The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents

Abstract

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase-DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial-bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.

Keywords: DNA gyrase; DNA topoisomerases; TOMMs; microbial toxins.

Graphical abstract

Fig. 1

Oxazole- and thiazole-containing bioactive molecules: drugs containing oxazole and/or thiazole ring; the target and the indication are indicated in brackets. COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; CYP3A4, cytochrome P450 3A4; CDK, cyclin-dependent kinase.

Fig. 2

MccB17 and other LAPs.

Fig. 3

The MccB17 gene cluster, showing the McbA gene product and how it is processed by the microcin synthase (McbBCD) and protease (TldD/E). McbE and McbF control efflux, and McbG is responsible for immunity.

Fig. 4

DNA gyrase catalytic cycle showing points at which inhibitors interact. The GyrA dimer and the GyrB monomers bind to DNA as an A₂B₂ complex. DNA wrapped around the C-terminal domains of GyrA presents the T (transported) segment over the G (gate) segment. The T segment is captured by closure of the N-gate (N-terminal domains of GyrB). Cleavage of the G segment and passage of the T segment through the G segment leads to the introduction of two negative supercoils. Catalysis requires the binding and hydrolysis of 2 molecules of ATP. The sites of action of three antibiotics are shown: simocyclinones prevent the binding of DNA; aminocoumarins prevent the binding of ATP; quinolones interrupt the DNA breakage-reunion cycle by gyrase. Stars indicate the active-site region for DNA cleavage, and the circle indicates the ATP-binding pocket. Adapted from Costenaro and Maxwell with permission.

Fig. 5

McbA protein and the nomenclature of MccB17 heterocycles. (A) McbA, the mcbA gene product and the nomenclature of the heterocycles contained in MccB17 used in this publication. (B) By-products of MccB17 biosynthesis found in MccB17-producer strain cell extract. Only domains that are different from MccB17 are shown, “…” stand for the rest of the peptide that is identical to MccB17. Corresponding residues in MccB17 and by-products are connected by blue dashed lines. (C) Ps_MccB17, a MccB17 variant produced by P. syringae.

Fig. 6

Effect of variation in the core, N-terminal and C-terminal region of MccB17. The core region containing the heterocyclic moieties of MccB17 is represented in the middle of the figure with heterocycle variations reported in the literature. On the left of the core region, N-terminal variations are depicted, and on the right, C-terminal variations. For each variant, the percentage of MccB17 cleavage activity is shown in blue, and the percentage of MccB17 antibacterial activity is shown in green. The region in PS_MccB17 different from MccB17 is shown in magenta. The black arrow represents deletion of residues up to the dashed line. The dashed red arrows represent unprocessed serines leading to MccB17 variants lacking oxazole (OAZ) heterocycles. ND indicates not determined.

Fig. 7

Effect of point mutations on MccB17 activity. The figure shows amino acids constituting MccB17 with their numbering; post-translationally modified residues are highlighted in orange. Mutations are show underneath the corresponding residue. The effect of each mutation is shown in brackets as a percentage of MccB17 cleavage activity in blue, and a percentage of MccB17 antibacterial activity in green. Similarly, mutants of McbI, a shorter version of MccB17, are shown. Ps_MccB17 is shown to illustrate the effect of the substitution of residues S52 N53 with GGG.

Fig. 8

Evaluation of McbI mutants: McbI is a MccB17 variant lacking G28 to G37; due to its improved cleavage activity, McbI was used as the reference to evaluate the effect of mutation of amide residues Q44 and N59, and the C-terminal I69. Upper panel: E. coli DNA–gyrase cleavage assays with McbI mutants. Lower panel: activity of various MccB17 derivatives. Inhibition of E. coli growth: Q44A and N59A have a marginal negative effect on the ability to stabilize the cleavage complex or on the inhibition of bacterial growth. McbI I69A and McbI I69G retain the ability to stabilize the cleavage complex; however, they lose their ability to inhibit bacterial growth. Assays were carried out as described previously , DNA gyrase was used according to the supplier's protocol (Inspiralis Ltd).

