(Figure Presented) The thiocillins are natural-product antibiotics derived from ribosomally encoded peptides that undergo extensive posttranslational modifications to yield the mature trithiazolylpyridine-containing macrocyclic compound. Poor pharmacokinetic properties have prevented the clinical use of these highly potent antibiotics. Through in vivo manipulation of the gene responsible for production of the thiocillin precursor peptide, we have generated 65 novel thiocillin variants, allowing us to explore structure-activity relationships involved in both precursor peptide maturation and antibiotic activity

Acker, M.G.

Bowers, A.A.

Walsh, C.T.

PubMed

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Generation of Thiocillin Variants by Prepeptide GeneReplacement and In Vivo Processing by Bacillus cereusMichael G. Acker, Albert A. Bowers, and Christopher T. Walsh*Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,Massachusetts 02115The thiazolyl peptide antibiotics comprise a family of >80 members with the commoncharacteristics of a central pyridine/piperidine ring typically decorated by three thiazolesubstituents and a macrocyclic peptide ring containing additional thiazoles1 (Figure 1). Somemembers of this antibiotic group, such as thiostrepton and nosiheptide, have a secondmacrocyclic ring. The thiazolyl peptide natural products target one of two sequential steps inbacterial protein synthesis. Molecules such as GE2270 and thiomuracin bind tightly to EF-Tuand abrogate its aminoacyl-tRNA delivery function.2a-c In contrast, thiostrepton and thethiocillins bind directly to the 50S ribosomal subunit, interacting both with 23S rRNA loopsand the amino acid side chains of protein L11, with the effect of disrupting EF-G activity andtherefore preventing tRNA translocation on the ribosome.2d-e While thiazolyl peptides displaypotent antibiotic activity against gram-positive bacteria such as methicillin-resistantStaphylococcus aureus (MRSA)2f poor aqueous solubility and pharmacokinetics have limitedtheir clinical use. Furthermore, total syntheses of thiazolyl peptide compounds whilerepresenting remarkable achievements still present formidable challenges for structure-activityvariations,3a-f limiting the production of novel compounds with improved pharmacokineticproperties.Whether biosynthesis of the highly modified thiazolyl peptides occurs via nonribosomal orribosomal assembly has long been debated;4a ribosomally-encoded natural products4b areknown to contain dehydro-amino acids (the lantibiotics4c-d) and thiazoles (microcin B17,patellamide4e-f) like the thiocillins, but pyridine formation has not been seen in other ribosomalpeptide scaffolds. Four recent reports have disclosed that GE2270, thiomuracin,2b nosiheptide,5a thiostrepton,5b-c and the thiocillins5c-d all arise by posttranslational modification ofribosomally generated prepeptides of 50-60 residues. The sequences that show up in the matureantibiotic scaffolds are derived from the C-terminus of these microbial prepeptides.In the thiocillins from the producer Bacillus cereus ATCC 14579 at least 10, and up to 13, ofthe C-terminal 14 residues undergo posttranslational modification to generate a set of eightrelated antibiotics. Based on stable isotope feeding studies6a-b, the ten core transformations arethought to involve dehydrations of Ser17 and Ser10 on the way to pyridine ring formation,dehydration of Thr4 and Thr13 to dehydrobutyrine residues, and cyclizations of Cys2, 5, 7, 9,11, 12 to six thiazoles. Three additional posttranslational modifications appear to occurstochastically: hydroxylation at Val6, O-methylation at Thr8 and/or ketone/alcoholinterconversion of the C-terminal residue arising from decarboxylation of Thr14, giving riseto 2 × 2 × 2 = 8 possible thiocillins. We note that four of the eight thiocillins producedabundantly by B. cereus display similar efficacy against Bacillus subtilis and two MRSAChristopher_Walsh@hms.harvard.edu.Supporting Information Available. SI Figures (23), SI Tables (3), experimental procedures, and spectral data.NIH Public AccessAuthor ManuscriptJ Am Chem Soc. Author manuscript; available in PMC 2010 December 9.Published in final edited form as:J Am Chem Soc. 2009 December 9; 131(48): 17563–17565. doi:10.1021/ja908777t.