douglasm@illinois.edu
room 3105 IGB (main office) and room 345 RAL
Office: (217) 333-1345
Mail to:
Box 22-5, RAL
M/C 712
Urbana, IL 61801
Douglas A. Mitchell
Assistant Professor of Chemistry
Affiliate, Department of Microbiology
Education
Professor Mitchell received his undergraduate degree in chemistry from Carnegie Mellon University in 2002. After a short internship in medicinal chemistry at Merck Research Laboratories, he joined the group of Michael Marletta at the University of California, Berkeley. Doug’s thesis centered on protein S-nitrosation for which he received a Ph.D. in chemistry in 2006. For postdoctoral studies, he worked with Jack Dixon at the University of California, San Diego. There, Doug studied the biosynthesis of a cytolytic virulence factor known as streptolysin S. Professor Mitchell joined the University of Illinois faculty in 2009.
Molecular underpinnings of bacterial virulence
Our primary objective is to better understand the molecular underpinnings of bacterial virulence. By understanding these processes in greater detail, we seek to develop strategies for the next generation of antimicrobial drugs. The Mitchell laboratory is a multidisciplinary research team that draws methodology from the fields of chemical biology, structural biology, bioinformatics, microbiology, and pharmacology.
The prototypic virulence factor for our studies is the broadly active cytolysin known as streptolysin S (SLS). This toxin is secreted by the human pathogen, Streptococcus pyogenes. Commonly known as “strep”, these bacteria are directly responsible for the death of over 500,000 people annually. Production of SLS allows the pathogen to flourish and initiate more invasive infections such as necrotizing fasciitis (the flesh-eating bacterial disease) and toxic shock syndrome. In fact, SLS is so important during the early stages of infection that S. pyogenes mutants deficient in SLS production are easily defeated by the host immune response and are non-pathogenic in animal models of infection. Chemically, SLS is an extensively posttranslationally modified microcin. Microcins are small peptides (<10 kDa) produced by ribosomes and nearly all exhibit antibiotic activity. The modifications that distinguish SLS from other microcins, with the exception of microcin B17, are thiazole and oxazole heterocycles. These moieties are derived from cysteine and serine residues and serve to restrict peptide backbone flexibility. This endows SLS with robust cytolytic activity. The genes that encode this SLS maturation machinery are neatly organized into a nine open reading frame biosynthetic operon.
During the course of studying the SLS biosynthetic pathway, genome mining efforts have yielded over 200 orthologous gene clusters in organisms that span the prokaryotic phyla. With the recent flood of genomic information, novel SLS-like biosynthetic clusters are being identified in exotic organisms on a weekly basis. These include the “Who’s who” of human pathogens, extremophiles, bacteria renowned for the production of antibiotics, and cannibalistic bacteria. It is important to note that even though these organisms harbor SLS-like biosynthetic clusters, and thus produce thiazoles and oxazoles by utilizing conserved machinery, only a handful are known or expected to produce cytolysins. Most of these biosynthetic clusters contain ancillary tailoring enzymes that ensure thiazoles and oxazoles are not the only modifications present in the final product. We have named the products produced by this genetic superfamily, “TOMM” for Thiazole and Oxazole Modified Microcins. Indeed, one TOMM biosynthetic cluster has been demonstrated to target DNA gyrase and recently, others have been shown to target the ribosome. The overwhelming majority remain functionally unclassified and await experimental interrogation.
Current research in the Mitchell Laboratory can be broadly divided into two categories. I.) We aim to characterize the structure and function of unique TOMM natural products and the enzymes that produce them. To achieve this, we employ chemical and biological approaches including high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, X-ray crystallography, in vitro reconstitution, and genetic manipulation techniques. II.) We aim to utilize what we have learned about TOMM natural product structure and function to therapeutically exploit biosynthetic weaknesses.
It is anticipated that these studies will shed light on a new family of natural products with diverse molecular structure and mechanism of action. In the cases were human pathogens produce a TOMM natural product, the development of biosynthetic inhibitors not only aids the study of toxin maturation but could also be clinically useful. Elucidation of the chemical structure of such toxins also lays the foundation for the development of a structure-based vaccine. Darwinian selection theory predicts that antibiotics targeting virulence, instead of essential life processes, will delay the development of drug resistance. Major progress in this area is needed if we are to avoid a medical catastrophe in the 21st century.
Representative Publications
Mitchell, D.A.; Lee, S.W.; Pence, M.A.; Markley, A.L.; Limm, J.D.; Nizet, V.; Dixon, J.E., “Structural and Functional Dissection of the Heterocyclic Peptide Cytotoxin Streptolysin S”, J. Biol. Chem., 284:13004-13012 (2009)
Ghosh, A.; Mitchell, D.A.; Chanda, A.; Ryabov, A.D.; Popescu, D.L.; Upham, E.; Collins, T.J., “Catalase-Peroxidase Activity of Iron(III)-TAML Activators of Hydrogen Peroxide” J. Am. Chem. Soc., 130:15116-15126 (2008)
Walsh, C.T.; Nolan, E.M., “Morphing Peptide Backbones into Heterocycles” Proc. Natl. Acad. Sci. USA, 105: 5655-5656 (2008)
Lee, S.W.* and Mitchell, D.A.*; Markley, A.L.; Hensler, M.E.; Gonzalez, D.; Wohlrab, A.; Dorrestein, P.C.; Nizet, V.; Dixon, J.E., “Discovery of a Widely Distributed Toxin Biosynthetic Gene Cluster” Proc. Natl. Acad. Sci. USA, 105: 5879-5884 (2008)
*These authors contributed equally to this work
Mitchell, D.A.; Morton, S.U.; Fernhoff, N.B.; Marletta, M.A., “Thioredoxin is Required for S-Nitrosation of Procaspase-3 and Inhibits Apoptosis in Jurkat Cells” Proc. Natl. Acad. Sci. USA, 104: 11609-11614 (2007)
Mitchell, D.A., Marletta, M.A., Michel, T., “Effects of S-Nitrosation of Nitric Oxide Synthase Isoforms” (Review) Adv. Exp. Biol, Nitric Oxide 1, Ch. 7.
Chahbane, N.; Popescu, D.L.; Mitchell, D.A.; Chanda, A.; Lenoir, D.; Ryabov, A.D.; Schramm, K.W.; Collins, T.J., “Fe(III)-TAML Catalyzed Green Oxidative Degradation of the Azo Dye Orange II by H2O2 and Organic Peroxides: Products, Toxicity, Kinetics, and Mechanisms” Green Chem., 9: 1-10 (2007)
Mitchell, D.A.; Morton, S.U.; Marletta, M.A., “Design and Characterization of an Active Site Selective Caspase-3 Transnitrosating Agent” ACS Chem. Biol., 1: 659-665 (2006)
Tannenbaum, S.R. and White, F.M., “Regulation and Specificity of S-Nitrosylation and Denitrosylation” ACS Chem. Biol., 1: 615-618 (2006)
Erwin, P.A.; Mitchell, D.A.; Sartoretto, J.; Marletta, M.A.; Michel, T., “Subcellular Targeting and Differential S-Nitrosylation of Endothelial Nitric Oxide Synthase” J. Biol. Chem. 281: 151-157 (2006)