Bacitracin by Feodor Belov, Dorothea Bickert, Eric Bräuchle, Paul Arras

Bacitracin By Feodor Belov, Dorothea Bickert, Eric Bräuchle, Paul Arras

Content: History and Name Structure and synthesis of the polypeptide antibiotic Bacitracin-A - structure - non ribosomal biosynthesis - bioactivity What Bacitracin it used for and how does it work? - General way of action

- Target: C55- Isoprenyl pyrophosphate and place of action - Details of Binding - Medical use

History and Name

In 1945 John T. Goorley discovered a strain of bacillus subtillis in the knee wound of a girl named Margaret Treacy. From these bacteria bacitracin was isolated and described by Johnson et al. in Science Magazine (Vol 102, 12. October 1945). The Name of the girl, Treacy, is the origin of the name Bacitracin, although the “e” was left out for terms of simplicity.1

Structure and synthesis of the polypeptide antibiotic Bacitracin-A

Bacitracin is a polypeptide antibiotic which, for industrial use, is produced through the use of bacteria cultures of Bacillus subtilis. Therefore the bacterium uses a non-ribosomal peptide synthetase, a multi-enzyme-complex separated in different modules.

Structure

It is important to differentiate between the many derivatives of bacitracin during the synthesis. While only Bacitracin-A possesses the antibiotic effect, Bacitracin-F is the reason for toxic by-effects. Although all of the derivatives just differ from each other through small alterations in their molecular structure, most of them have no real use for the bacteria killing effect.

1 structure of Bacitracin-A with its specific thiazolin-ring 2 Bacitracin-F which is the reason for Bacitracin’s toxic effect

1 Mechanism of Action of Antibacterial Agents herausgegeben von Fred E. Hahn; Springer Science & Business Media, 06.12.2012 2

More important than the variety of the derivatives is the specificity of hetero-cycles which each derivative possesses. Especially the condensation among the first two amino acids of the covered molecule determines its later importance and application. This can be seen in the bottom left corner of the above image of Bacitracin-A or the top left corner of the above image of Bacitracin-F. Overall, each bacitracin molecule is built up by twelve amino-acids. Isoleucine and cysteine are essential for the hetero-cycle.

3 3D-structure of Bacitracin-A  you can see the sulphur atom marked yellow as a part of the thioazolin-ring

Non-ribosomal pepdtidsythesis

“Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which are responsible for the synthesis of structural diverse peptide products that feature distinguished pharmacological profiles. One important structural aspect of many nonribosomal peptides is the introduction of heterocyclic elements into the peptide by the attack of an amino acid sidechain nucleophile onto the peptide backbone. To constrain the peptide into its bioactive conformation, additional macrocyclization can be observed, which is catalyzed in the last step of the biosynthesis by cyclase activity. […]” (Wagner, 2006, preface).

The non-ribosomal way of peptide synthesis is justified due to its enormous amount of multiplicity compared to the ribosomal way. This relies on the variety of non-ribosomal peptid synthetases (NRPS). Those NRPS have a defined and unique setup in which they condensate, link, bind and define the macromolecule. Each NRPS is segmented in modules which serve to assemble of one amino acid, and again are segmented in (mostly) three domains: A-domain, C-domain and PCP-domain.

The A-Domain (as for adenylation) is required in a module and activates the substrate. In presence of Mg2+ ions an activated acyl-adenyl stage will be able to bind the A-domain. This process requires energy in form of ATP. For each amino acid there is one unique A-domain.

4 activation of the substrate via A-domain (Wagner, B. (2006))

The PCP-domain (Peptide Carrier Protein) is also required in every single module. Its main function is to bind the activated substrates (after A-Domain) by thiolation to the enzyme region. This makes it possible for the next domain to connect two amino acids.

5 loading of the PCP-domain through thiolation (Wagner, B. (2006))

The C-domain (or condensation domain) is as well as the both shown domains required in every single module. By catalysing the attack from the alpha amino-group at the thioester carbonyl-C and forming the amide bond of both amino acids, it is the main character at chain elongation.

6 chain elongation through the catalysed amide bond (Wagner, B. (2006))

Although every module in a NRPS requires the three domains (A-, PCP- and C-domain), there are more optional domains that are important for the impact of the finished molecules. One domain that is only found once in a NRPS is the termination domain, which separates the finished molecule (after all A-domains are used) from the enzyme. In bacitracin this domain initializes the folding of the macro cyclic ring at the end of the molecule.

