Characterization of the toxin Plx2A, a RhoA‐targeting ADP‐ribosyltransferase produced by the honey bee pathogen Paenibacillus larvae

Abstract

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 biological role of the enigmatic C3larvinAB toxin of the honey bee pathogenic bacterium Paenibacillus larvae

“…B) with similarity to the one found in Plx2A (Fig. B), another C3‐like toxin of P. larvae , which was shown to be able to enter insect cells and for which a cell entry mechanism differing from other described C3‐like toxins was suggested (Ebeling et al ., ). Whether the extended α‐helix‐1 confers cell entry activity to C3larvinA needs to be verified by further analyses.…”

“…Hence, these toxins consist of the catalytic domain of AB toxins only and their cell entry mechanism is largely unknown. In contrast to this generally accepted view of C3‐like toxins, P. larvae has recently been shown to express the C3‐like toxin Plx2A, which seems to have an associated B subunit, Plx2B, together forming the enigmatic AB toxin Plx2 (Fünfhaus et al ., ; Ebeling et al ., ). Both Plx2A and Plx2B have experimentally been demonstrated to be relevant virulence factors of P. larvae ERIC I (Fünfhaus et al ., ).…”

“…A and B subunits can either be encoded by a single open reading frame (ORF) and translated into one protein as it is the case for P. larvae Plx1; or they can be encoded by two separate ORFs and expressed as two proteins, which form a binary AB toxinlike Plx2 of P. larvae (Fünfhaus et al, 2013). The enzymatically active A subunit of Plx2, Plx2A, has recently been characterized in detail and was shown to be a C3-like toxin with ADP-ribosyltransferase activity targeting the small GTPase RhoA thereby causing vacuolization and inhibition of cytokinesis in insect cells (Ebeling et al, 2017).…”

Targeting ADP-ribosylation as an antimicrobial strategy

“…Bacterial as well as eukaryotic ARTCs are well known to MARylate protein substrates onto basic amino acid residue arginine through Nglycosidic bonds, however other residues have been identified as targets of bARTC, such as cysteine, threonine, asparagine and glutamate [136][137][138][139][140][141][142]. Instead, bARTDs selectively MARylate post-translationally modified histidine residues, termed diphtamide, in protein substrates (please refer to 4.1.2 for further details).…”

“…Members of the C3-like group encompass exoenzymes from C. botulinum, Staphylococcus aureus, B. cereus, C. limosum and Paenibacillus larvae. C3-like exoenzymes selectively modify the small GTP-binding proteins RhoA/B/C at a conserved Asn residue 41 [140,[219][220][221]. RhoA/B/C GTPases are small-molecular weight G-proteins, which modulate the state of actin cytoskeleton polymerisation.…”