many of which are not frequently expressed under standard laboratory conditions. They are commonly found in soil, where they are exposed to root exudates and microbial communities. To investigate the role of plant signals on eliciting production of NPs that affect other soil bacteria, Streptomyces venezuelae was grown in plain media or media supplemented with the plant hormones salicylic acid (SA) or jasmonic acid (JA). These cultures were filter sterilized, and either Pseudomonas syringae, a plant pathogen, Burkholderia thailandensis, a plant growth promoting bacteria, or Chromobacterium subtsugae, an antimicrobial producer, were grown in the spent media or a non-spent control. All showed reduced growth in Streptomyces spent media supplemented with SA relative to the controls. This indicates that exposure to plant hormones could trigger production of a bacterial growth inhibitor that is not produced in standard media. To study how these changes in NPs impact the growth and NP production of other microbes, S. venezuelae was grown on plain or SA/JA supplemented plates either alone or in pairwise co-cultures with the above bacteria. Both the morphology of S. venezuelaeand the metabolomic profiles using molecular networks from LC-MS/MS were evaluated. These results indicate that NP production and growth can be reshaped by plant interactions. Signaling between plants and microbial communities is complex, but by studying how plant hormones influence NP production in soil microbes, we can better understand how plants shape interactions in microbial communities.

POSTER 33

Metal Meets Microbe: Investigating Zinc-Induced Stress on the Synthesis of Antibiotics of Soil Bacteria

Aneri Mehta¹, Ethan Kunston¹, Kate Vare¹ & Ellie Arbeiter¹

¹Tiny Earth Lab, Plant Pathology, Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI, USA

Given the severity of the antibiotic resistance crisis, developing or discovering more effective antibiotics is a critical priority. The widespread rise of antimicrobial resistance (AMR) has created an urgent need for novel antibiotics. This study investigates whether zinc treatment influences the ability of soil bacteria to inhibit ESKAPE pathogens, a group of clinically relevant, antibiotic-resistant bacteria. Zinc is an essential micronutrient, involved in intracellular signaling. In this experiment, soil bacteria were treated with 25 mg of zinc, and a library plate was constructed from colonies that survived treatment. ESKAPE pathogens were introduced to strains that were not resistant to zinc to examine zones of inhibition and thereby identify specific antibiotic-producing bacterial strains. Results showed that 7 out of 86 colonies in the control group (no zinc) exhibited zones of inhibition, compared to 2 out of 87 colonies in the zinc-treated group. Statistical analysis using a two-tailed Fisher’s exact test yielded a p-value of 0.0992, indicating no statistically significant difference between the groups. These findings suggest that, under the conditions tested, zinc treatment does not significantly enhance the antimicrobial activity of soil bacteria and may not improve their ability to inhibit ESKAPE-related bacteria. However, given the limitations of this study, zinc may still hold future potential as a tool in combating antibiotic resistance.

POSTER 34

Understanding and Characterizing the Increased Lytic Efficiency of a Kayvirus on Methicillin Resistant Staphylococcus aureus

Anna Moyer¹, Charlie Y. Mo¹

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

Phage therapy uses bacteriophages (phages; bacterial viruses) as a treatment method to target and kill antimicrobial-resistant pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA). While sequencing of staphylococcal phage genomes has rapidly increased, the genes themselves remain relatively undescribed, and mechanisms governing infectivity efficiency remain unclear. Understanding the mechanisms that improve phage infectivity will increase our knowledge in basic phage biology, and aid in engineering a reliable treatment against MRSA. In our lab, we found a mutant Kayvirus (Evo2) that showed increased infectivity against more than 30 MRSA clinical isolates compared to its predecessor, ɸStaph1N. A sequenced Evo2 clone revealed a single nonsense point mutation in ORF141 (ORF141mut)–a hypothesized DNA-binding protein. However, ORF141’s function remains unknown and how the genomic variation impacts infectivity efficiency remains unclear.

To address this, we first hypothesized complementing ORF141 in an Evo2 infection will reduce infectivity efficiency, phenocopying ɸStaph1N. However, we found that ORF141 alone is not sufficient to restore the ɸStaph1N phenotype, potentially indicative of an additional influencing factor on the Evo2 infection. We then explored ORF141’s hypothesized DNA-binding function by quantifying the phage transcriptional profile via RT-qPCR. Results showed that Evo2 produces up to 8 times more major capsid protein than ɸStaph1N during one lifecycle, suggesting Evo2 proliferates at a higher rate. In addition, average burst size assays revealed Evo2 has a 10-fold greater burst size than ɸStaph1N after two lifecycles. Collectively, these data indicate that Evo2 is producing more viral particles than ɸStaph1N. Moving forward, we will address this directly by using qPCR to quantify intracellular Evo2 genomic DNA present during an infection compared to ɸStaph1N. Additional future directions will include a bioinformatic approach to characterize the independent protein structure of the ORF141 and any predicted protein-protein interactions. Altogether, results from this research will improve our understanding of fundamental staphylococcal phage biology and can assist in the development of more effective phage therapies.

POSTER 35

The Role of Peptidoglycan in Neisseria gonorrhoeae Biofilm Formation

Andrew Moysis¹, JP Dillard¹

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

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a Gram-negative diplococcus of significant public health concern due to its robust antibiotic resistance. A key contributor to its pathogenesis is its ability to form biofilms in the human urogenital tract, on the epithelium of primary cervical and urethral cells. A feature of the gonococcal biofilm matrix is the release of peptidoglycan (PG) fragments into the extracellular space. The functional role of PG in biofilm development has not yet been investigated. To study this, we utilized two strains of N. gonorrhoeae: a wild-type strain that acetylates its PG, and a ΔpacA mutant strain. PacA is the gene that controls N. gonorrhoeae PG acetylation, so the mutant does not acetylate its PG. We compared the biofilm-forming capacity of these two strains using a safranin-stain biofilm assay. We found the non-acetylating mutant produced significantly more biofilm than the acetylating wild-type. To rule out the possibility of differential cell lysis causing this phenotype, we performed further assays with magnesium supplementation, which is known to stabilize the outer membrane and reduce autolysis. The difference in biofilm formation persisted, indicating that autolysis is not the underlying cause. Next, to directly test the influence of PG in biofilm formation, we performed assays with supplemented mutanolysin, an N-acetylmuramidase that digests secreted PG into monomers. Mutanolysin treatment almost entirely prevented biofilm formation in both the wild-type and mutant strains, demonstrating that PG integrity is necessary for biofilm formation. This is the first time that peptidoglycan has been shown to be essential to biofilm formation in N. gonorrhoeae. To generate results more directly relevant to human infections, future work will examine the effect of human neutrophil lysozyme on biofilm formation in both strains. Lysozyme does not cleave peptidoglycan in places where it is acetylated, which is a difference from the mutanolysin that will further elucidate the role of the acetylated peptidoglycan in N. gonorrhoeae biofilm formation, and inform future treatment avenues.