7 termination domain (TE-domain) and the add-on folding to the macro cycle (Wagner, B. (2006))

Bioactivity

Another very important domain, which is as well optional, is the Cy-domain. It is part of the first module in every NRPS (that produces bacitracin) and replaces the C-domain. At this domain the cyclisation of the thiazolin-ring takes place. The first amino acid is isoleucine which will be bond with the next amino acid cysteine.

8 isoleucine and cysteine are coupled and form the hetero-cycle which later will be important for the antibiotic effect (Wagner, B. (2006))

The hetero-cycle is the most specific part of bacitracin and in the end will be responsible for the antibiotic effect. It is able to bind bivalent zinc and in effect interacts with our target molecule.

9 concept of how the hole Bacitracin-A molecule is out of all twelve amino 6 acids, the Cy-domain in the beginning and the folding after it is separated from the enzyme (Wagner, B. (2006))

What Bacitracin it used for and how does it work?

The mixture of cyclic polypeptide, which are harvested for Bacillus subtilis are used as a potent antibiotic against gram-positive bacteria. General way of action

The most potent of the different Bacitracines is Bacitracine A2. The molecule wraps around the target (C55-Isoprenylpyrophosphate) and “[…] interrupts the flow of peptidoglycan [=murein] precursors to the site of cell-wall synthesis, weakening the cell wall and ultimately leading to bacterial death[…]” (Economou, N. J., Cocklin, S., & Loll, P. J. (2013), PNAS | August 27, 2013 | vol. 110 | no. 35 | 14207). . This specialisation on a target exclusively needed for the murein-cell wall explains why Bacitracin is only effective against gram-positive bacteria, since the not existence of a cell wall is the main difference between the two kinds of bacteria (gram-positive and gram-negative)

Bacitracin is believed to have some (smaller) secondary effects on bacteria3 which may explain why it is effective against some gram –negative germs to: - It is able to work as a redox agent (because it binds divalent metal ions) and can produce DNA cleaving reagents - Without the metal it can inhibit some bacterial proteases - It can inhibit protein disulphide isomerases

Target: C55- Isoprenyl pyrophosphate and place of action For a better understanding of the effect of Bacitracin we are going to have a closer look on the molecular target of Bacitracin and the place of action in the cell.

Bacitracin enters the cell and affects the cell form the inside. It binds to the so called C55- Isoprenylpyrophosphate (IPP) molecule located in the membrane of the bacteria.

Place of action (membrane)

10 Comparison Gram-positive & Gram-negative (changed, http://micro.digitalproteus.com/pics/grambacterium.jpg)

2 Stone, K. J., & Strominger, J. L. (1971). Mechanism of Action of Bacitracin: Complexation with Metal Ion and C55-Isoprenyl Pyrophosphate. Proceedings of the National Academy of Sciences of the United States of America, 68(12), 3223–3227. 3 Economou, N. J., Cocklin, S., & Loll, P. J. (2013). High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14207–14212. http://doi.org/10.1073/pnas.1308268110 7

11 C55 Isoprenylpyrophosphate

IPP is a lipid transporter which is synthesized in the cytoplasm then is the connected to the bacteria cell membrane, where it, via phosphorylation, bind to peptidoglycan precursors. It does a flip and takes its load to the other side, here the phosphor bond is broken and the peptidoglycan is used to create the murein cell envelope. If there is no interfering substance present the IPP is again dephosphorylated by an enzyme and the cycle begins again. In case of bacitracin being near, this molecule “wraps” around the pyro-group of the IPP, prevents the enzymatic dephosphorisation, thus inactivating it an removes it from the transport cycle. One after another the transporters are taken out and the cell wall synthesis can no longer be maintained. The peptidoglycan layer gets instable and the bacterium dies.

12 Reaction cycle of peptidoglycan snynthesis (Gerhard Siewert and Jack L. Strominger)

Details of Binding

While the target of bacitracin was identified soon after its discovery, the identification of the mechanism took until 1971, when K. John Stone and Jack L. Strominger published a paper, which suggested that the essential part of IPP binding was the complexion with a metal ion4. In 2013 a group of researchers (Nicoleta J. Economou, Simon Cocklin, and Patrick J. Loll) in Philadelphia were able to crystalize the complex formed by Bacitracin and geranyl (diprenyl) pyrophosphate (very similar to IPP, just a bit shorter and easier to work with)5 . The resulting high- resolution structures give answers on how the antibiotic recognizes the Pyrophosphate target and what bacitracin actually does to inactivate it.

