, the native toxinis produced in an inactive form which canbe activated by the proteolytic enzyme trypsin in the presence of thiol(reducing agent). The enzymatic activity of Fragment A is masked in theintact toxin. Fragment B is required to enable Fragment A toreach the cytoplasm of susceptible cells. The C terminal end ofFragmentB is hydrophilic and contains determinants that interact with specificmembrane receptors on sensitive cell membranes and the N-terminal endofFragment B (called the T domain) is strongly hydrophobic. The specificmembrane receptor forthe B fragment has been shown to be a transmembranousheparin-bindingprotein on the susceptible cell's surface.
The best known and studied bacterial toxin is the diphtheria toxin,produced by . Diphtheria toxin is abacterialexotoxin of the A/B prototype. It is produced as single polypeptidechainwith a molecular weight of 60,000 daltons. The function of the proteinis distinguishable into two parts: subunit A, with a m.w. of 21,000daltons,contains the enzymatic activity for inhibition of elongation factor-2involvedin host protein synthesis; subunit B, with a m.w. of 39,000 daltons, isresponsible for binding to the membrane of a susceptible host cell. TheB subunit possesses a region T (translocation) domain which insertsinto the endosome membrane thus securing the release of the enzymaticcomponent into the cytoplasm.
The effect of streptothricin F on macromolecular syntheses in intact cells and cell-free protein synthesis of was studied. The results indicate that protein synthesis is the primary site of inhibition by streptothricin F in growing cells. Cell-free polypeptide synthesis from directed by poly (U) was inhibited, while poly (A) and poly (C) directed polypeptide syntheses were both stimulated by the drug. Furthermore, streptothricin F caused misreading of translation of poly (U), poly (A) and poly (C) directed protein syntheses in systems. The extent of misreading by streptothricin F increases with increasing drug concentrations. The results are compared with those of other miscoding antibiotics. In rat liver extracts protein synthesis directed by poly (U) or endogenous mRNA was not inhibited.
A summary of bacterial protein toxins and their activities is givenin Tables 4. Details of the mechanisms of action of these toxins andtheirinvolvementin the pathogenesis of disease is discussed in chapters with thespecificbacterial pathogens.
Many protein toxins, notably those that act intracellularly (withregardto host cells), consist of two components: one component (subunit A)is responsible for the enzymatic activity of the toxin; theothercomponent (subunit B) is concerned with binding to aspecificreceptor on the host cell membrane and transferring the enzyme acrossthemembrane. The enzymatic component is not active until it is releasedfromthe native (A+B) toxin. Isolated A subunits are enzymaticallyactivebut lack binding and cell entry capability. Isolated B subunits maybindto target cells (and even block the binding of the native toxin), buttheyare nontoxic.
This activity will help students understand the role of DNA, mRNA, tRNA, and amino acids in protein synthesis. This activity will also introduce the concept of mutations.
Bacterial protein toxins are strongly antigenic., specificantibody neutralizes the toxicity of these bacterialexotoxins (antitoxin). However, specific antitoxin may not fully inhibit theiractivity. This suggests that the antigenic determinant of thetoxin may be distinct from the active portion of theproteinmolecule. The degree of neutralization of the active site may dependon the distance from the antigenic site on the molecule. However, sincethe toxin is fully neutralized , this suggests that other hostfactorsmust play a role in toxin neutralization in nature.