Regulation of cellular antimicrobial activity by a single microbiota metabolite

1. Regulation of cellular antimicrobial activity by a single microbiota metabolite Zeni Crisp1, Nandita Kohli2, Carrie Mueller1, Arul Jayaraman1,2, Robert C. Alaniz1 1 Microbial & Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center 2Chemical 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. How do the bacteria that naturally live in our gut protect us from oral infection with pathogenic microbes? Methods Results Introduction Phagocytes are immune cells that digest and kill bacteria. Cytotoxicity Assay • 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. Figure 6. Cytotoxicity is determined by the Lactase Dehydrogenase release from the cytoplasm. Results • Figure 3. • (Left) Macrophage is potent antimicrobial phagocyte. • (Right) Dendritic cells activate immune system (Tcells) Figure 9. Indole-treated Dendritic cells reduce Salmonella- induced Cytotoxicity. Conclusions Salmonella Pathogenesis Hypothesis [make this text bullets] Microbiota produce molecules that augment phagocyte killing of bacteria. • These data suggest that indole is an important microbiota-derived endogenous mediator that protects against gut pathogens such as Salmonella. Dendritic Cell Indole Salmonella Dendritic Cell Salmonella + + + Discussions • Indoleis 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. Figure 4. Host cells conditioned with Indole are more resistant to Salmonella infection. Figure 7. Indole-treated Dendritic cells resist Salmonella invasion • 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 Methods Gentamicin Protection Assay + Microbiota: Beneficial microbes in our gut + Gentamicin 1 hour Infection MOI = 10 1 hour Incubation with Gentamicin Acknowledgments • Dr. Robert C. Alaniz • NanditaKohli • Carrie Mueller • Dr. Arul Jayaraman Lyse = Invasion T = 0 • MadhuKatepalli • Shelby Steinmeyer • Dr. Andrews-Polymenis • NSF and NIH Figure 8. Indole-treated Dendritic cells restrict Salmonella intracellular survival and replication. Lyse = Survival/ Replication T = 4 References • Figure 5. • Dendritic cells (DC2.4) are conditioned with Indole over night. Infect DC 2.4 with S. typhimuriumfor 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. • TarunBansal, Robert C. Alaniz, Thomas K. Wood, Arul Jayaraman. The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation. doi: 10.1073/pnas.0906112107. • Andrea Haraga, Maikke B. Ohlson, and Samuel l. Miller. Salmonella interplay with host cells. 2008 January. Doi: 10.1038/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. 2011 December; 13(12): 1858-1869. doi:  10.111/j.1462-5822.2011.01701.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. 2008 March. doi: 10.1073/pnas.0707370105. • http://iconsinmedicine.wordpress.com/tag/salmonella/ • http://s2.hubimg.com/u/1366057_f520.jpg Figure 2. Colonization Resistance is a phenomenon where microbiota protect the host from infection by pathogens.