POSTER 36

Deciphering Cryptic Genes : A Continued Effort to Combat Antibiotic Resistance

Nischala Nadig^1,2, Amy B. Banta^1,3, Ashley N. Hall^,3, Jason M. Peters^1,3,4,5,6

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

Antibiotic-resistant bacteria have become increasingly prevalent over time due to the misuse and overuse of antibiotics. Scientists have discovered many genes involved in antibiotic resistance; however, some are challenging to identify as they do not exhibit resistance phenotypes under typical laboratory conditions. These genes may have functions not normally associated with antibiotic resistance or may show resistance phenotypes only when overexpressed. Our lab has developed a tool for targeted overexpression of genes using CRISPR-associated transposition called CRISPRtOE (Clustered Regularly Interspaced Short Palindromic Repeats transposition and OverExpression). We generated a genome-scale CRISPRtOE library in Escherichia coli K-12 and used it to identify cryptic genes associated with resistance towards the antibiotic bicyclomycin (BCM), which is the only known selective inhibitor of the Rho transcription termination factor. Our screen showed that setA, coding for a sugar efflux transporter protein, and cnu, coding for a nucleoid-associated protein, conferred resistance to BCM when overexpressed. As these genes are not normally associated with antibiotic resistance, we aim to decipher the mechanisms by which overexpression could protect cells against BCM inhibition. Our study provides a proof of concept for systematic discovery of cryptic antibiotic resistance genes in E. coli and other bacteria.

POSTER 37

Genome-Wide Characterization of Sulfite Stress Regulation in Salmonella Typhimurium

Vivian Nguyen¹, Trina Westerman¹, Steffen Porwollik², Michael McClelland², Patricia Tran³, Kevin Myers⁴, Madchen Ewing¹, Johanna Elfenbein¹

¹University of Wisconsin-Madison, Department of Pathobiological Sciences, Madison, WI, USA, ²University of California-Irvine, Department of Microbiology and Molecular Genetics, Irvine, CA, USA,³University of Wisconsin-Madison, Department of Bacteriology, Madison, WI, USA, ⁴University of Wisconsin-Madison, Great Lakes Bioenergy Research Center, Madison, WI

Sulfite is a ubiquitous reactive sulfur species that imposes significant stress on bacteria. However, the exact regulatory mechanism by which bacteria respond to sulfite stress remains uncertain. Bacteria can encounter sulfite in both environmental and host-associated contexts. Sulfite is used as a food additive to control microbial growth and prevent browning. In the host, sulfite can arise from dietary intake, amino acid catabolism, and during infection as a byproduct of the neutrophil oxidative burst. Salmonella Typhimurium (STm) encounters host-derived sulfite and can survive in the presence of sulfite, making it a relevant model to investigate the underlying regulatory mechanism of sulfite stress tolerance. We previously demonstrated that YeiE, a LysR-type transcriptional regulator, is required for survival to sulfite stress in STm. In this work, we investigated Salmonella’s genome-wide transcriptional response to sulfite stress, defined the role of YeiE in shaping this response, and mapped YeiE binding sites to characterize its regulatory network. Our data show that YeiE activates a distinct gene network in response to short-term sulfite exposure by upregulating sulfur metabolism while downregulating amino acid metabolism. Unexpectedly, sulfite stress is activating the sulfur assimilation pathway under conditions where it is typically repressed, suggesting a noncanonical regulatory mechanism. To identify direct regulatory targets, we performed chromatin immunoprecipitation to define YeiE targets at baseline and under sulfite treatment. Under sulfite stress, we identified 94 unique YeiE binding sites and uncovered previously unrecognized DNA-binding motifs, providing important insight into the regulatory mechanism of YeiE. Together, our data provide the first genome-wide characterization of bacterial transcriptional responses to sulfite stress and define how YeiE regulates this process, offering new insight into bacterial stress responses.

POSTER 38

Discovery and Identification of Effective Antimicrobial Compounds Against the ESKAPE Pathogens from Isolated Soil Bacterium

Shay Nolen¹, Danielle Schaal¹

¹Department of Math and Sciences, Marian University, Fond du Lac, Wisconsin, USA

Each day, growing numbers of pathogenic bacteria are becoming resistant to commonly used antibiotics. Of interest, the ESKAPEpathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are known to be resistant to multiple antibiotics, and are the number one cause of nosocomial infections. As antibiotic resistance is on the rise, the demand for new antimicrobial agents is high. Many of the current antimicrobial agents were discovered from naturally occurring microbes, such as bacteria and fungi. In this study, we looked at soil bacteria as a reservoir for effective antimicrobial compounds against the ESKAPE pathogens. We measured these selected soil microbes’ antimicrobial activity via a swab-patch assay and measured ZOI (Zones of inhibition) against each of the ESKAPE pathogens. Eight soil bacteria were found to produce an effective antibiotic against one or more of the ESKAPE pathogens. Following this, we isolated the metabolites produced by these unique soil microbes. Once the metabolites were isolated, via an ethyl-acetate extraction, a GC-MS was used to identify specific metabolites responsible for antimicrobial activity. With the antimicrobial compounds identified, future work aims to elucidate the mechanism of action for the individual antimicrobial compounds produced by the eight isolated soil bacteria. Altogether these findings suggest that naturally occurring microbes may be a potential solution to the growing antibiotic resistance crisis.

