Molecular basis of antibiotic self-resistance in a bee larvae pathogen.

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays 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 Paenibacillus larvae by deactivation of paenilamicin. Using tandem mass spectrometry, nuclear magnetic resonance 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 ribosome inhibitor. Here, we further determined 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 Paenibacillus larvae to fight American Foulbrood.

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

Paenilamicins are a group of complex polycationic peptide secondary metabolites with antibacterial and antifungal activities produced by the devastating honey bee brood pathogen Paenibacillus larvae causing the lethal brood disease American Foulbrood (AFB). Here, we report the convergent total synthesis and structural revision of paenilamicin B2. Specific stereoisomers of paenilamicin B2 were synthesized for unambiguous confirmation of the natural product structure and for evaluation of biological activities. These studies revealed the N-terminal fragment of paenilamicin as an important pharmacophore. Infection assays using bee larvae and the insect pathogen Bacillus thuringiensis demonstrated that paenilamicins outcompete bacterial competitors in the ecological niche of P. larvae. Finally, we show first data that classifies paenilamicins as potential ribosome inhibitors. Hence, our synthesis route is a further step for understanding the pathogenicity of P. larvae and for thorough structure-activity-relationship as well as mode-of-action studies in the near future.

Biological Role of Paenilarvins, Iturin-Like Lipopeptide Secondary Metabolites Produced by the Honey Bee Pathogen Paenibacillus larvae.

The Gram-positive bacterium Paenibacillus larvae (P. larvae) is the causative agent of a deadly honey bee brood disease called American Foulbrood (AFB). AFB is a notifiable epizootic in most countries and, hence, P. larvae is of considerable relevance for veterinarians and apiculturists alike. Over the last decade, much progress has been made in the understanding of the (patho)biology of P. larvae. Recently, several non-ribosomally produced peptides (NRP) and peptide/polyketide (NRP/PK) hybrids produced by P. larvae were identified. Among these NRPs were iturin-like lipopeptides, the paenilarvins A-C. Iturins are known to exhibit strong anti-fungal activity; for some iturins, cytotoxic activity towards mammalian erythrocytes and human cancer cell lines are described. We here present our results on the analysis of the natural function of the paenilarvins during pathogenesis of P. larvae infections. We demonstrated production of paenilarvins in infected larvae. However, we could neither demonstrate cytotoxicity of paenilarvins towards cultured insect cells nor towards larvae in feeding assays. Accordingly, exposure bioassays performed with larvae infected by wild-type P. larvae and a knockout mutant of P. larvae lacking production of paenilarvins did not substantiate a role for the paenilarvins as virulence factor. Further experiments are necessary to analyze the relevance of the paenilarvins' anti-fungal activity for P. larvae infections in the presence of fungal competitors in the larval midgut or cadaver.