Exotoxin A Gene Fragments

Exotoxin A (ExoA) is the most toxic of the numerous extracellular proteins (LasA and LasB elastases, alkaline protease, protease IV, hemolytic and nonhemolytic phospholipase C exoenzyme S, and cytotoxin) produced by the opportunistic pathogen Pseudomonas aeruginosa [1]. The molecule consists of three distinct structural domains: Domain I, II, and III.

Deletion and mutation analysis of the ExoA molecule has revealed that Domain l contains signals that direct the secretion of ExoA from the bacterial cell; it is also involved in the binding of ExoA to a specific eukaryotic receptor. Domain II is involved in the internalization and translocation of ExoA to the cytosol of the eukaryotic cell, and Domain III possesses the ADP-ribosyltransferase and NAD-glycohydrolase activities [1].

NAD glycohydrolase activity refers to the cleavage of NAD+. ADP ribosylation refers to the transfer of an ADP molecule from NAD+ to EF-2. ExoA’s catalytic domain uses NAD⁺ as a donor and transfers an ADP-ribose group onto a special histidine derivative on eukaryotic elongation factor 2 (EF-2) called diphthamide. Once EF-2 is ADP-ribosylated, it can no longer drive the translocation step of translation, so protein synthesis stalls and the cell undergoes stress responses that can lead to death [2].

We want to express a non-toxic mutated ExoA, because we want to test if the minibinders we designed can bind to Domain 1a of ExoA using SPR. We also want to see if we can design minibinders for other regions of ExoA that might be easier. As a result, we also made nontoxic gene fragments with Domains 2 and 3. In addition, we designed ExoA constructs fused to mCherry and sfGFP to visualize Exotoxin A uptake in cells. The mCherry and sfGFP combination allows us to see the lysosomal degradation of Exotoxin A as it is being uptaken.

Information about GenScript Gene Fragments

We now have six gene fragments: three variants of ExoA and two recombinant ExoA fused with mCherry and sfGFP. This work has been guided by our mentors, Adam Chazin-Gray from the IPD, and Dr. David Fitzgerald, a leading Exotoxin A scientist in the NIH.

1. Domain Ia construct (residues ~1–252)

   This fragment contains only the receptor-binding region of ExoA (Domain I). It is fully outside the catalytic region (Domain III) and has no ADP-ribosyltransferase activity.

2. Truncated Domain 1a (residues ~1–232)

   This fragment is slightly smaller than the entire Domain 1a construct and is fully outside the catalytic region. As explained above, it is merely a receptor-binding protein and lacks any catalytic activity that makes it a toxic enzyme. We wanted to test if it is still able to bind LRP1 with just these residues. If it is not able to, then it indicates that the region with amino acids 232-252 is important in LRP1 binding.

3. Catalytic domain mutant: H440A and E553Δ

   Mutation of His-440 reduces ADP-ribosyltransferase activity by ~1000-fold without affecting NAD binding [3]. His-440 is essential for transferring ADP-ribose to elongation factor-2, the key step that causes translation arrest and cell death. With this residue mutated, the toxin cannot carry out its cytotoxic function.

   Glu-553 is an active-site residue identified by photoaffinity labeling [4]. Deletion at this position abolishes ADP-ribosyltransferase activity by preventing catalytic transfer to EF-2. Without this residue, the protein is catalytically inactive and unable to kill mammalian cells.

   

4. Domain 1a fused to mCherry and sfGFP at the C terminus

   This construct consists of Domain 1a of Exotoxin A fused at its C-terminus to the fluorescent proteins mCherry and sfGFP. The fluorescent tags allow visualization of ExoA uptake into eukaryotic cells, with sfGFP providing pH-sensitive fluorescence (it loses its signal in acidic compartments) and mCherry providing a stable fluorophore. This design allows monitoring of internalization, trafficking, and lysosomal degradation of ExoA in flow cytometry and live-cell imaging experiments.

5. Domain 1 and 2 fused to mCherry and sfGFP at the C terminus

   This construct includes Domains 1 and 2 of Exotoxin A fused to mCherry and sfGFP at the C-terminus. The fluorescent tags allow visualization of protein uptake and trafficking using flow cytometry or live-cell imaging. This construct allows comparison of the internalization of ExoA Domain I and Domain II. The fluorescent proteins provide two-color readouts for cellular localization and lysosomal processing.

Together, these five gene fragments represent completely non-toxic derivatives of ExoA: 4 that lack the catalytic domain entirely and 1 with well-characterized point mutations that eliminate toxic activity, as shown in the referenced literature below.

All of these parts are non-toxic and can be assembled into plasmids using BsaI-mediated Golden Gate Assembly. They were made to be compatible with the plasmid LM1425.

References

• [1] S. E. H. West, “Pseudomonas aeruginosa Exotoxin A: Structure/Function, Production, and Intoxication of Eukaryotic Cells,” in Bacterial Protein Toxins, K. Aktories and I. Just, Eds., Handbook of Experimental Pharmacology, vol. 145. Berlin, Heidelberg: Springer, 2000. doi: https://doi.org/10.1007/978-3-662-05971-5_4.
• [2] K. Rutault, M. J. Vacheron, M. Guinand, and G. Michel, “Comparative immunochemistry of two fragments from domains Ib and III of Pseudomonas aeruginosa Exotoxin A,” Infection and Immunity, vol. 61, no. 12, pp. 5417–5420, 1993. doi: https://doi.org/10.1128/iai.61.12.5417-5420.1993.
• [3] X. Y. Han and D. R. Galloway, “Active site mutations of Pseudomonas aeruginosa Exotoxin A: Analysis of the His440 residue,” Journal of Biological Chemistry, vol. 270, no. 2, pp. 679–684, 1995. doi: https://doi.org/10.1074/jbc.270.2.679.
• [4] S. F. Carroll and R. J. Collier, “Active site of Pseudomonas aeruginosa Exotoxin A: Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin,” Journal of Biological Chemistry, vol. 262, no. 18, pp. 8707–8711, 1987. doi: https://doi.org/10.1016/S0021-9258(18)47472-8.