POSTER 39

Modeling the Impact of Low Dose and High Dose Radiation on Mammalian Immune cells

Tochukwu Olie¹, Jayaraman Tharmalingam^4,5, Mark Harvey³, Elebeoba May²^4,,5

¹Department of Biology, Texas Southern University, Houston, TX, USA, ²Department of Biomedical Engineering, University of Houston, TX, USA, ³Department of Physics, Texas Southern University, Houston, TX, USA, ⁴Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA, ⁵Wisconsin Institutes for Discovery, Madison, WI, USA

Advances in medical imaging have increased patient exposure to low-dose ionizing radiation (LDR), with individuals receiving ~6.2 mSv annually, nearly half from diagnostic procedures. LDR can cause DNA damage and cell death through both direct ionization and indirect pathways. One key indirect mechanism is water radiolysis, where radiation interacts with water to generate reactive oxygen species (ROS). In this study, we developed a computational model to simulate the chemical cascade initiated by water excitation, focusing on hydrogen peroxide (H₂O₂), a central ROS involved in cellular stress responses. To evaluate macrophage behavior, we designed the “Buzz Experiment,” where cells are exposed to varying H₂O₂ concentrations and monitored over time (0, 24, 48, 72 hours).We assess both phenotypic changes and cell viability (alive vs dead) using microscopy and GFP-based microplate analysis. Preliminary observations show that at low H₂O₂ levels, cells exhibit unexpected proliferation patterns and appear capable of temporarily containing ROS before reaching a threshold that leads to cellular rupture and lysis. Experimental data will be used to parameterize and validate the model. This framework connects predicted H₂O₂ production from water radiolysis to observed cellular outcomes and integrates these dynamics into a biological model of cell death pathways, including apoptosis and necrosis.

POSTER 40

The Nucleotide (p)ppGpp Mediates Protection from Bacteriophages in Gram-positive Bacteria

Aditya Kumar Pal¹, Wanyang Huang², Jue D. Wang¹

¹Department of Bacteriology, University of Wisconsin- Madison, Madison, USA, ²Department of Integrative Biology, University of Wisconsin- Madison, Madison, USA

Bacteria encounter diverse environmental stresses, including nutrient limitation, amino acid starvation, and other metabolic constraints, and adapt to these conditions through the stringent response mediated by the alarmone guanosine pentaphosphate known as (p)ppGpp. This conserved signaling pathway reprograms cellular physiology to promote survival under stress. While bacteriophage infection is a pervasive and potent pressure in bacterial ecosystems, it is less understood how global stress responses shape susceptibility to phage and influence infection outcomes over time.

Here, we identify (p)ppGpp as a key regulator of bacteriophage susceptibility in Gram-positive bacteria. Using Bacillus subtilis, we show that cells with elevated (p)ppGpp levels, such as those in the stationary phase or under nutrient stress, exhibit increased resistance to lytic phage infection. In contrast, loss of (p)ppGpp synthesis leads to rapid and catastrophic phage propagation. Mechanistically, we find that (p)ppGpp-mediated depletion of intracellular GTP is a key determinant of this phenotype: cells with reduced GTP levels display enhanced tolerance to phage infection and slower phage replication dynamics. Consistently, disruption of GTP-responsive regulation via CodY further increases phage tolerance, supporting a model in which the (p)ppGpp-GTP axis governs host permissiveness to phage replication.

Importantly, we observe that this phenomenon is conserved in Staphylococcus aureus, including methicillin-resistant strains (MRSA), suggesting a broadly conserved role for metabolic state in determining phage infection outcomes. Together, our findings reveal that bacteriophage infection can be viewed as a metabolic stress and that bacterial adaptation through (p)ppGpp signaling fundamentally alters phage-host dynamics. This work provides a framework for understanding how intrinsic physiological states shape viral propagation and highlights the importance of metabolic regulation in governing bacteria-phage interactions.

POSTER 41

Listeriolysin O-Dependent Phagosomal Ca²⁺ Flux and ERK2 Signaling Induce Prostaglandin E₂synthesis in Listeria monocytogenes Infected Phagocytes

Kevin R. Parducho¹, Zachary T. Morrow¹, John-Demian Sauer¹

¹Department of Medical Microbiology and Immunology, University of Wisconsin – Madison School of Medicine and Public Health

Prostaglandin E₂ (PGE₂) is an eicosanoid that contributes to a variety of processes ranging from induction of labor to regulation of tumor growth. Listeria monocytogenes (Listeria) infection induces phagocyte-dependent PGE₂ production that is essential for CD8⁺ T-cell priming¹. However, our understanding of the host pathways and bacterial factors that regulate PGE₂ production remains incomplete. Published data have shown that two of the canonical host enzymes for PGE₂ synthesis (MPGES1, COX-2) and the bacterial protein listeriolysin O (LLO) – which forms pores in the phagosomal membrane – are required for Listeria-induced PGE₂synthesis^1,2. The calcium-sensitive cPLA2, which canonically initiates PGE₂synthesis via releasing arachidonic acid (a PGE₂ precursor) from membrane phospholipids has remained understudied in this response to bacteria. Our results show that the cPLA2 inhibitor, pyrrophenone, decreased PGE₂synthesis from cells infected with Listeria, demonstrating the importance of cPLA2. Given the Ca²⁺-sensitivity of cPLA2, we hypothesized that the essential role of LLO in Listeria PGE₂ induction is via its ability to induce Ca²⁺ flux from infected phagosomes³. To test this hypothesis, we treated macrophages infected with LLO deficientListeria (∆hly) with the phagosomal Ca²⁺ channel agonist ML-SA5, and found that induction of Ca²⁺ flux with ML-SA5 rescued the ability of ∆hly Listeria to induce PGE₂. Similarly, phagosome-constricted B. subtilis⁴ did not induce PGE₂ synthesis until after ML-SA5 treatment or ectopic expression of LLO. Notably, treatment with either ML-SA5 or Δhly Listeria alone did not induce PGE₂, suggesting synergy between Ca²⁺ flux and a bacterially-derived factor. cPLA2 activity is modulated by ERK2⁵, a kinase activated downstream of various pattern recognition receptors (PRRs). Consistent with this model, treatment with U0126, an inhibitor of the ERK2 activator, MEK1, prevented PGE₂synthesis after infection with WT Listeria. Furthermore, stimulation using heat-killed B. subtilis or TLR agonists in the presence of ML-SA5 synergistically induced PGE₂synthesis. We hypothesized that TLR signalling from the phagosome could synergize with phagosomal Ca²⁺flux to activate PGE₂ production, however, primary macrophages from Unc93b^-/- mice (which lack the proper localization and signalling from endosomal TLRs)⁶ still produce PGE₂in response to either wild type or ΔhlyListeria plus ML-SA5, suggesting redundant functions of other PRRs. Collectively, these data suggest that Listeria infection induces phagocyte PGE₂ synthesis through LLO-mediated phagosomal Ca²⁺ flux and activation of other ERK2-dependent pathway(s). Further research on the modulation of Ca²⁺ and ERK2 signalling may help improve our ability to control PGE₂production and develop more effective bacteria-based immunotherapies.

