Structural engineering of a phage lysin that targets Gram-negative pathogens Petra Lukacika, Travis J. Barnarda, Paul W. Kellerb, Kaveri S. Chaturvedic, Nadir Seddikia,JamesW.Fairmana, Nicholas Noinaja, Tara L. Kirbya, Jeffrey P. Hendersonc, Alasdair C. Stevenb, B. Joseph Hinnebuschd, and Susan K. Buchanana,1 aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; bLaboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda,MD 20892; cCenter for Women’s Infectious Diseases Research, Washington University School of Medicine, St. Louis, MO 63110; and dLaboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840 Edited by* Brian W. Matthews, University of Oregon, Eugene, OR, and approved April 18, 2012 (received for review February 27, 2012) Bacterial pathogens are becoming increasingly resistant to antibio- ∼10 kb plasmid called pPCP1 (7). Pla facilitates invasion in bu- tics. As an alternative therapeutic strategy, phage therapy reagents bonic plague and, as such, is an important virulence factor (8, 9). containing purified viral lysins have been developed against Gram- When strains lose the pPCP1 plasmid, they are killed by pesticin positive organisms but not against Gram-negative organisms due thus ensuring maximal virulence in the bacterial population. to the inability of these types of drugs to cross the bacterial outer Bacteriocins belong to two classes. Type A bacteriocins depend membrane. We solved the crystal structures of a Yersinia pestis on the Tolsystem to traverse the outer membrane, whereas type B outer membrane transporter called FyuA and a bacterial toxin bacteriocins require the Ton system. For both classes, the primary called pesticin that targets this transporter. FyuA is a β-barrel mem- interaction that triggers the translocation process occurs between brane protein belonging to the family of TonB dependent transpor- the bacteriocin and a specific outer membrane receptor protein. ters, whereas pesticin is a soluble protein with two domains, one Pesticin has been classified as a type B bacteriocin because killing that binds to FyuA and another that is structurally similar to phage depends on the products of the tonB, exbB, and exbD genes and, T4 lysozyme. The structure of pesticin allowed us to design a phage like other type B bacteriocins, pesticin has a TonB box motif therapy reagent comprised of the FyuA binding domain of pesticin located near its N-terminus (10). In addition to tonB, exbB, and fused to the N-terminus of T4 lysozyme. This hybrid toxin kills exbD, these bacteria must express a receptor gene—fyuA—in specific Yersinia and pathogenic E. coli strains and, importantly, can order to be killed by pesticin. FyuA is an integral outer membrane evade the pesticin immunity protein (Pim) giving it a distinct advan- protein that acts as the receptor for pesticin (11). It is also a major tage over pesticin. Furthermore, because FyuA is required for viru- virulence factor for some Yersinia strains (12, 13) and certain lence and is more common in pathogenic bacteria, the hybrid toxin pathogenic E. coli (14, 15). Although Y. pestis also expresses FyuA also has the advantage of targeting primarily disease-causing bac- in its outer membrane, it is protected from pesticin-induced cell teria rather than indiscriminately eliminating natural gut flora. death by the product of the pesticin immunity gene that is always found adjacent to the pesticin gene (10). Interaction between colicin ∣ muramidase ∣ TonB-dependent transport ∣ plague FyuA and pesticin is necessary to trigger the import of pesticin across the outer membrane into the periplasm. Once inside the acteriophages are viruses that have a prolific ability to infect, periplasm, pesticin kills the cell by degrading peptidoglycan (16) Bmultiply within, and subsequently eliminate large numbers of through a muramidase (lysozyme) activity (17). bacteria. Their potential for use in microbial therapy was recog- Here, we report the first production of an engineered lysin that nized shortly after their discovery in the late 1910s (1, 2). Phage can directly kill Gram-negative bacteria. We solved the 2.1 Å therapy was initially developed, but later fell out of favor, when structure of pesticin and used the structure to guide engineering small molecule antibiotics became cheaper and easier to manu- of a “hybrid lysin.” Pesticin consists of two domains: one that facture in large homogeneous quantities. More recently, the targets and binds to FyuA, and the other that degrades peptido- emergence of antibiotic resistant bacterial strains has renewed glycan. The engineered hybrid consists of T4 lysozyme, which is interest in using phages or their components in therapy, agricul- an archetypal lysin, attached to the FyuA-targeting domain de- ture, food, and water treatment (3). Reagents