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptstrains, with minimum inhibitory concentrations (MIC) of 0.2 - 0.9 μg/mL and < 0.03 – 0.1μg/mL, respectively (see SI).With the recent discovery of the thiocillin gene cluster from Bacillus cereus5d, we set out togenetically manipulate the biosynthetic machinery by site-directed mutagenesis to makemodifications to the thiocillins, perhaps improving their pharmacokinetic properties withoutthe need for new synthetic strategies. The thiocillin gene cluster contains four contiguousidentical copies of a gene encoding a purported 52-residue precursor peptide (tclE-H), whichis thought to be post-translationally modified to yield the mature antibiotic scaffold. To confirmthat the tandem genes tclE-H are responsible for generating the thiocillin prepeptide, wegenerated a B. cereustclE-H knockout strain (tclΔE-H) by homologous recombination with aplasmid containing sequence homology to the tcl gene cluster but lacking tclE-H (see SI).Cultures of Bacillus cereus ATCC 14579 (WT) and tclΔE-H were extracted for compound andanalyzed by reverse phase HPLC. WT B. cereus extracts contained thiocillins as observed byUV absorption at 350 nm and LC-MS (Figure 2). In contrast, extracts of tclΔE-H failed to yieldany of the eight thiocillin compounds. To rescue production of the thiocillins and confirm tclEas the prepeptide responsible for thiocillin production, we inserted a single, plasmid-basedcopy of tclE into the chromosome of tclΔE-H by Campbell integration. Cultures of the knock-in strain (tclE KI) were extracted and analyzed by reverse phase HPLC and LC-MS to revealthat tclE KI rescued production of thiocillins to near WT levels (Figure 2).The ability to rescue thiocillin production with a single, plasmid-based copy of tclE enablesmutasynthesis of novel thiocillin compounds in B. cereus by use of variant tclE genes to initiatestructure/activity relationship studies of the thiazolyl peptide antibiotics. Initially, we havefocused on residues 3, 4, 6, 8, and 13 which are not involved in setting the trithiazolylpyridinecore in the mature scaffold, reasoning that amino acid substitutions at these positions probethe promiscuity of the thiocillin tailoring enzymes while minimizing disruption to the coreframework. In all, 14 single amino acid substitutions were made in the thiocillin prepeptidetclE at these 5 positions, with 12 resulting in production of one or more thiocillin variants(Table 1 and SI Table 6.1). The combination of possible post-translational modifications atpositions 6, 8 and/or 14, gave rise to 65 thiocillin molecular variants detected by LC and highresolution MS (SI Table 6.1).In a first set of site-directed mutants of the tclE gene, amino acids T3, V6, T8 and T13 weresubstituted with alanine, and T4 with valine, relatively conservative changes, minimizing theexisting side chains to methyl or isopropyl groups. The tclE mutant plasmids were eachtransformed into B. cereus tclΔE-H. HPLC analysis of extracts from 0.5 L cultures confirmedthe presence of a number of thiocillins. The T3A variant produced six of eight expectedthiocillins, while four thiocillins were observed in T8A, where A8 cannot be methylated. T4Vgenerated four of eight expected compounds plus two additional derivatives with massescorresponding to the addition of