POSTER 42

Determining the Impacts of the Mammalian Lung Environment on Cryptococcus Spore Germination

Nicolas Pereira^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

Cryptococcus is an environmental yeast capable of causing fatal meningitis in humans. Cryptococcus causes disease from inhalation 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 occurs early, but the kinetics of this process and their effect on dissemination and disease are unknown. We aim to understand how both spore properties and germination environment impact germination kinetics and dissemination in mammalian disease. To evaluate Cryptococcus germination in vitro we developed a high throughput, quantitative germination assay (QGA). Using QGAs, we determined 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 understand germination in a lung environment, we are developing a germination medium for QGAs from mouse lung homogenate (LH). Preliminary data show that spores germinate in LH media, but do so slower and less synchronously than in synthetic glucose medium. RNA-seq data from germination in LH medium show that germinating spores induce transcripts associated with host environment responses, such as the host environment associated transcription factor Pdr802. Simultaneously, we are using a mouse model of intranasal infection to determine germination timing in vivo. We discovered that in vivo germination kinetics are slower than in vitro, and approximately 20% of spores fail to germinate. Overall, these findings indicate that host lung and lung-like environments lead to germination profiles that differ substantially from standard in vitro germination. Studying the characteristics of spore germination in physiologically relevant environments promises to inform our understanding of early events in Cryptococcusinfection and provide insights into how spores mediate fatal cryptococcal disease.

POSTER 43

The McbR Transcription Factor Links Intracellular Folate Pool to Virulence in Enterohemorrhagic E. coli O157:H7

Quentin Perraud¹, Vanessa Sperandio¹

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

Management of enterohemorrhagic E. coli (EHEC) infection is symptomatic due to the increased risk of hemolytic uremic syndrome when antibiotics are administered. A different approach for the handling of EHEC infection would be to target virulence factors controlling the attachment of bacteria to the intestinal epithelium, such as the type III secretion system (TIIISS). The development of such approaches requires better knowledge of virulence regulation in EHEC.

A retrospective analysis on approaches developed to inhibit virulence in diverse pathogens led us to identify the sulfonamide scaffold as a common feature of a large number of anti-virulence compounds. Some sulfonamide drugs can act as antimicrobials by disrupting the tetrahydrofolate biosynthesis pathway, leading us to explore whether or not tetrahydrofolate starvation could lead to a decrease of virulence.

We showed that pharmacological disruption of tetrahydrofolate biosynthesis led to a decrease of TIIISS expression even when the drug is used below its minimum inhibitory concentration. Through RNAseq, mutagenesis, and ligand-binding experiments, we were able to demonstrate that this effect is directly dependent on the binding of a tetrahydrofolate pathway metabolite to the previously orphan McbR transcription factor, impacting its ability to bind to the DNA region controlling TIIISS transcription. The discovery of the natural ligand for this virulence-regulating protein opens the way for the rational design of anti-virulence drugs that do not present the undesirable off-target effects of sulfonamides.

POSTER 44

A Vital Role for B Cells in Vaccine-Induced Pulmonary T Cell Immunity to Influenza Virus

Cynthia C. Pryde-Aguilar^1,2, Hongtae Park¹, Thomas Cleven¹, Marulasiddappa R. Suresh¹

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

Current influenza vaccines primarily provide protection through subtype-specific antibodies targeting the mutation-prone surface proteins hemagglutinin and neuraminidase. However, these vaccines must be reformulated annually to match evolving seasonal strains. Broad immunity against heterosubtypic influenza A virus (IAV) infections, which could offer more durable protection, has been linked to tissue-resident memory (T_RM) CD4 and CD8 T cells in mucosal sites. To address this, we developed a nano-emulsion-adjuvanted IAV nucleoprotein (NP)-based subunit vaccine that induces robust NP-specific CD4 and CD8 T_RMs in the lungs and airways, providing broad protection against multiple IAV strains. The cellular and molecular mechanisms driving the development and function of these vaccine-induced T_RMs remain poorly understood. Our findings show that NP-specific B cells are induced in the lungs and mediastinal lymph nodes (MLNs) after vaccination. Depletion of B cells significantly reduced the activation and/or expansion of NP-specific CD4 T cells in the lymph nodes, spleen, lungs, and airways. In contrast, B cell depletion had a minimal effect on NP-specific CD8 T cell numbers in the MLNs and spleen but impaired the trafficking and/or accumulation of effector CD8 T cells in the lungs and airways. These results suggest that B cells might play a critical role in supporting CD4 T cell activation and the trafficking of CD8 T cells to mucosal sites. Future studies will explore the complex interactions between B cells, CD4 T cells, and CD8 T cells in vaccine-induced mucosal immunity to IAV.

