Question

Exotoxin A from Pseudomonas aeruginosa has the exact same activity and host cell target as diphtheria toxin. Yet, Pseudomonas aeruginosa infection does not result in the same symptoms or clinical manifestation as diphtheria. Can you think of any reason(s) why this might be the case?

Rat cells are known to be resistant to diphtheria toxin. Yet, like humans, EF-2 from rats contains diphthamide. Propose a hypothesis to explain why rat cells are resistant to diphtheria. Can you think of a way to experimentally test your hypothesis?

Answer 1

1. Both diphtheria toxin and exotoxin A affect protein synthesis. Diphtheria toxin produced by Corynebacterium diphtheria is composed of two fragments-A and B. The fragment B is required for entry into host cells while enzymatic activities lie with A fragment. The toxin binds to cell membrane via R domain (receptor binding domain). R domain binds to precursor of heparin binding epidermal growth factor. Furin and furin like proteases then cleave the toxin and protein is then internalized in clathrin coated pits. It then reaches the endosome, where there is conformation change in T domain (translocation domain). Hydrophobic areas are exposed, which insert into membrane. A channel is formed through which a catalytic domain (A fragment) passes. The A fragment will enter the cytoplasm. It transfers ADP-ribose from NAD molecule to dipthamide, a histidine residue present on eEF2 (eukaryotic translation elongation factor 2). The eEF2 is inhibited As a result, protein synthesis is inhibited.

Exotoxin A is secreted by pseudomonas aeruginosa. It is also an ADP-ribosylating toxin as diphtheria toxin. A c-terminal lysine at position if 613 is first cleaved by carboxypeptidase in plasm. It then bonds to alpha2‑macroglobulin receptor/low-density lipoprotein receptor-related protein (α2MR/LRP) present on cell membrane via its cell membrane binding domain. The toxin is internalized and enters early endosome similar to diphtheria toxin via formation of clathrin coated pits. It is however, cleaved by furin or furin like protease in the endosomes. Here, a conformation change occurs prior to cleavage. The single disulphide bond is reduce and an active C’ 37 fragment is release. This active C’ 37 fragment travels to the trans-Golgi network. The C-terminal KDEL sequence is exposed which binds to KDEL sorting receptor. This leads to transport to the ER. The translocation domain II in the toxin fragment will cause translocation of the C’37 fragment into cytoplasm. This will allow it to escape ER the ER-associated degradation (ERAD) pathway. Further, the retrograde transport via the Sec61p translocon is also subversed. The toxin then acts in similar fashion to diphtheria toxin to inactivate eEF2.

The differences in symptoms shown by exotoxin A and diphtheria toxin may be linked to the different routes their catalytic domains take to enter the cytoplasm. The diphtheria toxin will directly enter the cytoplasm from endosome. The exotoxin however has to pass from endosome to trans-Golgi network and then ER to reach the cytoplasm. Thus, it will bypass degradation pathways, making them more potent.

2. Rat cells contain dipthamide. Hence, diphtheria toxin can cause ADP-ribosylation where ADP-ribose is transferred from NAD to diphthamide. Hence, technically they should be sensitive to diphtheria toxin. The resistance of rate cells toe diphtheria toxin is not due to catalytic domain. However, rat cell are not able to bind to the diphtheria toxin B fragment or does not allow the toxin to enter the cells. The rat HBEGF do not bind to diphtheria toxin A fragment, thereby not allowing the toxin inside the cytoplasm for catalytic activity. There are differences in four amino acids that are critical for binding to diphtheria toxin. These amino acids at position 133, 124, 126 and 147 are altered in rat HB-EGF, making them insensitive to the toxin.

The rat HB-EFG sequence can be transfected in human cell lines (such as B-cell hematological cell lines) that lack HB-EGF in the cell membrane. This DNA construct can be over expressed and then the cell exposed to diphtheria toxin. Binding of the B fragment can be tested on isolated plasma membrane of these cells using antibodies to B fragment. Further, apoptosis can be checked as well as inhibition of protein synthesis. Similarly, human HB-EGF can be transfected in rat cells and similar studies can be performed. If the rat sequence, prevents apoptosis in human cells, then the binding of B fragment to HB-EGF is defective in rats.