have been devel- rived from pesticin. To visualize the import machinery better, oped using phage derived proteins called lysins to clear in vivo we solved the structure of FyuA at 3.2 Å and the hybrid toxin infections of Gram-positive bacteria such as Streptococcus pneu- at 2.6 Å resolution. FyuA is a TonB-dependent iron transporter moniae (4) and Bacillus anthracis (5). Clearance occurs because (18) consisting of a 22-strand β-barrel with a plug domain inserted lysins hydrolyze the surface exposed peptidoglycan that forms the in the pore. The hybrid toxin resembles pesticin and retains BIOPHYSICS AND cell wall of Gram-positive bacteria causing cell rupture ; however, the same two-domain architecture. We show that the hybrid lysin COMPUTATIONAL BIOLOGY a major limitation of this approach is that it cannot be applied crosses the outer membrane and kills cells not only in a model to Gram-negative bacteria because viral lysins cannot cross the E. coli system but also in bacterial pathogens. Importantly, killing outer membrane without help from accessory proteins or mem- only affects cells that produce FyuA (11). The action of the hybrid brane-disrupting agents. The outer membrane effectively shields lysin is unaffected by Pim (10), which is a protein that some peptidoglycan from degradation by the externally added lysin. Yersiniae produce to inhibit peptidoglycan degrading enzymes. We solved the crystal structure of a protein toxin from Y. pestis that showed us how to engineer a phage lysin capable of killing Author contributions: P.L., T.J.B., N.N., T.L.K., J.P.H., A.C.S., B.J.H., and S.K.B. designed Gram-negative bacteria. This protein, called pesticin, belongs to a research; P.L., T.J.B., P.W.K., K.S.C., N.S., J.W.F., N.N., T.L.K., J.P.H., and S.K.B. performed class of bacterial proteins known as bacteriocins (referred to as research; P.L., T.J.B., P.W.K., J.W.F., N.N., J.P.H., A.C.S., B.J.H., and S.K.B. analyzed data; and colicins when they target E. coli). Bacteriocins are produced by P.L., T.J.B., and S.K.B. wrote the paper. bacteria to kill related bacterial strains in times of stress. Bacteria The authors declare no conflict of interest. producing the bacteriocin protect themselves by expressing an *This Direct Submission article had a prearranged editor. immunity protein to inhibit activity of the toxin (6). Y. pestis pro- 1To whom correspondence should be addressed. E-mail: [email protected]. duces pesticin to promote virulence. Pesticin, pesticin immunity This article contains supporting information online at www.pnas.org/lookup/suppl/ protein, and plasminogen activator (Pla) are encoded on a doi:10.1073/pnas.1203472109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203472109 PNAS ∣ June 19, 2012 ∣ vol. 109 ∣ no. 25 ∣ 9857–9862 Downloaded by guest on October 1, 2021 The hybrid lysin can, therefore, potentially be used against any strands 5 and 6 and an additional α-helix following strand 7. strain expressing FyuA. The C-terminal domain of pesticin is primarily α-helical, except for a small irregular antiparallel three-strand β-sheet (Fig. 1B). Results The N-terminal 13 residues are disordered in the structure. This Structure Determination of Y. pestis FyuA and Pesticin. We began this region includes the TonB box motif (DTMVV, residues 3–7) (10) project by determining the structure of FyuA, a 71 kDa outer which is necessary for type B bacteriocins to be translocated membrane transporter that normally transports ferric yersinia- across the outer membrane. Other regions that could not be bactin (Fe-Ybt) and is important for virulence in certain Yersinia resolved include residues 25–34 (25–32 in noncrystallographic and E. coli strains. FyuA was expressed in E. coli, purified and symmetry related copy B) and the C-terminal residue. crystallized using the detergents LDAO and C8E4. Crystals dif- fracted to 3.2 Å resolution, and the structure was solved by single Pesticin Shares Structural Similarity with Phage T4 Lysozyme. Pesticin wavelength anomalous dispersion (Table S1). The structure of does not resemble any other known bacteriocin in sequence or FyuA has features typical of the TonB-dependent transporter structure (6); so, we analyzed the pesticin fold using the DALI family (Fig. 1A) (18) including a membrane-spanning 22-strand server (20) to search for other structural homologs. DALI ana- β-barrel whose pore is blocked by a plug domain. On the periplas- lysis revealed a striking similarity between the C-terminal domain mic side of the membrane, the individual β-strands of the barrel of pesticin and phage T4 lysozyme (21) that had not been postu- are connected by short turns, whereas on the extracellular side lated prior to the structure determination because the sequence strands are connected by long extracellular loops L1–L11.