Stone and Strominger already proposed the importance of the divalent metal ions by observing that the antibiotic abilities of Bacitracin (i.e. the prevention of dephosphorylation of the C55-IPP lipid carrier) where completely deactivated if metal chelating agents were added before adding ether Bacitracin or IPP. But on the other hand if Bacitracin and IPP were added first, the addition of metal chelators did affect the activity of bacitracin much lesser. They did also notice the effect only occurs if the divalent metals where chelated. They conclude that divalent ions are essential in the formation of the complex between Antibotic and target.6 While many divalent metal ions are able to support the complex, the most potent one is zinc, which is also used in most of the pharmaceutic formulations.

The crystal structure X-Ray analysis in 2013 could finally explain the interaction of the three parts of the complex. In the complex bacitracin folds snake like around the phosphate group and the zinc ion: Economou et al. where abele to notice the following:

“In our crystal structure, bacitracin A adopts a compact configuration, with the antibiotic wrapping tightly around the lipid pyrophosphate and zinc ion [...]. For the first eight residues of the antibiotic, the backbone curves around the pyrophosphate group; at residue 9, the backbone forms a reverse turn, so that residues 10 to 12 lie above residues 6 to 8, in an antiparallel orientation. The antibiotic forms a highly curved C-shaped “wall” that encloses the ligand. The two ends of this curved structure— the antibiotic’s N terminus on one side, and the reverse turn containing residues 8 to 10 on the other—do not interact directly with each other, but rather are bridged by the lipid pyrophosphate ligand, which acts as a clasp to maintain the antibiotic in a closed form. Side chains from either end of the antibiotic (Ile-1, Phe-9, and His-10) wrap around the opening, completing the sequestration of the target from its environment. This results in the almost complete burial of the target’s pyrophosphate group—less than 2% of the pyrophosphate’s surface area remains solvent- accessible in the complex [..].” (Economou, N. J., Cocklin, S., & Loll, P. J. (2013), PNAS | August 27, 2013 | vol. 110 | no. 35 | 14208)

4 See 1 5 See 2 6 See 1 9

The also suggest that the lipid tail of IPP in vivo says in the bacterial membrane, while the hydrophobic part of the complex (the hole where the lipid tail can exit) associates with the membrane.

13 Stereo view of bacitracin complexation (Economou et al.)

14 Hydrophobic (red) interactions (The Protein Data Bank)

The binding is achieved by five hydrogen bonds between the amides of the bacitracin amino acid backbone, and a sixth by the side chain amine, and the oxygen atoms of the phosphate groups of IPP. The Zinc and another ion (e.g. Sodium) bind to two oxygens, one from each phosphate group. 7

15 Binding and recognition of the pyrophosphate group (Econumou et al.)

After all this bacitracin sits like a compact hat on the phosphate groups and the target is completely useless for transporting peptidoglycans cell wall synthesis, because no enzyme can reach the phosphate groups to dephosphorylate it.

Medical use

The main use for bacitracin is the use as an antibiotic. It is used in human medicine as well as for veterinary purposes. Shortly after its discovery(1945) , patients were treated externally as well as internally, to fight a various number of infections, for example against gonorrhoea. But 1949 it was discovered that the substance as a toxic effect on the kidneys. This happens because the degradation products of bacitracin (which are identical to bacitracin F) are able to inactivate catabolic enzymes in the kidneys which leads to proteinuria. 89 Despite the toxic effects it is still possible to apply bacitracin (-zinc salt) topically as ointment, typically in combination with a second antibiotic like neomycin. It is often used to tread burns, wounds or infections of the middle ear.

