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.

Anti-Virulence Strategy against the Honey Bee Pathogenic Bacterium Paenibacillus larvae via Small Molecule Inhibitors of the Bacterial Toxin Plx2A.

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

Swarming motility and biofilm formation of Paenibacillus larvae, the etiological agent of American Foulbrood of honey bees (Apis mellifera).

American Foulbrood is a worldwide distributed, fatal disease of the brood of the Western honey bee (Apis mellifera). The causative agent of this fatal brood disease is the Gram-positive, spore-forming bacterium Paenibacillus larvae, which can be classified into four different genotypes (ERIC I-IV), with ERIC I and II being the ones isolated from contemporary AFB outbreaks. P. larvae is a peritrichously flagellated bacterium and, hence, we hypothesized that P. larvae is capable of coordinated and cooperative multicellular behaviors like swarming motility and biofilm formation. In order to analyze these behaviors of P. larvae, we firstly established appropriate functional assays. Using these assays we demonstrated that P. larvae ERIC II, but not P. larvae ERIC I, was capable of swarming. Swarming motility was hampered in a P. larvae ERIC II-mutant lacking production of paenilarvin, an iturin-like lipopeptide exclusively expressed by this genotype. Both genotypes were able to form free floating biofilm aggregates loosely attached to the walls of the culture wells. Visualizing the biofilms by Congo red and thioflavin S staining suggested structural differences between the biofilms formed. Biofilm formation was shown to be independent from paenilarvin production because the paenilarvin deficient mutant was comparably able to form a biofilm.

Octopamine Underlies the Counter-Regulatory Response to a Glucose Deficit in Honeybees (Apis mellifera).

An animal's internal state is a critical parameter required for adaptation to a given environment. An important aspect of an animal's internal state is the energy state that is adjusted to the needs of an animal by energy homeostasis. Glucose is one essential source of energy, especially for the brain. A shortage of glucose therefore triggers a complex response to restore the animal's glucose supply. This counter-regulatory response to a glucose deficit includes metabolic responses like the mobilization of glucose from internal glucose stores and behavioral responses like increased foraging and a rapid intake of food. In mammals, the catecholamines adrenalin and noradrenalin take part in mediating these counter-regulatory responses to a glucose deficit. One candidate molecule that might play a role in these processes in insects is octopamine (OA). It is an invertebrate biogenic amine and has been suggested to derive from an ancestral pathway shared with adrenalin and noradrenalin. Thus, it could be hypothesized that OA plays a role in the insect's counter-regulatory response to a glucose deficit. Here we tested this hypothesis in the honeybee (Apis mellifera), an insect that, as an adult, mainly feeds on carbohydrates and uses these as its main source of energy. We investigated alterations of the hemolymph glucose concentration, survival, and feeding behavior after starvation and examined the impact of OA on these processes in pharmacological experiments. We demonstrate an involvement of OA in these three processes in honeybees and conclude there is an involvement of OA in regulating a bee's metabolic, physiological, and behavioral response following a phase of prolonged glucose deficit. Thus, OA in honeybees acts similarly to adrenalin and noradrenalin in mammals in regulating an animal's counter-regulatory response.

Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus.

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees(Apis mellifera)we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.