Characterization of C3larvinA, a novel RhoA-targeting ADP-ribosyltransferase toxin produced by the honey bee pathogen,Paenibacillus larvae

Abstract

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.

The N-terminus ofPaenibacillus larvaeC3larvinA modulates catalytic efficiency

“…C3larvinA homology model -The C3larvinA homology model was built as described earlier (29). Briefly, the 1.65 Å crystal structure of Plx2A (PDB:5URP; 55% sequence identity) was used as a template to model the C3larvinA structure in Phyre2 (45).…”

“…Human pathogens, including Vibrio cholera, Clostridium botulinum and Corynebacterium diphtheriae have been previously described to use mART toxins (4,(24)(25)(26)(27); however, more recently mART toxins were discovered in the agricultural pathogen, Paenibacillus larvae (28)(29)(30)(31)(32).…”

“…This also applies to the associated mART toxins expressed by each genotype. To date, three mART toxins have been characterized in P. larvae : Plx2A in ERIC I, C3larvin trunc in ERIC I and II, and C3larvinA in ERIC III [ 28 , 29 , 32 ].…”

“…It was determined that the protein had a truncated N-terminal α 1 -helix which led to an inability to gain entry to host cells and cause intracellular damage [ 32 ]. The full-length protein, C3larvinA, was later identified in the ERIC III genotype [ 29 , 30 ]. Like C3larvin trunc , C3larvinA targets RhoA for ADP-ribosylation through a catalytic Q-X-E motif, and possesses GH activity; however, the rate of reaction was 12-fold, and 200-fold higher than that of C3larvin trunc , respectively.…”

“…Like C3larvin trunc , C3larvinA targets RhoA for ADP-ribosylation through a catalytic Q-X-E motif, and possesses GH activity; however, the rate of reaction was 12-fold, and 200-fold higher than that of C3larvin trunc , respectively. Additionally, C3larvinA has the necessary N-terminal machinery (α-helix 1) to translocate the host cell membrane and initiate infection [ 29 ]. These findings further confirm the importance of the N-terminus in cell entry as well as enzymatic activity.…”