two hydroxyl groups, suggesting that tclD, the V6 hydroxylaseis also able to hydroxylate a valine at position 4. Further evidence of the promiscuity of tclDis the identification of forms with hydroxylated Cβ of the alanine side chain among the sixcompounds isolated from the V6A variant.To determine antimicrobial activity, the extracts from each compound were subjected to normalphase chromatography on silica gel and fractions containing compounds absorbing at 350 nmwere collected. Because the individual thiocillin derivatives produced by WT B. cereusinhibited bacterial growth with similar MICs, the tclE variants from a given mutant were pooledfor antibiotic activity assays by disk diffusion on LB plates containing B. subtilis strain 168.V6A, T8A and T13A variants maintain similar levels of antibiotic activity to the wild typethiocillin set (Table 1). In contrast, T3A and T4V derivatives failed to inhibit growth at amountsup to 8 μg suggesting that these particular variations disrupt binding or positioning of theAcker et al. Page 2J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptcompounds at the L11/23S rRNA binding interface on the large ribosomal subunit. To quantifyantibiotic activity MICs were determined in serial dilution liquid culture assays with both B.subtilis and S. aureus. V6A and T13A variants showed 2-4 fold improved activity against B.subtilis and Methicillin-resistant Staphylococcus aureus (MRSA) strain COL and were equallyactive against MRSA strain MW2. In contrast, T8A was slightly decreased in antibiotic activity.To introduce charge, T3D and V6D were then generated to determine if the side chaincarboxylate anions, which could potentially improve the solubility of the thiocillin scaffold,can be accepted in these positions by the nine posttranslational modification ORFstclDJKLMNOPS. Cultures of the V6D mutant failed to produce any thiocillin compounds asdetermined by LC and high res MS. In contrast, T3D produced four of eight expectedcompounds in sufficient quantity to be purified; however at amounts up to 8 μg, the T3Dvariants failed to inhibit growth of B. subtilis. Positions T3 and V6 were also substituted withlysine. No thiocillin compounds were identified from extracts of the V6K mutant. Althoughproduction levels were significantly reduced from those of WT thiocillin, requiring growth ina 5L fermenter, the T3K variant produced two thiocillin compounds. As with T3D, T3K wasinactive against B. subtilis in disk diffusion assays containing up to 8 μg of compound.LC-MS analysis of extracts of T3K identified multiple compounds with additional massincreases of 100.106 Da, suggesting C4H4O3 as the added functional group. MS/MS analysisconfirmed the extra mass in all fragments containing lysine 3 and N-succinylation wasconfirmed by MS/MS and NMR (see SI). N-succinylation of T3K and T8K, presumably bysuccinyl-CoA may be a self-protection strategy by the producer organism. N-succinylated T3Kderivatives with the extended carboxylate side chain were as inactive as the unmodified T3Kthiocillin scaffold in disk diffusion assays. The corresponding T4K and T8K mutants in thetclE gene were next explored. T8K generated all four expected compounds (no 8 O-methylation) as well as three N-succinylated derivatives. T4K produced four compounds, allof which were methylated at position T8. Interestingly, not a single N-succinylated derivativeof T4K was observed. Production of T4K was insufficient for antibiotic activity analysis andT8K failed to inhibit growth of both B. subtilis and MRSA.As a third test of the thiocillin biosynthetic processing machinery to accept tclE prepeptidemodifications, we introduced a cysteine substitution as variant T8C. In the maturation of WTthiocillin, all 6 cysteine residues in the 14-amino acid C-terminus of tclE