Our structures of Y. pestis FyuA and pesticin allowed us to engineer a novel phage therapy drug against a Gram-negative pathogen: We solved the structure of FyuA, a TonB-dependent iron transporter required for virulence in bubonic plague, with and without its cognate siderophore, ferric yersiniabactin. At the same time, we determined the structure of a bacteriocin called pesticin that uses FyuA to cross the outer membrane. Once inside the periplasm, pesticin kills the cell by degrading the peptidoglycan layer. From our structure we discovered that the killing domain of pesticin resembles phage T4 lysozyme, so we engineered a hybrid bacterial-phage toxin that contains a bacterial targeting domain (to FyuA) and a phage killing domain. We showed that the hybrid lysine evades the natural protection mechanism of toxin-producing strains and kills all Yersinia strains tested, both in vitro and in vivo (mouse model of bubonic plague). This is the first demonstration of phage therapy for Gram-negative pathogens because until now, no one knew how to transport the toxin across the outer membrane. This work was recently published at PNAS* and has received considerable attention, including a Nature Microbiology Reviews Highlight. We have recently begun a project to identify and characterize outer membrane proteins that are either essential or required for virulence in a variety of Gram-negative pathogens, since these proteins may make good vaccine components and drug targets. Structural characterization of several candidate proteins is currently underway. *Lukacik, P., Barnard, T.J., Keller, P.W., Chaturvedi, K., Seddiki, N., Fairman, J.W., Noinaj, N., Kirby, T.L., Henderson, J.P., Steven, A.C., Hinnebusch, B.J. &Buchanan, S.K. (2012). Structural engineering of a phage lysin that targets Gram-negative pathogens. Proc. Natl. Acad. Sci. USA, 109, 9857-9862. PMCID: PMC3382549 Research Highlighted: In this Issue Proc. Natl. Acad. Sci. USA, 109, 9667-9668 (2012). Research Highlighted: Nat. Rev. Microbiol. 10, 520-521 (2012). Research Highlighted: SciBX 5(27);doi:10.1038/scibx.2012.706 (2012) (Nature publishing group).

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$617,337
Indirect Cost
City
State
Country
Zip Code
Noinaj, Nicholas; Mayclin, Stephen; Stanley, Ann M et al. (2016) From Constructs to Crystals - Towards Structure Determination of β-barrel Outer Membrane Proteins. J Vis Exp :
Botos, Istvan; Majdalani, Nadim; Mayclin, Stephen J et al. (2016) Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens. Structure 24:965-76
Cash, Devin R; Noinaj, Nicholas; Buchanan, Susan K et al. (2015) Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae. Infect Immun 83:4438-49
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Noinaj, Nicholas; Cornelissen, Cynthia Nau; Buchanan, Susan K (2013) Structural insight into the lactoferrin receptors from pathogenic Neisseria. J Struct Biol 184:83-92
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Madden, Jeremy T; Toth, Scott J; Dettmar, Christopher M et al. (2013) Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline. J Synchrotron Radiat 20:531-40
Lukacik, Petra; Barnard, Travis J; Keller, Paul W et al. (2012) Structural engineering of a phage lysin that targets gram-negative pathogens. Proc Natl Acad Sci U S A 109:9857-62
Fairman, James W; Dautin, Nathalie; Wojtowicz, Damian et al. (2012) Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis. Structure 20:1233-43