Molecular Microbiology and Biosynthesis of Peptide Antibiotics
B.A., University of California Berkeley
Ph.D., Oregon Health Science University
Postdoctoral Fellow, HHMI, University of California San Diego
325 Galvin Life Science Center
Office Phone: (574) 631-7197
Bacteriocins are a large class of ribosomally synthesized toxins that serve as effective antibiotics for the producing organism against similar species (narrow spectrum) or across genera (broad spectrum). It is believed that about half of all bacteria and archaea produce at least one bacteriocin, and current efforts in genome analysis will likely lead to a tremendous increase in the number and diversity of this class of antibiotics.
It is our primary research goal to gain a better understanding of the biosynthetic mechanisms underlying bacteriocin production. Our focus is on a particular class of bacteriocins that use a conserved mechanism of posttranslational modification to produce the active toxin. One important member of this family is the highly active cytolysin Streptolysin S (SLS), an important virulence factor produced by the human pathogen Group A Streptococcus pyogenes (GAS). GAS is a leading human pathogen causing common infections such as pharyngitis and impetigo, as well as invasive syndromes such as necrotizing fasciitis and toxic shock syndrome. Worldwide estimates of 18 million cases of severe GAS disease with 500,000 deaths are reported each year.
In vitro reconstitution of the SLS toxin has demonstrated that a precursor peptide and three conserved enzymes work in concert to modify the precursor into an active toxin (Figure 1). Gene clusters that resemble the SLS biosynthesis complex are present in several important human pathogens, such as E. coli, Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. Importantly, this class of peptide antibiotics are produced ribosomally, and thus will be amenable to genetic engineering strategies. Furthermore, inhibitors of the posttranslational modifying enzymes will offer new targets for drugs which could be effective against a variety of pathogens. In addition, antibodies raised against modified versions of these peptide antibiotics can be explored as potential strategies for vaccine development. Finally, in vitro reconstitution studies demonstrate that we can exploit this mechanism of bacteriocin biosynthesis to generate a library of artificial peptide antibiotics, many of which will undoubtedly have novel therapeutic value. It is likely that the discovery of similar peptidic antibiotics will rapidly expand as more genomes are sequenced.
Efforts in our laboratory involve important multidisciplinary avenues— chemical approaches for structural identification of bacteriocins, large-scale screening methods to identify active antibiotic candidates, as well as molecular and microbiology-based approaches to better understand how microorganisms biosynthesize and utilize these bacteriocins.
Figure 1.In vitro reconstitution of Streptolysin S. The SLS genetic cluster contains a precursor peptide (SagA) that is posttranslationally modified by the action of the SLS synthetase complex SagBCD, encoded by the genes adjacent to SagA. In vitro reconstitution of SLS cytolytic activity is measured using lysed erythrocytes. (Upper right). Hemolytic assays of SagA plus SagBCD synthetase reactions in microtiter wells containing defibrinated sheep blood. Bars indicate lysis normalized to a positive control (Triton X-100). Levels indicated are 1:1, 3:4, 1:2, and 1:4 ratios of synthetase reaction to blood (left to right) of 16-h reactions (n = 3). Lane 1, SagA plus SagBCD; lane 2, SagA alone; lane 3, SagBCD alone; lane 4, SagA plus SagBC; lane 5, SagA plus SagCD; lane 6, SagA plus SagBD; lane 7, vehicle. Inset demonstrates typical appearance of lytic (L) and nonlytic (N) reactions. (Lower right). Fluorescence microscopy and DIC images of HEK293a cells treated as indicated. Actin filaments (red) and cytoplasm (green) are merged in Upper, and DIC images are in Lower.
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).
Lee, S.W. *, 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
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