Clostridial Neurotoxins: Structure, Function and Implications to Other Bacterial Toxins

Abstract

Gram-positive bacteria are ancient organisms. Many bacteria, including Gram-positive bacteria, produce toxins to manipulate the host, leading to various diseases. While the targets of Gram-positive bacterial toxins are diverse, many of those toxins use a similar mechanism to invade host cells and exert their functions. Clostridial neurotoxins produced by Clostridial tetani and Clostridial botulinum provide a classical example to illustrate the structure-function relationship of bacterial toxins. Here, we critically review the recent progress of the structure-function relationship of clostridial neurotoxins, including the diversity of the clostridial neurotoxins, the mode of actions, and the flexible structures required for the activation of toxins. The mechanism clostridial neurotoxins use for triggering their activity is shared with many other Gram-positive bacterial toxins, especially molten globule-type structures. This review also summarizes the implications of the molten globule-type flexible structures to other Gram-positive bacterial toxins. Understanding these highly dynamic flexible structures in solution and their role in the function of bacterial toxins not only fills in the missing link of the high-resolution structures from X-ray crystallography but also provides vital information for better designing antidotes against those toxins.

Keywords: Gram-positive bacterial toxins; anthrax toxin; botulinum neurotoxin; clostridial neurotoxin; diphtheria toxin; molten globule; pore-forming toxins; proteolytic activation; tetanus neurotoxin.

Figure 1

Mechanisms of bacterial pathogenesis: bacteria-induced toxicity and host-mediated damage [2].

Figure 2

(A) Schematic diagram of a BoNT (BoNT/A) showing different domains. (B) Three-dimensional representation of the backbone BoNT/A [66]. The catalytic domain LC is indicated in blue, the translocation domain HN is in green, and the receptor-binding domain HCC and HCN are in yellow and red, respectively.

Figure 3

The proposed model for BoNT across the intestinal epithelial barrier (modified from Fujinaga et al., 2012 [77]). Step 1: NAPs/hemagglutinin proteins facilitate the toxin transcytosis through the epithelial cells. Step 2: HA and/or other NAPs mediate the disruption of the epithelial barrier (BoNT/A and /B disrupt the tight junction, while BoNT/C is cytotoxic to the epithelial cells. Step 3: BoNT is absorbed from the disrupted cellular barriers.

Figure 4

Schematic models of the neurotransmitter release and the actions of botulinum and tetanus toxins [108]. (A) Synaptic vesicles containing neurotransmitters dock with the plasma membrane through SNARE proteins (synaptobrevin, syntaxin, and SNAP-25). SNARE proteins remain in random coil conformations until associated with the SNARE complex at docking, where they form a helical bundle. (B) Botulinum or tetanus toxin binds to the presynaptic membrane through gangliosides and a protein receptor (step 1) internalized through endocytosis (step 2a), and its LC is translocated across the membrane (step 2b). The LC acts as a specific endopeptidase against either synaptobrevin (on synaptic vesicles), syntaxin (on the plasma membrane), or SNAP-25 (on the plasma membrane) (step 3). BoNTs (or TeNT) cleave their substrates before the SNARE complex is formed.

Figure 5

Target neurons of BoNTs and TeNT and their trafficking (modified from Connan and Popoff 2017 [99]). BoNTs target motor neuron endings at the neuromuscular junction, while TeNT enters the motoneuron and sensory neuron endings, moves retrogradely along the axon, is transcytosed, and reenters the central inhibitory neurons.

Figure 6

Positions of the SNARE motif in three substrates responsible for the target specificity of clostridial neurotoxins [200]. The motif consists of nine residues that are common to all three substrates: hydrophobic residue (H), Asp or Glu residue (-), polar residue (P), and any residue (X).

Figure 7

Schematic model of the reduction-induced molten globule state of BoNT/A facilitating the binding of SNAP-25 [212].

Figure 8

Schematic model representing the PRIME state and the molten globule state in BoNT/A LC facilitating the binding of substrate SNAP-25. The blue balls represent the nonpolar side chains, which are tightly packed in the native conformation. (a) Heating to 37 °C significantly alters the polypeptide folding, and the protein core becomes slightly loosened compared to the native state (b) and facilitates the binding of SNAP-25 to form the enzyme–substrate complex (c). BoNT/A LC exhibits the optimum activity in this PRIME state. The further heating of the BoNT/A LC leads to the formation of a molten globule intermediate, which is represented by a loose packing of the side chains in the protein core and partial unfolding of loops with a slightly more expanded structure than PRIME (d). While less optimal, this state of the BoNT/A LC also binds to SNAP-25, forming the enzyme–substrate complex, (e) and displays less enzymatic activity than the optimal PRIME state [209].