are converted tothiazoles. The addition of a seventh cysteine could give distinct outcomes: 1) completeconversion to a seventh thiazole like the six native cycteines, via an intermediate thiazoline;2) S-methylation of C8 in analogy to O-methylation of T8 in the native tclE; or 3) nomodification. Extracts of the T8C variant contained near WT levels of compounds, and LC-MS analysis identified seven thiocillins. No thiazole at residue 8 was detected, however threeof four expected thiazoline derivatives were identified by MS/MS; the S-methyl Cys8 formspredominated. Disk diffusion assays confirmed the antibiotic activity of T8C compoundsagainst B. subtilis, and MIC values against B. subtilis, MRSA COL and MRSA MW2 indicateretention of almost full antibiotic efficacy.The results presented herein begin to decipher the functional requirements for processing ofthe 52-residue tclE prepeptide to the mature thiocillin scaffold and the stochastic variants atside chains 6, 8, and 14. This is also a beginning to map the antibiotic activity of thiocillinvariants. In all, 65 novel thiazolyl peptide compounds were generated. While conservativevariants at positions 6, 8 and 13 maintained considerable or equivalent antibiotic activity, thoseat positions 3 and 4 as well as more drastic charge insertion mutants were completely inactivein the concentration ranges tested. These results correlate well with the position of micrococcinmodeled into the complex with the 50S ribosome by Harms and colleagues2e. Threonine-3 anddehydrobutyrine-4 appear in close proximity to ribosomal protein L11; disrupting theseAcker et al. Page 3J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptcontacts could result in perturbation of micrococcin binding to the ribosome and loss ofantibiotic activity.The ability to express tclE gene mutants in a B. cereus strain deleted of its four tandemendogenous copies of tclE-H sets the stage for more extensive structure-activity evaluations.These include alterations that still allow processing to the mature trithiazolylpyridine core inthis highly morphed ribosomal peptide antibiotic framework (e.g., the requirement for threethiazoles surrounding the pyridine core and the size and flexibility of the macrocyclic ringconnecting thiazole 2 and thiazole 9). Fermentation of the variant thiocillins will also allowevaluation of the subset of scaffolds that retain antibiotic activity and show improvements insuch parameters as aqueous solubility.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank Michael Fischbach, Laura Wieland Brown, David Rudner and Daniel Lopez for helpful discussions andJonathan Swoboda and Jenny O'Neill for guidance with MIC assays. This work was supported by NIH NIGMS GrantNo. 20011 and NERCE Grant No. NIAID U54 AI057159 (C.T.W.). Reagents were prepared with the assistance ofthe NERCE Biomolecule Production Core (NIAID U54 AI057159). A.A.B. is supported by NIH National CancerInstitute Postdoctoral Fellowship Grant No. CA136283.References1. Bagley MC, Dale JW, Merritt EA, Xiong X. Chem. Rev 2005;105:685. [PubMed: 15700961]2. (a) Parmeggiani A, Krab IM, Okamura S, Nielsen RC, Nyborg J, Nissen P. Biochemistry 2006;45:6846.[PubMed: 16734421] (b) Morris RP, Leeds JA, Naegeli HU, Oberer L, Memmert K, Weber E,LaMarche MJ, Parker CN, Burrer N, Esterow S, Hein AE, Schmitt EK, Krastel P. J. Am. Chem. Soc2009;131:5946. [PubMed: 19338336] (c) Heffron SE, Jurnak F. Biochemistry 2000;39:37. [PubMed:10625477] (d) Cameron DM, Thompson J, March PE, Dahlberg AE. J. Mol. Biol 2002;319:27.