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.

C3larvinA is a putative virulence factor produced by Paenibacillus larvae enterobacterial-repetitive-intergenic-consensus (ERIC) III/IV (strain 11-8051). Biochemical, functional and structural analyses of C3larvinA revealed that it belongs to the C3-like mono-ADP-ribosylating toxin subgroup. Mammalian RhoA was the target substrate for its transferase activity suggesting that it may be the biological target of C3larvinA. The kinetic parameters of the NAD+ substrate for the transferase (KM = 75 ± 10 µM) and glycohydrolase (GH) (KM = 107 ± 20 µM) reactions were typical for a C3-like bacterial toxin, including the Plx2A virulence factor from Paenibacillus larvae ERIC I. Upon cytoplasmic expression in yeast, C3larvinA caused a growth-defective phenotype indicating that it is an active C3-like toxin and is cytotoxic to eukaryotic cells. The catalytic variant of the Q187-X-E189 motif in C3larvinA showed no cytotoxicity toward yeast confirming that the cytotoxicity of this factor depends on its enzymatic activity. A homology consensus model of C3larvinA with NAD+ substrate was built on the structure of Plx2A, provided additional confirmation that C3larvinA is a member of the C3-like mono-ADP-ribosylating toxin subgroup. A homology model of C3larvinA with NADH and RhoA was built on the structure of the C3cer-NADH-RhoA complex which provided further evidence that C3larvinA is a C3-like toxin that shares an identical catalytic mechanism with C3cer from Bacillus cereus. C3larvinA induced actin cytoskeleton reorganization in murine macrophages, whereas in insect cells, vacuolization and bi-nucleated cells were observed. These cellular effects are consistent with C3larvinA disrupting RhoA function by covalent modification that is shared among C3-like bacterial toxins.

No abstract

C3larvinA was recently described as a mono-ADP-ribosyltransferase (mART) toxin from the enterobacterial repetitive intergenic consensus (ERIC) III genotype of the agricultural pathogen, Paenibacillus larvae. It was shown to be the full-length, functional version of the previously described C3larvintrunc toxin, due to a 33-residue extension of the N-terminus of the protein. In the present study, a series of deletions and substitutions were made to the N-terminus of C3larvinA to assess the contribution of the α1-helix to toxin structure and function. Catalytic characterization of these variants identified Asp23 and Ala31 residues as supportive to enzymatic function. A third residue, Lys36, was also found to contribute to the catalytic activity of the enzyme. Analysis of the C3larvinA homology model revealed that these three residues were participating in a series of interactions to properly orient both the Q-X-E and S-T-S motifs. Ala31 and Lys36 were found to associate with a structural network of residues previously identified in silico, whereas Asp23 forms novel interactions not previously described. At last, the membrane translocation activity into host target cells of each variant was assessed, highlighting a possible relationship between protein dipole and target cell entry.

No abstract