The toxin Plx2A is an important virulence factor of Paenibacillus larvae, the etiological agent of American Foulbrood, the most destructive bacterial disease of honey bees. Biochemical and functional analyses as well as the crystal structure of Plx2A revealed that it belongs to the C3 mono-ADP-ribosylating toxin subgroup. RhoA was identified as the cellular target of Plx2A activity. The kinetic parameters (K , k ) were established for both the transferase and glycohydrolase reactions. When expressed in yeast, Plx2A was cytotoxic for eukaryotic cells and catalytic variants confirmed that the cytotoxicity of Plx2A depends on its enzymatic activity. The crystal structure of Plx2A was solved to 1.65 Å and confirmed that it is a C3-like toxin, although with a new molecular twist, it has a B-domain. A molecular model of the 'active' enzyme conformation in complex with NAD was produced by computational methods based on the recent structure of C3bot1 with RhoA. In murine macrophages, Plx2A induced actin cytoskeleton reorganization while in insect cells, vacuolization and the occurrence of bi-nucleated cells was observed. The latter is indicative of an inhibition of cytokinesis. All these cellular effects are consistent with Plx2A inhibiting the activity of RhoA by covalent modification.

The gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood of honey bees, a notifiable disease in many countries. Hence, P. larvae can be considered as an entomopathogen of considerable relevance in veterinary medicine. P. larvae is a highly specialized pathogen with only one established host, the honey bee larva. No other natural environment supporting germination and proliferation of P. larvae is known. Over the last decade, tremendous progress in the understanding of P. larvae and its interactions with honey bee larvae at a molecular level has been made. In this review, we will present the recent highlights and developments in P. larvae research and discuss the impact of some of the findings in a broader context to demonstrate what we can learn from studying "exotic" pathogens.

Summary Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I‐IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3‐like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full‐length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus.

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.

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.