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
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.
1. Bao, Y.j. Liang, Z., Booyjzsen, C. Mayfield, J.A. Li, Y. Lee, S.W., Ploplis, V.A., Song, H. Castellino, F.J. Unique Genomic Arrangements in an Invasive Serotype M23 Strain of Streptococcus pyogenes that Induce Hypervirulence. 2014. Manuscript in revision.
2. Ugrinov, K.U., Freed, S.D. and Lee, S.W. A multiparametric algorithm to assess genetic mutations in Mucopolysaccharidosis type IIIA amenable to pharmacological-chaperone based therapies. 2014. Manuscript in revision.
3. Flaherty, R.A., Freed, S.D., Lee, S.W. The wide world of Bacteriocins: Ribosomally encoded bacterial peptides. 2014. PLOS Pathogens.10(7): e1004221.
4. Mayfield,J.A. Liang, Z., Agrahari, G., Lee, S.W., Ploplis, V.A., Castellino, F.J. Random mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal variants with altered gene regulatory activity and virulence. 2014. PLOS One. 9(6):e1000698.
5. Thomas, C.L. and Lee, S.W. Knowing is half the battle: Targeting virulence factors of Group A Streptococcus for vaccine and therapeutics. 2012. Curr Drug Targets. 13(3):308-22.
6. Markley, A.L., Jensen, E.R., Lee, S.W. A novel E. coli-based bioengineering strategy to study Streptolysin S biosynthesis. 2012. Anal Biochem. 420(2):191-3.
7. Gonzalez, D.J, Lee, S.W., Hensler, M. Dahesh, S. Markley, A.L. Mitchell, D.A., Banderia, N., Nizet V. , Dixon, J.E., Dorrestein, P.C. Clostridiolysin S: A posttranslationally modified biotoxin from Clostridium botulinum. 2010. J Biol Chem. 285(36):28220-8.
8. Mitchell, D.A., Lee, S.W., Markley, A.L., Limm, J., Pence, M.A., Gonzalez, D., Dorrestein, P.C., Nizet, V., Dixon, J.E. Structural and functional dissection of the heterocyclic peptide cytotoxin streptolysin S. 2009. J Biol Chem. 284(19):13004-12.
9. 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. 2008. Proc. Natl. Acac. Sci. U.S.A.105:(15), 5879-84.
10. Yooseph S., Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li W, Jaroszewski L, Cieplak P, Miller CS, Li H, Mashiyama ST, Joachimiak MP, van Belle C, Chandonia JM, Soergel DA, Zhai Y, Natarajan K, Lee S, Raphael BJ, Bafna V, Friedman R, Brenner SE, Godzik A, Eisenberg D, Dixon JE, Taylor SS, Strausberg RL, Frazier M, Venter JC. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families. 2007. PLoS Biol. Mar;5(3):e16.
11. Higashi, D.L., Lee, S.W., Snyder, A., Weyand, N.J., Bakke, A, So, M. Dynamics of Neisseria gonorrhoeae attachment: microcolony development, cortical plaque formation, and cytoprotection. 2007. Infect Immun. Oct;75(10):4743-53.
12. Weyand, N., Lee, S.W., Higashi, D.L. , Cawley, D., Yoshihara, P., and So, M. Monoclonal antibody detection of CD46 clustering beneath Neisseria gonorrhoeae microcolonies. 2006. Infect Immun. Apr;74(4):2428-35.
13. Lee, S.W., Higashi, D.L., Snyder A., Merz, A.J., Potter, L., and So, M. PilT is required for PI(3,4,5)P3- mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. 2005. Cell Micro. Sep;7(9):1271- 84.
14. Lee, S.W., Bonnah, R.A., Higashi, D.L., Atkinson, J.P., Milgram, S.L., and So, M. CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae. 2002. J Cell Biol, 156(6): 951-957.
15. Bonnah, R.A., Lee, S.W., Vasquez, B.L., Enns, C.A., and So, M. Alteration of epithelial cell transferrin- iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae. 2000. Cell Micro. 2:207-218.