Structure-function analysis of the photorhabdus Tc toxin complex

Abstract: Photorhabdus are gram negative, bioluminescent, entomopathogenic bacteria. They are mostly found in symbiosis with entomopathogenic nematodes (EPNs). As pathogens, they produce a variety of toxins, among which are the large tripartite ABC-type Photorhabdus toxin complexes (PTCs), also known as Tc toxins. PTCs show efficacy against insect, mammalian and human cells. The complexes consist of three proteins, TcA, TcB and TcC, which combine for biological activity. TcA is the binding and translocation component. TcB links TcA to TcC, which contains the enzymatic component responsible for cytopathic and cytotoxic effects (TcChvr).

This study aimed to elucidate the structure-functional characteristics of the Photorhabdus Tc toxin. Specifically, we aimed to (1) elucidate the binding and activation characteristics of TcdA1, (2) to characterize the translocation mechanism of protein cargo through the complex, and (3) to determine and analyse putative receptors of the complex. In this regard, we show that the metalloproteases PrtA1 and collagenase can activate TcdA1. This activation leads to increased binding on both human and insect cells and increases intoxication kinetics 8 – 10-fold. Unlike uncleaved TcdA1, processed and thereby activated TcdA1 is not inhibited by the protease inhibitor MG132. Moreover, bafilomycin A1 and dynasore, which inhibit the action of unprocessed toxin, most likely by impairing uptake into cells, have no effects on the processed toxin.

We also show that the PTC-injection machinery can be used to deliver foreign peptides into target cells. Here, the native TcChvr was replaced with other toxic and non-toxic enzymes, or enzyme fragments, including the Clostridium botulinum C3 toxin, Yersinia enterocolitica effector, YopT and Metridia longa luciferase, Mluc7. The tripartite complexes of the toxin chimeras preserved enzyme activity and delivered the cargoes into target cells. Furthermore, Mluc7 enabled quantification of the efficiency of PTC. Overall, using these toxin chimeras, we determine the size and charge of the cargo optimal for efficient delivery through the injection machinery.

Finally, in the search for cell-surface receptors of PTC, a glycan microarray shows that the TcA protein (TcdA1) interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-glycan deficient CHO-Lec1 cells, which are resistant to intoxication by PTC3. Additionally, exogenous addition of a Lewis X trisaccharide inhibits intoxication. Furthermore, the glycosaminoglycans, heparin and heparan sulfate, also interact with TcdA1. They too inhibit intoxication. This data suggests that TcdA1-binding is multivalent and these sugars probably work cooperatively to facilitate binding and intoxication

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2020

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2020
• Creator: Ng'ang'a, Peter Njenga
• Contributor: Aktories, Klaus ; Schmidt, Gudula ; Deutscher Akademischer Austauschdienst ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie

Further information

• DOI: 10.6094/UNIFR/166648
• URN: urn:nbn:de:bsz:25-freidok-1666486
• Rights: Kein Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:30 AM CEST