Fig. 9

(a) Alignment of pentapeptide proteins related to McbG: the reference is E. coli McbG, and the second is McbG from P. syringae that produce a variant of MccB17. AlbG is the immunity protein to Albicidin, an NRP topoisomerase poison produced by X. albilineans, the causal agent of leaf scald disease. CysO is the immunity protein to cystobactamide, a compound related to Albicidin produced by Myxobacterium cystobacter. Qnr is produced by K. pneumoniae and confers resistance to quinolones drugs. MfpA familly proteins confer quinolone resistance to mycobacteria; M. tuberculosis and M. smegmatis are presented. Alignment generated by Clustal Omega and displayed with Mview 1.63, Copyright © 1997–2018 Nigel P. Brown. Despite their sequence differences, all these proteins have multiple repeating sequences of five amino acids in length and probably adopt a right-handed β-helix fold with a roughly square-shaped cross section as shown by AlbG. (b) the crystal structure of AlbG PDB 2XT4.

Fig. 10

Mutations conferring resistance to MccB17 and quinolones in the context of the cleavage complex: the gyrase cleavage complex with the topoisomerase poison etoposide is shown with the residues important for MccB17 action. Two GyrB C-terminal domains are shown, respectively, in smudge green and deep teal; two GyrA N-terminal domains are shown in blue and dark green. The DNA is shown as white sticks. Catalytic tyrosines (GyrA Y122) that form a covalent bond with DNA are shown as spheres in red. GyrB W751 (E. coli), the residue responsible for specific resistance to MccB17, is shown as violet spheres. GyrB K447 (E. coli) shown as orange spheres and GyrA S83 (E. coli) shown as yellow spheres are two residues involved in quinolone resistance that also influence MccB17 activity . Etoposide is shown as magenta sticks inserted in one of the cleavage site (only one of its two binding sites is shown for clarity). a and b show, respectively, the crystal structure of Staphylococcusaureus gyrase cleavage complex with Etoposide (PDB: 5CDN) seen from the side and from the top. A′ and B′ are the same views but shown as molecular surfaces. c and d are the side and top view of an hypothetical open conformation of the gyrase DNA gate constructed from S. aureus gyrase cleavage complex with Etoposide (PDB: 5CDN) and the B. subtilis GyrA-N-terminal domain dimer in an open gate conformation (PDB: 4DDQ) . C′ and D′ are the corresponding views shown as molecular surfaces. The position of different residues influencing MccB17 activity on DNA gyrase combined with the position of the topoisomerase poison Etoposide in the cleavage site, particularly its interaction with DNA, shows the potential area of MccB17 interaction with the gyrase–DNA cleavage complex.

Fig. 11

Structure of the MccB17 synthase . (a) MccB17 synthase is organized as an octamer formed through 2-fold symmetry of the tetramer composed of two McbBs in different conformations, McbB1 (in blue) and McbB2 (in purple), one McbC (in pink or violet) and one McbD (in light green). McbB1 and McbB2 form a dimer in a head to tail arrangement. McbB's structural Zn atoms are show as red spheres. MccB17 is shown in yellow, and the leader peptide (labeled LP) is shown as a yellow β-sheet connected to a α-helix bound to the McbB dimer, mainly interacting with McbB1 (blue). Four heterocycles of MccB17 are shown as yellow spheres labeled with the heterocycle name: OTZ39 TOZ54 OAZ64. MccB17 C-terminal residues G66, S67 and H68 are shown as yellow sticks. No interactions between with the cyclodehydratase McbD and MccB17 are visible, and McbD converts MccB17 Ser and Cys residues into the corresponding azoline. The active site of McbD is highlighted by P396 shown as orange spheres; P396 has been shown to have an important role in the catalysis of the heterocyclization . The dehydrogenase McbC (in pink or violet) converts azolines synthesized by McbD to azole, OAZ64 is visible bound in the active site of McbC, interacting with the FMN co-factor (shown as green spheres). McbC is arranged as a homodimer at the center of the octamer. The dimer is stabilized by one monomer embracing the other monomer with a 50-residue-long loop seen in violet pointed out by a violet arrow on one monomer, and seen in pink pointed out by a pink arrow in the other. For a better visibility MccB17 fragments, FMN, and P396 are only shown for one McbB1B2CD tetramer, but the other is similarly occupied. (b) NMR structure of the leader peptide with a C-terminal amide in solution (PDB: 2MLP) suggests a change of conformation between unbound and bound state.