POSTER 45

Role of Secondary Metabolites in Microbial Community Invasion

Josephine Putnam^1,2,3, Margaret Thairu^2,3, Alex Cheong^2,3, Jo Handelsman^2,3, Marc Chevrette^2,3,4

¹Microbiology Doctoral Training Program, University of Wisconsin–Madison, Madison, WI, USA, ²Department of Plant Pathology, University of Wisconsin–Madison, Madison, WI, USA, ³Wisconsin Institute for Discovery, Madison, WI, USA, ⁴Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA Despite decades of research on probiotics that improve human health and biocontrol bacteria that suppress plant disease, these microbial additives often fail to reliably establish themselves in resident microbial communities. Understanding the mechanisms underlying resistance to invasion is made difficult by the inherent ecological complexity of microbial communities. Here, we use a model microbial community called THOR (The Hitchhikers of the Rhizosphere), a well-characterized, genetically tractable community comprised of three bacterial species originally isolated from soybean roots. Many of THOR’s interactions are driven by secondary metabolites including antibiotics and siderophores, making it a good model to explore the role of secondary metabolites in mediating invasion. In this work, we examined how THOR population ratios respond to invasion and investigated what differentiates a successful invader from an unsuccessful one. Of 13 phylogenetically diverse rhizosphere bacterial isolates tested, only five successfully invaded THOR, and THOR population ratios were robust to invasion over time. The successful invader Delftia sp. CI11 produces a zone of inhibition against each THOR member, suggesting successful invasion may depend on the production of antibiotics or other inhibitory compounds. Ongoing work seeks to identify mutants of each THOR member with resistance to inhibition by Delftia sp. CI11 to elucidate the mechanism(s) of action of the inhibition and to determine whether Delftia sp. CI11 can invade a resistant THOR community. Overall, our work aims to deepen our understanding of the role of secondary metabolites in microbiome stability and invasion success to inform the rational design of better probiotics and biocontrol agents as well as provide new strategies for antibiotic discovery based on ecological principles.

POSTER 46

The Remediation of Antibiotic Resistance and Overusage Through Antibiotic-Producing Bacteria from Naturally Occurring Samples

Jamie Reilly¹, Manuel Wuest¹, Danielle Schaal¹

¹Department of Math and Natural Sciences, Marian University, Fond du Lac, Wisconsin, USA

Exposure to antibiotics in everyday life has exponentially increased over the past several decades, leading to an increase in bacterial survival and antibiotic resistance. When antibiotics are rendered ineffective, bacteria can contribute to nosocomial infections, surgical complications, and increased mortality upon infection. Of interest, E. Faecium, S. Aureus, K. Pneumoniae, A. Baumannii, P. Orzyihabitans, and E. Aerogenes are the leading causes of nosocomial infections due to their antibiotic resistance. This research is centered around providing relief from the antibiotic resistance crisis by searching for novel antibiotics produced by naturally occurring bacteria in the environment. Soil samples were collected and diluted to isolate individual bacterial colonies. Soil bacteria were initially screened for antibiotic production against B. Sub and E. Coli, which exhibited zones of inhibition (ZOI). Soil bacteria that produced a ZOI were then tested against the antibiotic-resistant ESKAPE pathogens using the swab-patch assay. Additionally, antibiotics were extracted from their bacterial sample with ethyl acetate, and different concentrations were tested against the ESKAPE pathogens. To characterize the bacteria, they were Gram-stained and tested against a series of commercial antibiotics, and sequenced to reveal Pseudomonas species. Subsequent experimentation will employ gas chromatography-mass spectrometry (GC-MS) as a means of identifying metabolites within the extracts to determine the prospect of a potential novel antibiotic.

POSTER 47

Identifying and Characterizing E. coli Oxidative Stress Responses

Viola Rintoul¹, Daniel Ajuzie¹, Elebeoba May^1,2

¹Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, USA, ²Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI, USA

Bacterial growth, reactions, and adaptations change drastically under different environments and stressors, including oxidative stress. Multiple different gene pathways can result in unexpected bacterial responses and different gene expressions that would not otherwise occur in a non-stressful environment, and the understanding of these responses are instrumental in identifying how bacteria overcome or succumb to stress. To study the effects of oxidative stress, we are utilizing different levels of iron against varied concentrations of hydrogen peroxide in fluorescently labeled E. coli cells, gene responses can be captured via quantification of GFP-labeled operons and characterized in different environments. We have found that activation of genes depends on not only the environment and nutrient availability, but also the starting growth levels before being put under stress and are continuing to investigate the effects of different growth conditions prior to additions of hydrogen peroxide. In addition to investigating oxidative stress, we have also been investigating the effects of how non-specific nutrient availability and different media types generate different levels of growth and gene activation following bolus additions of hydrogen peroxide, giving us a broader understanding of the many components that go into cells either overcoming or succumbing to external stresses. With a current focus on ihfB, a housekeeping gene, and oxyR, a gene that triggers a cascade of superoxide responses, we have begun to identify differences in stress responses that stem from multiple factors.

POSTER 48

Turmeric Exposure Increases Antimicrobial Activity in Soil-Derived Bacteria

Claire Roup¹, Claire Mathison,¹ Aurelia Meza¹, Tyson Engel¹

¹Tiny Earth, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Antibiotic resistance is a growing global health concern, increasing the need for new antimicrobial compounds.Turmeric was chosen because it contains bioactive compounds like curcumin that have been shown to have antimicrobial properties and may stimulate secondary metabolite production in bacteria. In this study, turmeric was added to soil-derived bacterial cultures to investigate whether exposure to turmeric increased the frequency of antimicrobial activity in bacterial isolates. Soil bacteria were cultured on media with and without turmeric exposure, and isolates were tested for antimicrobial activity using cross-streak assays against saferelatives of pathogenic bacteria. A Fisher’s Exact Test was used to compare antimicrobial production between treatment groups. Turmeric-exposed isolates produced greater antimicrobial activity than control isolates, with 30 producers in the turmeric groupcompared to 16 in the control group (p = 0.019).This research contributes to understanding how plant compounds influence microbial metabolism and may have implications for improving the antibiotic resistance issues.

