Molecular Basis of Antibiotic Self-Resistance in a Bee Larvae Pathogen

Abstract

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays high antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in P. larvae by deactivation of paenilamicin. Using tandem MS, NMR spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a new ribosome inhibitor. Here, we further elucidated the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of P. larvae to fight American Foulbrood.

Total Synthesis and Biological Evaluation of Paenilamicins from the Honey Bee Pathogen Paenibacillus larvae

“…We further concluded that the (6 R )-configuration of the terminal amino group in Aga plays a functional role, which is more important for exerting antifungal rather than for antibacterial activity. In our parallel study, we show that the self-resistance factor PamZ acetylates the 6-amino group of Aga and thereby inactivates paenilamicins. Moreover, this study demonstrated that PamZ requires the correct (6 R )-configuration of Aga, being unable to convert the non-native diastereomers PamB2_1 and PamB2_2 .…”

“…This rather small difference in inhibitory potency toward the Gram-negative E. coli ribosome fairly reflected the reasonable conservation of anti-Gram-positive activity despite the non-native 6 S -configuration of Aga in PamB2_2 (Table ). Again, the functional importance of the N-terminal residue Aga was much more evident given the approximately 100-fold weaker ribosome inhibition, when its 6-amino group became N -acetylated ( N -acetyl- PamB2_3 , IC 50 ∼31.9 μM) by PamZ (Figure b; see the Supporting Information for results of the in vitro activation assay of PamZ).…”

“…The inset shows the translation efficiency at low substrate concentrations. b) Effects of N -acetylation of PamB2_3 on ribosome inhibition: PamB2_3 (orange) and PamB2_3 incubated with acetyl-CoA only (blue); PamB2_3 incubated with PamZ and acetyl-CoA (green). Nonlinear regression curves are depicted as continuous lines (Supporting Information).…”

“…The supply with superior amounts of synthetic PamB2 enabled us to conduct the first mode-of-action studies identifying the bacterial ribosome as one potential molecular target of paenilamicins. In our parallel study, we were able to relate this activity to the free amino group at position 6 of Aga in the R -configuration, since regio- and stereoselective N -acetylation by the self-resistance factor PamZ of P. larvae impeded the antibacterial activity, as well as the inhibition of ribosomal biosynthesis. The synthesis of diastereomers thus allowed us to establish first structure–activity relationships.…”

Paenilamicins from the honey bee pathogenPaenibacillus larvaeare context-specific translocation inhibitors of protein synthesis

“…1b), a compound originally isolated from a soil sample from New Guinea in the 1970's 6 . A recent structural revision of PamB2 revealed that the (6R)-configuration of the terminal amino group in Agm is important for maximal activity and plays a role in self-resistance, being required for acetylation, and thereby inactivation, by the self-resistance factor PamZ 4,7 . The protection of the N-terminus during biosynthesis by attachment of an Acyl-D-Asn moiety is also a prominent prodrug resistance mechanism 8 .…”