[PubMed: 12051934] (e) Harms JM, Wilson DN, Schluenzen F, Connell SR, Stachelhaus T,Zaborowska Z, Spahn CM, Fucini P. Mol. Cell 2008;30:26. [PubMed: 18406324] (f) Kamigiri K,Watanabe M, Nagai K, Arao N, Suzumura K, Suzuki K, Kurane R, Yamaoka M, Kawano Y.Thiopeptide compounds suitable for treatment of multidrug resistant bacteria infection.2002WO20020726173. (a) Nicolaou KC, Dethe DH, Leung GY, Zou B, Chen DY. Chem. Asian J 2008;3:413. [PubMed:18188863] (b) Nicolaou KC, Zak M, Rahimipour S, Estrada AA, Lee SH, O'Brate A, Giannakakou P,Ghadiri MR. J. Am. Chem. Soc 2005;127:15042. [PubMed: 16248640] (c) Nicolaou KC, Zou B, DetheDH, Li DB, Chen DY. Angew. Chem. Int. Ed. Engl 2006;45:7786. [PubMed: 17061302] (d) DelgadoO, Muller HM, Bach T. Chemistry 2008;14:2322. [PubMed: 18270986] (e) Muller HM, Delgado O,Bach T. Angew. Chem. Int. Ed. Engl 2007;46:4771. [PubMed: 17503407] (f) Lefranc D, CiufoliniMA. Angew. Chem. Int. Ed. Engl 2009;48:4198. [PubMed: 19408268]4. (a) Carnio MC, Stachelhaus T, Francis KP, Scherer S. Eur. J. Biochem 2001;268:6390. [PubMed:11737193] (b) McIntosh JA, Donia MS, Schmidt EW. Nat. Prod. Rep 2009;26:537. [PubMed:19642421] (c) Xie L, Miller LM, Chatterjee C, Averin O, Kelleher NL, van der Donk WA. Science2004;303:679. [PubMed: 14752162] (d) Willey JM, van der Donk WA. Annu. Rev. Microbiol2007;61:477. [PubMed: 17506681] (e) Li YM, Milne JC, Madison LL, Kolter R, Walsh CT. Science1996;274:1188. [PubMed: 8895467] (f) Donia MS, Hathaway BJ, Sudek S, Haygood MG, RosovitzMJ, Ravel J, Schmidt EW. Nat. Chem. Biol 2006;2:729. [PubMed: 17086177]5. (a) Kelly WL, Pan L, Li C. J. Am. Chem. Soc 2009;131:4327. [PubMed: 19265401] (b) Yu Y, DuanL, Zhang Q, Liao R, Ding Y, Pan H, Wendt-Pienkowski E, Tang G, Shen B, Liu W. ACS Chem. Biol.2009epub ahead of print (c) Liao R, Duan L, Lei C, Pan H, Ding Y, Zhang Q, Chen D, Shen B, Yu Y,Liu W. Chem. Biol 2009;16:141. [PubMed: 19246004] (d) Brown LC, Acker MG, Clardy J, WalshCT, Fischbach MA. Proc. Natl. Acad. Sci. U. S. A 2009;106:2549. [PubMed: 19196969]Acker et al. Page 4J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript6. (a) Mocek U, Knaggs AR, Tsuchiya R, Nguyen T, Beale JM, Floss HG. J. Am. Chem. Soc1993;115:7557. (b) Mocek U, Zeng Z, O'Hagan D, Zhou P, Fan LDG, Beale JM, Floss HG. J. Am.Chem. Soc 1993;115:7992.7. thiocillin numbering: Ser1-Thr14 where Ser1 is Ser39 of the prepeptideAcker et al. Page 5J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Annotated gene cluster responsible for the production of the thiocillins. Structure of thiocillindepicts the three positions of stochastic modification (R1, R2, R3) in green.Acker et al. Page 6J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.HPLC trace of thiocillin extractions from B. cereus ATCC 14579 (WT), tclE-H knockouttclEΔE-H (KO) and tclE KI (KI).Acker et al. Page 7J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcker et al. Page 8Table 1Summary of thiocillin variants produced in this study and their antibiotic efficacy against B. subtilis and methicillin-resistant S. aureus.MIC (μg/mL)aMutantCompoundsObservedbAdditionalModifications168cCOLdMW2e1T3K4N-succinylation>8>8>82T3D4-->8>8>83T3A6-->100>100>1004T4K4--ntfntnt5T4V6-->100>100>1006V6DN/A--ntntnt7V6KN/A--ntntnt8V6A6Ala-hydroxylation0.130.060.069T8C7S-methylation,Cys-cyclization0.30.150.0810T8A4--10.250.2511T8K6N-succinylation>4>4>412T13A7--0.130.060.0613WTg6--0.50.060.13a Minimum inhibitory concentrations determined by overnight culture in 96-well plate format.b details available in SI.c Bacillus subtilus 168.d Methicillin-resistant Staphylococcus aureus COL.e Methicillin-resistant Staphylococcus aureus MW2.f nt = not tested.g WT = wild type.J Am Chem Soc. Author manuscript; available in PMC 2010 December 9.

Generation of thiocillin variants by prepeptide gene replacement and in vivo processing by Bacillus cereus

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Generation of thiocillin variants by prepeptide gene replacement and in vivo processing by Bacillus cereus

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