POSTER 49

Comparative Genomics Reveals Decoupling of Secondary Metabolism and Antimicrobial Resistance across Bacterial Lifestyles

Murrel Saldanha^1,4,5, Jordi Weijers³, José D.D. Cediel-Becerra², Francisco Barona-Gómez³ Marc Chevrette^{4,5, †}

¹Department of Biophysics, University of Wisconsin-Madison, Madison, WI, USA, ²Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA, ³Institute of Biology Leiden, Leiden University, Leiden, South Holland, Netherlands, ⁴Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI, USA, ^5†Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA

Microbial secondary metabolism (SM) is widely assumed to be tightly coupled with antimicrobial resistance (AMR), with resistance genes protecting producer organisms from self-toxicity and forming the basis of resistance-guided antibiotic discovery. Despite this paradigm, the discovery of new antibiotic classes has been limited. This highlights a lack of understanding of the relationship between secondary metabolism and resistance across bacteria. To systematically evaluate this assumption, we analyzed ~100,000 bacterial genomes spanning 10 major taxonomic groups to quantify the relationship between biosynthetic gene clusters (BGCs) and AMR genes. Genomes were mined using antiSMASH for BGCs and the Comprehensive Antibiotic Resistance Database’s Resistance Gene Identifier for resistance genes. Homologous BGCs were grouped into gene cluster families (GCFs) to enable large-scale comparisons. Across taxa, we observe a striking lack of global association: 90–99% of GCF–AMR gene pairs are statistically independent, and resistance genes rarely co-localize within BGCs. These findings challenge the assumption that resistance is a reliable proxy for biosynthetic activity. Generating detailed biosynthetic and resistance profiles and stratifying by lifestyle reveals that prolific natural product producers such as the Streptomyces genus encode limited resistomes, whereas pathogenic taxa exhibit diverse and expanded resistomes. To quantify the effect of possible lateral transfer in AMR genes that could skew its limited correlation with SM, a pan-taxon phylogenetic tree reconstruction was performed to quantify the statistical influence of phylogeny on resistance. Results show that antibiotic classes with high rediscovery rates in resistance-guided strategies, such as glycopeptides, exhibit strong lineage-associated signals, whereas classes like β-lactams, shows higher evidence of lateral transfer. Together, this work reframes the relationship between SM and AMR, highlights limitations of resistance-guided discovery strategies and employs an eco-evo lens to help accelerate the discovery of new antibiotics.

POSTER 50

Host-Age and Niche-Specific Role of O-Antigen in a Mouse Model of Enteric Infection

Aidan Schmidt¹, Gale Hakopian¹, Gustavo Caballero-Flores¹

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

Enteropathogenic Escherichia coli (EPEC) is one of the largest causes of diarrhea-induced mortality and morbidity in infants and young children. Due to the lack of adequate animal models for EPEC, the enteric murine pathogen Citrobacter rodentium is used as an alternative model. C. rodentium is an effective surrogate system because it is closely related with EPEC and shares the Locus of Enterocyte Effacement (LEE) –a pathogenicity island responsible for regulating a Type-3 Secretion System (T3SS) and the formation of Attaching-Effacing Lesions. Another virulence factor shared between EPEC, C. rodentium, and other Gram-negative bacteriais O-antigen (O-Ag). O-Ag is the outermost portion of lipopolysaccharide, a cell surface component of gram-negative bacteria. However, unlike the LEE-regulated virulence factors, the role of O-Ag in C. rodentium pathogenesis in vivo is not well defined. Furthermore, although in vitro or ex vivo O-Ag studies exist for other pathogens, they fail to account for the complex gut environment interactions.

The purpose of this project is to use in vivo assays to determine the role O-Ag plays in C. rodentium pathogenesis in mouse models with distinct age, microbiota composition and/or genetic backgrond. For this, we generated a C. rodentium mutant strain lacking O-Ag. This mutant showed differences in colonization associated with mouse age. To test whether this effect was due to differences in microbiome composition, we infected gnotobiotic mice transplanted with distinct age microbiomes and found significant differences in colonization ability between groups. Additionally, we found that the O-Ag-deficient strain displays increased mortality when colonizing immunodeficient mice as opposed to wild-type animals, even though colonization levels remained similar between mouse strains. Compared with WT C. rodentium, the mutant strain also caused lessened intestinal inflammation but similar systemic colonization in immunodeficient mice. This phenotype was not observed in immunocompetent mice, suggesting O-Ag is required for natural systemic disease establishment in the presence of a fully functional immune system. Colectively, our results highlight specific contributions of O-Ag in a host-age and niche-specific manner.

POSTER 51

Defining and Modeling Structure-Function Relationships in Immune Response to Mycobacteria

Marina Slawinski^1,3, Thomas Potts³, Jayaraman Tharmalingam³, 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, USATuberculosis (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 [1]. Immune response to TB infection generates granulomas, which are organized clusters of immune cells that surround bacteria in an attempt to physically contain and clear infection. The granulomas can either successfully eliminate infection or contain the bacteria with ~10% risk of dissemination and progression to active disease, with a major concern being the spread of multidrug resistant strains. The ability of an individual to clear infection is impacted by various risk factors including nutrition, habits such as smoking and drinking, and comorbidities with other diseases. Therefore, it is of great importance to investigate how these risk factors impact granuloma formation and disease outcome to gain an understanding of which populations are most at risk of developing active infection and target therapies accordingly. To this end, we have developed fluorescently labeled murine macrophage cell lines and infect them with fluorescently labeled mycobacteria 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 environmental conditions representing risk factors (vitamin D deficiency, alcoholism) with the goal of gaining an understanding of how cell line origin and risk factors impact the effectiveness of immune response. Bacterial uptake and elimination are inferred from quantification of intracellular and extracellular bacteria, and infection outcome is further determined from microscopy images. Cell segmentation with Imaris software is used to process confocal microscopy data and provide features related to macrophage infection status, morphology, and movement [2], which will be used to further characterize differences in infection dynamics and develop various types of computational models [3]. 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. Ideally, these immune models can serve as predictive tools, advancing the development of “digital twin” models that can be personalized to individuals for improved diagnostic and treatment outcomes in healthcare.

