Regulation of cellular antimicrobial activity by a single microbiota metabolite Zeni Crisp 1, Nandita Kohli 2, Carrie Mueller 1, Arul Jayaraman 1,2, Robert C. Alaniz 1 1 Microbial & Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center 2 Chemical Engineering, College of Engineering, Texas A&M University Abstract: The microbiota, commensal symbiotic microbes that naturally inhabit the gastrointestinal (GI) tract, contribute to immune & physiologic homeostasis in the host GI tract. The microbiota provide resistance to enteric pathogen invasion and colonization; an important function termed “colonization resistance” (CR). Despite this observation, the mechanisms mediating CR are poorly understood. We hypothesized the microbiota produce compounds that either directly or indirectly contribute to CR. Indole is an abundant GI tract metabolite produced from tryptophan strictly by the microbiota. In the GI tract, dendritic cells (DCs), professional phagocytes that sense the microbiota and provide a link to host immunity, are continuously exposed to indole. Our previous work demonstrated that indole reduces pathogenic bacteria chemotaxis, motility, and epithelial cell attachment. Here, we tested whether indole directly promotes DC antimicrobial properties. Our results demonstrate that DCs conditioned with indole restrict Salmonella typhimurium invasion, revealing a potential novel mechanism regulating CR. Results Conclusions Discussions Indole is a natural product, derived from microbiota, with low toxicity, and potential to develop new therapeutic compounds. With chemical engineering, we may develop indole analogues that may be superior to antibiotics, without the emergence of drug resistance. Results Figure 7. Indole-treated Dendritic cells resist Salmonella invasion Figure 8. Indole-treated Dendritic cells restrict Salmonella intracellular survival and replication. These data suggest that indole is an important microbiota-derived endogenous mediator that protects against gut pathogens such as Salmonella. References Figure 9. Indole-treated Dendritic cells reduce Salmonella- induced Cytotoxicity. Introduction Salmonellosis is one of the most common foodborne disease in the world. 93 million cases per year of gastroenteritis worldwide caused by Salmonella. The increase of multi-drug resistant Salmonella strain highlights the need for more research on this clinically relevant pathogen and for the discovery of new treatment options. Salmonella Pathogenesis + + Salmonella IndoleDendritic Cell Hypothesis Figure 4. Host cells conditioned with Indole are more resistant to Salmonella infection. Methods Phagocytes are immune cells that digest and kill bacteria. T = 0 Lyse = Invasion + 1 hour Infection MOI = 10 + Gentamicin T = 4 Lyse = Survival/ Replication 1 hour Incubation with Gentamicin Figure 5. Dendritic cells (DC2.4) are conditioned with Indole over night. Infect DC 2.4 with S. typhimurium for 1 hour. Add Gentamicin to kill the extracellular Salmonella for 1 hour. At T=0 hr, lyse DC 2.4 and determine CFU/mL for Salmonella invasion. At T=4 hr, lyse DC 2.4 and determine CFU/mL for Salmonella survival and intracellular replication. Figure 5. Dendritic cells (DC2.4) are conditioned with Indole over night. Infect DC 2.4 with S. typhimurium for 1 hour. Add Gentamicin to kill the extracellular Salmonella for 1 hour. At T=0 hr, lyse DC 2.4 and determine CFU/mL for Salmonella invasion. At T=4 hr, lyse DC 2.4 and determine CFU/mL for Salmonella survival and intracellular replication. Gentamicin Protection Assay [make this text bullets] Figure 1. Upon contact with intestinal epithelial cells, Salmonella activate Type 3 Secretion System I to translocate the virulence proteins (effectors) into the host cytoplasm. The effectors modulate rearrangement of actin cytoskeleton, which results in membrane ruffles and promote Salmonella uptake. After internalization, Salmonella modify host phagosome into Salmonella-Containing Vacuole (SCV). Changes in microenvironment of SCV trigger the activation of Type 3 Secretion System II (T3SSII). The effectors of T3SSII are necessary for positioning of SCV near the Golgi network. This allow Salmonella to intercept host vesicular trafficking and redirect nutrients to promote intracellular replication Microbiota: Beneficial microbes in our gut + Salmonella Dendritic Cell Microbiota produce molecules that augment phagocyte killing of bacteria. How do the bacteria that naturally live in our gut protect us from oral infection with pathogenic microbes? Methods Cytotoxicity Assay Acknowledgments Dr. Robert C. Alaniz Nandita Kohli Carrie Mueller Dr. Arul Jayaraman Madhu Katepalli Shelby Steinmeyer Dr. Andrews-Polymenis NSF and NIH Tarun Bansal, Robert C. Alaniz, Thomas K. Wood, Arul Jayaraman. The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation. doi: /pnas Andrea Haraga, Maikke B. Ohlson, and Samuel l. Miller. Salmonella interplay with host cells January. Doi: /nrmicro1788. Shannon E. Majowicz, Jennie Musto, Elaine Scallan. The Global Burden of Nontyphoidal Salmonella Gastroenteritis. International Collaboration on Enteric Disease “Burden of Illness” Studies. Terence A. Agbor, Beth A,. McCormick. Salmonella Effectors: Important players modulating host cell function during infection. Cell Microbiol December; 13(12): doi: /j x. Susan L. Fink, Tessa Bergbaken, Brad T. Cookson. Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms March. doi: /pnas Figure 3. (Left) Macrophage is potent antimicrobial phagocyte. (Right) Dendritic cells activate immune system (Tcells) Figure 2. Colonization Resistance is a phenomenon where microbiota protect the host from infection by pathogens. Figure 6. Cytotoxicity is determined by the Lactase Dehydrogenase release from the cytoplasm.