Structural Biochemistry/Bacterial Proteins
Bacterial proteins are the most powerful human poisons known and belong to two broad categories: lipopolysaccharides (Gram-negative bacteria) and proteins, which are released from bacterial cells. Endotoxins, which are structural components of bacteria, are cell-associated substances that a located in the cell envelope and can be released from growing bacteria or lysed cells as a consequence of effective host defense mechanisms or antibiotics. The extracellular diffusible toxins are referred to as exotoxins and are usually secreted by bacteria during exponential growth. Exotoxins are usually polypeptides that act at tissue sites remote from the original point of bacterial invasion or growth. The location for activity of a particular toxin, like Botulinium, is determined by the site of damage. Enterotoxin, neurotoxin, leukocidin and hemolsyin are terms that describe the target site of well-defined protein toxins. Although the tissues affected and the target site may be known, the exact mechanism by which toxins cause death is not clear and is subject to debate.
Botulinum neurotoxins (BoNTs), a family of bacterial proteins produced by the anaerobic bacteria Clotridium botulinu, and the causative agent of botulism, is acknowledged to be the most poisonous protein known. Botulism poisoning is a serious and life-threatening illness in humans and animals. BoNT proteases disable synpathic vesicle exocytosis by cleaving their cytosolic SNARE substrates. There are seven distinct BoNT isoforms (A-G), which show strong amino acid sequence similarity. Human botulism is caused by the BoNT serotypes A, B, E and F. Interestingly, type A is used for various cosmetic and medical procedures, more commonly known as Botox.
BoNTs exert their neurotoxic effect by a multistep mechanism: binding, internalization, membrane translocation, intracellular traffic and proteolytic degradation. The activated mature toxin consists of 3 parts: the N-terminal light chain (~50 kDa), the heavy chain (100 kDa) that encompases the light chain (HN) and the receptor-binding doman (HC). HC determines the cellular specificity with a protein receptor (SV2 or Syt depending on the isoform) and a ganglioside. HN is a helical bundle that chaperones the light chains across endosomes where it is driven by a transmembrane proton gradient. Then, BoNTs enter the cells via receptor-mediated endocytosis, induces a conformational change and the light chains (LCs) cleave the unique components of the synaptic vesicle docking-fusion complex known as SNARE. As a result, cleavage of SNARE nullifies vesicle fusion and synaptic transmission, which causes the severe paralysis characteristic of botulism.
Tetanus toxin is a very powerful neurotoxin produced by the vegetative cell of Clostridium tetani in conditions that lack oxygen (anaerobic). As the bacterium matures, it developed its characteristic terminal spores which also give them advantage by increasing the bacteria's resistance to heat and most antiseptics. The toxin cause tetanus, a fatal disease that involves unfavorable muscle spasms that can cause respiratory failure and even death. The LD50 of this toxin has been measured to be approximately 1 ng/kg, making it the second most deadliest toxin in the world after the Botulinium neurotoxins.
The mechanism of the toxin is it first travels through the vascular and lymphatic systems of the body, disrupting the neuromuscular junctions and the central nervous system. Tetanus toxin blocks the release of inhibitory gamma-aminobutyric acid (GABA) and glycine by degrading the protein synaptobrevin. This causes the failure of regulating motor reflexes by sensory stimulation, which leads to the muscle depolarizing even with the smallest of action potentials. This continued depolarization causes the antagonist and agonist muscles to contract simultaneously and this generalized contraction causes the symptom known as tetanic spasm.
Diphtheria Toxin is a bacterial exotoxin caused by Corynebacterium Diphtheriae. This toxin exists as a single polypeptide chain, about 60,000 daltons in molecular weight. Outside of the cell, the toxin is produced in its inactive form, later to be activated by trypsin, a proteolytic enzyme, in the presence of thiol. Thiol acts as a reducing agent during this activation process. The toxin consists of two parts: Fragment A and Fragment B. Fragment A, which is responsible for the catalytic activity of the toxin, is masked until it reaches the target cell. The hydrophobic portion of the toxin, named Fragment B, is responsible for interacting with cell membrane receptors on the target cell surface.
Diphtheria toxin may enter a target cell via direct entry or receptor mediated endocytosis. In direct entry, the toxin binds to a target cell surface receptor. This binding induces the formation of a pore on the cell membrane, allowing the catalytic chain of the toxin to enter the target cell’s cytoplasm. During receptor-mediated endocytosis, the toxin is placed in a vesicle, where the pH drops, allowing both Fragment A and Fragment B to unfold. The hydrophobic regions of both chains then enter the vesicle membrane. Next, reduction and proteolytic cleavage of the A chain is released into the cytoplasm, where it regains enzymatic conformation.
Diphtheria toxin utilizes NAD as a substrate, and catalyzes ADP ribosylation, where the ADP-ribose portion of NAD combines with elongation factor-2 (EF-2). This process inactivates protein synthesis (in animal cells), resulting in cell death.
Corynebacerium Diphtheriae consists of two subunits. Subunit A contains an NH3+ group and is responsible for the enzymatic activity during the inhibition of EF-2. This inhibition interferes with the protein synthesis, resulting in cell death. Subunit B contains a carboxylic acid and a Hinge loop, which permits movement of the regulatory domain. Subunit B allows the toxin to bind to the membrane of a target host cell. This subunit possesses a region, known as T (translocation) domain, which is inserted into the target cell’s membrane, thus ensuring the release of Fragment A (catalytic component of the toxin) into the cytoplasm of the host cell.
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2. Montal, Mauricio. “Botulinium Neurotoxin: A Marvel of Protein Design.” Annual Review of Biochemistry, Vol. 79: 591-617, 2010
4. M.J. Bennett and David Eisenberg, “Refined structure of monomeric diphtheria toxin at 2.3 Å resolution": www.doe-mbi.ucla.edu