7 See 2 8 Miller, J. H., McDonald, R. K., & Shock, N. W. (1950). THE EFFECT OF BACITRACIN ON RENAL FUNCTION. Journal of Clinical Investigation, 29(4), 389–395. 9 Mutschler, E. (2008). Mutschler Arzneimittelwirkungen : Lehrbuch der Pharmakologie und Toxikologie. Stuttgart: Wiss. Verl.-Ges.. ISBN: 9783804719521 11

Bacitracin is used also to treat livestock, as antibiotic as addition to the animal food to prevent infection with potentially pathogenic bacteria right away. Since there is very little absorption by the intestines there is no remains found in the meat after processing the animal to food10

10 Butaye, P., Devriese, L. A., & Haesebrouck, F. (2003). Antimicrobial Growth Promoters Used in Animal Feed: Effects of Less Well Known Antibiotics on Gram-Positive Bacteria. Clinical Microbiology Reviews, 16(2), 175–188. http://doi.org/10.1128/CMR.16.2.175- 188.2003 12

Pictures

1. structure of Bacitracin-A with its specific thiazolin-ring Made with chemsketch 2. Bacitracin-F which is the reason for Bacitracin’s toxic effect http://www.trc-canada.com/prod-img/B106530.png 3. 3D-structure of Bacitracin-A thioazolin-ring Made with chemsketch 4. activation of the substrate via A-domain Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 6 5. loading of the PCP-domain through thiolation Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 96) 6. chain elongation through the catalysed amide bond Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 7 7. termination domain (TE-domain) and the add-on folding to the macro cycle Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 10 8. isoleucine and cysteine are coupled and form the hetero-cycle which later will be important for the antibiotic effect Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 16 9. concept of how the hole Bacitracin-A molecule is out of all twelve amino acids, the Cy-domain in the beginning and the folding after it is separated from the enzyme Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation . | page 5 10. Comparison Gram-positive & Gram-negative (changed, http://micro.digitalproteus.com/pics/grambacterium.jpg) 11. C55 Isoprenylpyrophosphate: Made with Avogadro (http://avogadro.cc/) 12. Reaction cycle of peptidoglycan snynthesis (Gerhard Siewert and Jack L. Strominger): changed, Siewert, G., & Strominger, J. L. (1967). BACITRACIN: AN INHIBITOR OF THE DEPHOSPHORYLATION OF LIPID PYROPHOSPHATE, AN INTERMEDIATE IN THE BIOSYNTHESIS OF THE PEPTIDOGLYCAN OF BACTERIAL CELL WALLS. Proceedings of the National Academy of Sciences of the United States of America, 57(3), 767–773.

13. Stereo view of bacitracin complexation (Economou et al.): Economou, N. J., Cocklin, S., & Loll, P. J. (2013). High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14207–14212. http://doi.org/10.1073/pnas.1308268110 14. . Hydrophobic (red) interactions (The Protein Data Bank): PDB ID: 4K7T; Economou, N. J., Cocklin, S., & Loll, P. J. (2013). High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14207–14212. http://www.rcsb.org/pdb/results/results.do?qrid=FC973723&tabtoshow=Current 15. Binding and recognition of the pyrophosphate group (Econumou et al.): Economou, N. J., Cocklin, S., & Loll, P. J. (2013). High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14207–14212. http://doi.org/10.1073/pnas.1308268110

Sources and further information:

How bacitracin is synthesised: Wagner, B. (2006). Chemoenzymatische Synthese von Bacitracin-Derivativesn und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins. Unpublished doctoral dissertation .

Well done summary on bacitracin (and other antibiotics) Butaye, P., Devriese, L. A., & Haesebrouck, F. (2003). Antimicrobial Growth Promoters Used in Animal Feed: Effects of Less Well Known Antibiotics on Gram-Positive Bacteria. Clinical Microbiology Reviews, 16(2), 175–188. http://doi.org/10.1128/CMR.16.2.175-188.2003

Mechanism of Action of Antibacterial Agents published by Fred E. Hahn; Springer Science & Business Media (old but still informative)

Mechanism of bacitracin:

Stone, K. J., & Strominger, J. L. (1971). Mechanism of Action of Bacitracin: Complexation with Metal

Ion and C55-Isoprenyl Pyrophosphate. Proceedings of the National Academy of Sciences of the United States of America, 68(12), 3223–3227.

Economou, N. J., Cocklin, S., & Loll, P. J. (2013). High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14207–14212. http://doi.org/10.1073/pnas.1308268110

Models of bacitracin compex (and other peptides and proteins) RCSB PDB: The Protein Databank http://www.rcsb.org 14

Medical use

Mutschler, E. (2008). Mutschler Arzneimittelwirkungen : Lehrbuch der Pharmakologie und Toxikologie. Stuttgart: Wiss. Verl.-Ges.. ISBN: 9783804719521