POSTER 52

Cryptococcus Spore Germination Can Occur in the Absence of Oxygen

Spieles, J^1,2 Barnes, M¹ Hull, CM^1,2

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

Cryptococcus fungal spores must germinate into yeast to cause disease in mammalian hosts. Our lab used a high-throughput screen of small molecules to identify novel germination inhibitors. We discovered one class of compounds that presumably targets complex II of the electron transport chain (ETC) in a fungal-specific manner. Although yeast (obligate aerobes) cannot grow in the presence of ETC inhibitors or in anaerobic environments, we discovered that spores can germinate in the absence of detectable oxygen. This calls into question the need for ETC function and the mechanism of complex II inhibitors during germination. These paradoxical findings reveal a substantial knowledge gap in how respiration and metabolism operate during the energy-intensive process of germination. To evaluate the role of the ETC in germination, we tested inhibitors of ETC complexes and conducted transcriptomic analyses on spores germinating hypoxically. Our data indicate that ETC function is important for hypoxic germination, including a role for the cyanide-independent alternative oxidase (AOX1). Future experiments will determine the unique and potentially targetable mechanisms of cellular respiration in germinating Cryptococcus spores, including evaluating potential roles of non-oxygen terminal electron acceptor pathways.

POSTER 53

Ketogenic Diet Responsive Microbial and Host Metabolites Mediate Th17 Inhibition in a Multiple Sclerosis Model

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

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

The interactions between diet, the gut microbiota, and immune cells shape autoimmune diseases including multiple sclerosis (MS). In a mouse model of MS, both a ketogenic diet (KD) and supplementation of a KD-associated host metabolite, β-hydroxybutyrate (βHB), reduced disease severity in a microbiota-dependent manner. In further studies, a KD-enriched microbe, Lactobacillus murinus, and its tryptophan derived metabolite indole lactate (ILA) both independently conferred disease protection without a KD. However, whether ILA is required for Lactobacillus mediated Th17 inhibition and disease protection is unclear. To investigate this, we used isogenic strains of Lactobacillus reutericapable or incapable of ILA production through the deletion of the aromatic aminotransferase (ArAT) enzyme. ArAT catalyzes the first step in converting tryptophan into ILA. We assessed the Th17 inhibitory and disease protective effects of both mutant and wildtype strains in vitro and using the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS. The ILA-producing L. reuteri strain significantly inhibited IL-17A in vitro and reduced EAE disease severity compared to the ArAT knockout, indicating that ILA is required for microbial-mediated Th17 inhibition and disease protection. To further explore whether metabolite-driven immunomodulation could be therapeutically leveraged, additional EAE studies were conducted using the commercially available β-hydroxybutyrate (βHB) precursor, 1,3-butanediol, administered in drinking water either as a pre-treatment or during disease progression. 1,3-butanediol treatment ameliorated EAE, significantly increased circulating βHB levels, and reduced central nervous system T cell responses. Together, these findings demonstrate that distinct ketogenic-responsive microbial and host metabolites, ILA and βHB, suppress Th17-driven inflammation.

POSTER 54

Within THOR’s Roots: The Antimicrobial Koreenceine as a Measure of Community Metabolome Variation between Environmental Conditions

Willow Ungaro^1,2, Delaney Miller¹,Marc Chevrette^1,2, 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

Bacteria are capable of producing a large array of antibiotic compounds, but many are only elicited when associated with certain microbes or specific environmental niches. Model microbial communities can help us understand how community dynamics and environmental conditions change metabolic profiles. To investigate metabolomic shifts within plant roots, the rhizosphere, I utilized a synthetic microbial community named THOR (The Hitchhikers of the Rhizosphere), which is composed of Pseudomonas koreensis, Bacillus cereus, and Flavobacterium johnsoniae. Specifically, THOR was grown in hydroponically collected Medicago sativa(alfalfa) root exudate and compared to typical laboratory media conditions. Community density was quantified with CFU plating and metabolic analysis executed through filter-sterilized supernatants that were solid-phase extracted and ran through LC-MS/MS. A key feature that differed between treatments was koreenceine, an antibiotic responsible for a cascade of gene modulation within the THOR community as well as lowered F. johnsoniae populations. Koreenceine A, present in typical laboratory conditions, was also found when THOR was grown on alfalfa plant roots while root exudate collected hydroponically. These results indicate that THOR as a model microbial community grown within a laboratory is comparable to what may be seen within the rhizosphere and can lead us one step closer to fully understanding natural product elicitation within the rhizosphere.

POSTER 55

Gut Bacterial Metabolite Imidazole Propionate Exacerbates Neurodegeneration

Vaibhav Vemuganti^1,2, Jea Woo Kang³, Eric R. McGregor⁴, James R. Hilser⁵, Ruben Aquino-Martinez¹, Sandra Harding³, Katharina R. Beck⁶, Yuetiva Deming³, Rachel Studer⁷, Sterling C. Johnson^3,7, Sanjay Asthana^3,7, Henrik Zetterberg^{3,8,9,10,11,12,13}, Kaj Blennow^8,914,15, Corinne D. Engelman¹⁶, Hooman Allayee⁵, Rozalyn M. Anderson^4,17, Tyler K. Ulland^3,18, Fredrik Bäckhed^6,19,20, Barbara B. Bendlin^3,7*, Federico E. Rey^1,21*

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA, ²Cellular and Molecular Pathology Graduate Training Program, University of Wisconsin-Madison, Madison, WI, USA, ³Wisconsin Alzheimer’s Disease Research Center, University of Wisconsin-Madison, Madison, WI, USA, ⁴Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA, ⁵Division of Cardiology, Department of Medicine, David Geffen School of Medicine of UCLA, Los Angeles, California, USA, ⁶Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden, ⁷Wisconsin Alzheimer’s Institute, University of Wisconsin, Madison, WI, USA, ⁸Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden, ⁹Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden, ¹⁰Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK, ¹¹UK Dementia Research Institute at UCL, London, UK, ¹²Hong Kong Center for Neurodegenerative Diseases; Inn,6,oHK, Hong Kong, China,¹³Centre for Brain Research, Indian Institute of Science; Bangalore, India,¹⁴Paris Brain Institute, ICM, Pitié-Salpêtrière Hospital, Sorbonne University; Paris, France,¹⁵Neurodegenerative Disorder Research Center, Division of Life Sciences and Medicine, and Department of Neurology, Institute on Aging and Brain Disorders, University of Science and Technology of China and First Affiliated Hospital of USTC, Hefei, P.R. China, ¹⁶Department of Population Health Sciences, University of Wisconsin-Madison; Madison, WI, USA, ¹⁷Geriatric Research, Education, and Clinical Center, William S. Middleton Memorial Veterans Hospital, Madison, WI, USA, ¹⁸Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison; Madison, WI, USA, ¹⁹Region Västra Götaland, Sahlgrenska University Hospital, Department of Clinical Physiology; Gothenburg, Sweden, ²⁰Microbiome Health Initiative and the National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark, ²¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison; Madison, WI, USA

