Mode of Action and Mechanisms of Resistance to the Unusual Polyglycosylated Thiopeptide Antibiotic Persiathiacin A

Abstract

Persiathiacin A is a novel thiopeptide antibiotic produced by Actinokineospora species UTMC 2448. It has potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. Thiopeptides, including persiathiacin A, exhibit antibacterial activity by inhibiting protein synthesis. In this study, we characterize the mechanism of action of persiathiacin A and investigate how resistance to this antibiotic can emerge. In vitro assays revealed that persiathiacin A inhibits translation elongation, leading to ribosome stalling. Genetic analysis of resistant Bacillus subtilis mutants identified mutations primarily in the rplK gene encoding ribosomal protein L11, which is the binding site for other 26-membered macrocycle-containing thiopeptides. The resistant mutants showed growth impairment and an increased lag time, even in the absence of persiathiacin. Comparative proteomic analysis of a resistant mutant versus the parental strain revealed multiple changes, indicative of negative effects on protein synthesis. Thus, although persiathiacin-resistant mutants can arise readily by the loss of L11 function, it is likely that such mutants would be severely compromised in pathogenesis. Furthermore, bioinformatics analysis identified differences in the key amino acids within the thiopeptide-binding region of L11 in the persiathiacin producer. These probably prevent the antibiotic from associating with its target, providing a mechanism for self-resistance.

Keywords: RiPP; Ribosomal protein L11; Self-resistance; Translation inhibition.

Figure 1

A) Structure of the 26-membered thiopeptides persiathiacin A, nosiheptide, thiocillin I, and thiostrepton. B) X-ray structure of thiostrepton (left) and nosiheptide (right) bound at the interface of 23S rRNA (gold) and L11 (cyan) of the 50S ribosomal subunit of Deinococcus radiodurans. Structures were obtained from Protein Data Bank (accession numbers: 3CF5 (thiostrepton) and 2ZJP (nosiheptide)).

Figure 2

Concentration-dependent inhibition of E. coli ribosomes by persiathiacin A.

Figure 3

In vitro translation to investigate the effect of persiathiacin A on translation initiation and elongation. A) In vitro translation mRNA schematic. The coding region on the right-hand side is annotated with single letter amino acids names. B) Denaturing PAGE loaded with radiolabeled RNA products following the persiathiacin challenge during translation initiation and elongation. RelE was added post elongation. C) Denaturing PAGE loaded with radiolabeled RNA products following persiathiacin challenge during translation elongation.

Figure 4

Effects of persiathiacin A on mutant and wild type B. subtilis strains. A) Growth of strains: B. subtilis 168CA (left), B. subtilis 168CA MutD (middle), and B. subtilis ΔrplK (BKE01020) (right), in NB medium containing the indicated concentrations of persiathiacin A. B) Cultures of the indicated strains were serially diluted, and 5 μL samples were spotted on NA plates with (left) and without (right) 15 μg/μL persiathiacin.

Figure 5

Volcano plot of the comparative proteomics analysis between B. subtilis 168CA MutD and its parental strain B. subtilis 168CA. The cutoff values, q = 0.05 and fold change = 2, are indicated by dashed lines.

Figure 6

Superimposition of an AlphaFold model of L11 from Actinokineospora sp. UTMC 2448 (gold) with the X-ray crystal structure of L11 from D. radiodurans (cyan) with nosiheptide bound (PDB accession: 2ZJP). The side chains of the Pro21, Pro25 and Gln29 residues in the N-terminal helix of L11 from D. radiodurans, which contact thiopeptide antibiotics bound to the ribosome, and are mutated to Val, Lys, and Pro, respectively in L11 from Actinokineospora sp. UTMC 2448, are highlighted in stick representation.