Samenvatting
Toxin-antitoxin (TA) modules are small operons that are ubiquitous in bacterial genomes and are involved in stress response and persister cell formation. In this thesis, the effects of nanobodies raised against two TA toxins are studied: MazF from S. aureus (MazFsa) and Doc from the E. coli bacteriophage P1. The MazFsa toxin of the staphylococcus aureus MazEF TA module is a specific endoribonuclease that cleaves mRNA and modifies ribosomes to translate preferentially leaderless mRNA. It is neutralized by its corresponding antitoxin MazEsa antitoxin. The Doc toxin of the phd/poc addiction module of bacteriophage P1 on the other hand inhibits translation through phosphorylation of elongation factor Tu. We have produced and characterized nanobodies against Doc and MazFsa respectively. ITC results show a high binding affinity between these toxins and the respective nanobodies.??? nanobodies against MazFsa were characterized, ??? of which at least parially inhibit the ribonuclease activity of MazFsa and none that are able to activate this activity in presence of MazEsa. A single anti-MazFsa nanobody binds per monomer of the MazFsa homodimer. The crystal structure of one such co-crystallized complex shows that anti-MazFsa [WHICH ONE?] occupies part of the region of overlap of the MazEsa and the RNA binding sites on MazFsa, partially inhibiting MazFsa endoribonuclease activity as observed in the MazFsa RNase assay. Several complexes were crystallized and the corresponding crystal structures will provide more understanding of the nanobody molecular epitopes on the Doc and MazFsa proteins in rationalizing the inhibitory properties of these antibodies.
Similarly, of 7 anti-Doc nanobodies tested, 6 are capable of complete inhibition of Doc phosphorylation activity, but again none are activating These anti-Doc nanobodies. Our ITC results show a stoichiometry (n) of 1 depicting a single anti-Doc nanobody binding of the Doc. We observe Doc-anti-Doc binding affinity ranges between KD = 2.5 - 377 nM. This however is much lower than the Doc-Phd52-73 binding affinity (KD = 0.0017 nM). Agian, Doc could be crystallized in presence of several of the nanobodies. As there is currently no crystal structure available of Doc in a state where it is not inhibited by PhD, the non-inhibiting nanobodies may for the first time allow us to observe the active site of Doc in its free state.
| Datum prijs | 2 sep. 2014 |
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| Originele taal | English |
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