Host–microbe interactions are strongly influenced by diet and lifestyle. Imidazole propionate (ImP), a gut-derived metabolite of histidine, has been linked to cardiometabolic disease and unhealthy lifestyle choices that often potentiate neurodegenerative disorders. However, mechanistic links in the gut-brain axis remain largely unexplored. Therefore, we used a well characterized cohort of 1,196 individuals to examine links between plasma ImP, ImP-producing bacteria, and Alzheimer’s disease and related dementias (ADRD). Furthermore, ImP was independently associated with cognitive decline and blood markers of neurodegeneration, even after adjusting for age, sex, apolipoprotein-E genotype, and lifestyle. Additionally, genome wide integrative analysis identified a region on chromosome 12 linked to both ImP and ADRD, supporting a causal role. In mouse models, long-term ImP exposure worsened amyloid and tau pathology. Mechanistic studies showed that ImP disrupts the blood–brain barrier and increases neuronal phosphorylation, partly through interaction with glycogen synthase kinase-3-beta. These findings suggest that ImP-producing gut bacteria may be potential targets to reduce ADRD risk.

POSTER 56

Role of Gut Microbial-Derived Indole in Behavior and Central Nervous System Modulation in Mice

Wierschke H¹, Rosay T¹, Perraud Q¹, Zhao C², Riters LV², Kiraly D³, Sperandio V¹

¹Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA,²Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA ³Center for Addiction Research, Wake Forest University, Winston-Salem, North Carolina, USA

Gut microbial metabolites can influence host central nervous system (CNS) function, but the specific metabolites and interactions that shape reward- and anxiety-like behaviors from the gut remain unclear. We tested whether microbial indole production from the commensal Bacteroides thetaiotaomicron (B. theta) alters behavior and CNS activation in mice. After depletion of the gut microbiota with antibiotics, mice were mono-colonized with wild-type B. theta (indole-producing) or an isogenic ΔtnaA strain (indole-deficient), along with antibiotic-only and intact-microbiome controls. Behavioral testing focused on exploration- and reward-related phenotypes, including open-field testing, locomotor activity, and sucrose preference, and we quantified c-Fos immunofluorescent cell counts in select CNS regions relevant to arousal, affect, and reward. ΔtnaA-colonized mice showed a more exploratory phenotype, including increased distance traveled and increased rearing/vertical activity. These mice also gained more weight without a clear increase in food intake. In sucrose testing, ΔtnaA-colonized mice drank a greater percentage of sucrose water. In the CNS, several regions showed trending differences in c-Fos positive cell counts. Together, these preliminary findings suggest that the presence of the gut microbial metabolite indole can shift reward- and anxiety-related behavior in mice and may also alter CNS activation.

POSTER 57

Investigating Dietary Interventions in a Mouse Model of Early-Life Infectious Diarrhea

Victoria G. Zalutskiy¹, Aidan Schmidt¹, Federico Rey¹, Gustavo Caballero-Flores¹

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

Infant and early childhood enterohemorrhagic and enteropathogenic Escherichia coli (EHEC, EPEC) infections are major causes of severe pediatric diarrheal disease globally. Relative to adults, children are at greater risk for potentially lethal EHEC and EPEC complications such as hemolytic uremic syndrome (HUS) and severe, persistent diarrhea, respectively. Currently, there are no effective treatments for EHEC and EPEC infections in children, partially because antibiotic usage is not recommended as it triggers Shiga toxin release, HUS manifestation, and long-term microbiota dysbiosis. Therefore, there is an urgent need for the development of alternative strategies, such as dietary interventions, as potential therapies for early-life infectious diarrhea. In this project, I utilize an EHEC/EPEC-related, mouse-pathogen model organism, Citrobacter rodentium, to study the effect of a fibreless, simple-nutrient diet (SD) on host survival, intestinal inflammation, and microbiota composition in infected juvenile mice. Our preliminary results show the SD to improve juvenile mouse survival, weight gain, and infection resolution. Microbiome analysis suggests the SD enriches specific commensal bacteria that may promote C. rodentium eradication. Furthermore, fiber reconstitution in the SD diminished its protective effect. Further characterization of the gut microbiome and other host or pathogen specific effects of the SD will fully define the mechanism of protection involved. Overall, this work suggests dietary interventions as a possible alternative therapy for severe infectious diarrheal disease in early life.

POSTER 58

Investigating the Mechanism of Enterohemorrhagic Escherichia coli Virulence Repression by the QseE Histidine Kinase

Shenyan Zhang¹, Vanessa Sperandio¹

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

Enterohemorrhagic Escherichia coli (EHEC) is a Gram-negative pathogen that colonizes the human colon. The pathogenicity island termed locus of enterocyte effacement (LEE) is a major virulence factor in EHEC and is responsible for EHEC attachment to the intestinal epithelium. Deciphering how the LEE genes are regulated will help to understand virulence regulation in EHEC and develop potential therapeutic strategies. LEE gene expression is controlled by multiple regulatory systems including two component systems (TCSs), which consists of a membrane sensor histidine kinase (HK) and a cytoplasmic response regulator (RR). My research has shown that an HK termed QseE represses LEE expression under anaerobic conditions. While this mechanism is not fully understood, we identified the Cnu regulator to be involved in QseE regulation of LEE genes, which is independent of QseF, the cognate RR of QseE. My project aims to explore how QseE regulates LEE gene expression through Cnu. Firstly, I will screen for non-cognate RRs which can be phosphorylated by QseE and subsequently regulate cnu and LEE expression. Then I will explore the mechanisms of LEE regulation by Cnu. Finally, I will characterize the effect of QseE and Cnu on LEE expression and virulence in vivo using a murine infection model.