Cell biological implications of Vibrio cholerae MARTX autoprocessing
Final Report Abstract
Vibrio cholerae MARTX toxin is a large bacterial protein toxins autotranslocating functional effector domains to the eukaryotic cell cytosol, resulting in destruction of the actin cytoskeleton of eukaryotic cells that ultimately benefits the infecting pathogen. For posttranslocation autoprocessing that releases these functional domains from the large Holotoxin the toxin carries an embedded cysteine protease domain (CPD) that is activated for autoprocessing by binding inositol hexakisphosphate (InsP6) a molecule found exclusively in eukaryotic cells. Besides normal processing of CPD at the N-terminus, there was suggested a secondary processing event at the C-terminal side to release the CPD itself in the eukaryotic cell cytosol where it is available also to target eukaryotic cell proteins and, as previous studies showed, resulting in necrotic and apoptotic cells [13]. Different in vitro and in vivo studies were not able to confirm the hypothesis of a C-terminal cleavage event to release the CPD in the cytosol. Cleavage at the C-terminus has been seen in vitro and in vivo using a recombinant protein fragment. Although we ended up with a stable 33 kDa fragment, the cleavage event in vitro and in the cytosol of cells with an EGFP fusion protein occurred very slow and at multiple cleavage sites. Investigations on the C-terminal processing event with purified native MARTX toxin were not successful because of the low detection limit of our antibodies, but also a delivery of the CPD by the anthrax toxin delivery system didn´t show previous results of cytotoxicity in HeLa cells, supposing that the cleavage is not a natural event happening in vivo with native toxin. In the course of investigations on the C-terminal cleavage of the CPD we discovered a new phenotype of HeLa cells intoxicating cells with a V. cholerae strain just active in the αβ hydrolase and the CPD (CCO5). The cells lost the connection to surrounding cells and formed chain like formations after 48 hours postinfection which was linked to an active αβ hydrolase domain. At present nothing is known about the αβ hydrolase and its function in the toxin what makes further studies on this domain very interesting. Nor recombinant expression of the αβ hydrolase domain in eukaryotic cells neither in yeast showed cytotoxicity. Future investigations on this domain will include a 2-D DIGE to find proteins of the eukaryotic cell affected by this domain and further understand the connection of this domain in the intoxication process of V. cholerae. Also crystallization of the αβ hydrolase domain will help to get more information of a catalytic region in the domain and amino acids involved in the enzymatic activity.
Publications
- Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins. PLoS Pathog. 2010 Jul 8; 6 (7):e1000942
Egerer M., Satchell KJ.
