Modulation of the activity of the staphylococcus aureus toxin protein MazF by nanobodies

Scriptie/Masterproef: Master's Thesis


Antibiotics are often used incorrectly in the healthcare and livestock sector, and this has contributed to the fact that bacteria become resistant to several antibiotics very quickly. This results in limited availability of effective antibiotics for certain multi-drug resistant strains, like, for example, meticillin-resistant Staphylococcus aureus (MRSA). Next to this resistance, there is also persistence, whereby a percentage of the bacterial population can go into a dormant state. Antibiotics mainly act on metabolically active cells, these pathogenic bacteria can wait the course of antibiotics out, and the infection can reappear afterwards. It needs to be investigated whether toxin-antitoxin (TA) systems can respond to both forms of tolerance, in other words if they are helpful to develop novel strategies to counteract bacterial infections. I will focus on the sole chromosomal mazEF toxin-antitoxin from Staphylococcus aureus.
A panel of existing nanobodies raised against S. aureus MazF (SaMazF) was tested for their potential to inhibit or activate this enzyme. These data were correlated with the structural characteristics of the SaMazF- nanobody complexes. First, the nanobodies and SaMazF were purified. Afterwards, a fluorescence assay where a RNA/DNA hybrid substrate has on one end a fluorophore attached, and on the other end, a fluorescence quencher was performed to investigate the effect of the nanobodies on SaMazF activity. The kinetic parameters Vmax and KM were also determined through this assay. The values are 3.12 μM/min and 6.98 μM, respectively. The KM value indicates that the enzyme has a high affinity for the substrate, kcat could also be determined via KM and is 20.8 s-1 indicating a mediocre turnover number. After that, structures of SaMazF-nanobody complexes that form crystals were determined. The modes of binding of the different nanobodies to SaMazF were compared and correlated with their functional properties (inhibitor, activator, neutral).
Nanobodies 8, 10 and 11 seem to have no or minimal effect on SaMazF activity. It would need to be investigated further to see more precisely if they actually have no effect or if they perhaps activate SaMazF. The other nanobodies inhibit the enzyme. The structure of nanobody 10 and 11 differ in the CDR loops. PISA analysis revealed that the CDR3 loop is always involved in nanobody- SaMazF interaction. The CDR3 loops of nanobodies 10 and 3 both form an alpha helix. They both mimic a part of the BsMazE antitoxin, meaning they affect its binding affinity, but nanobody 10 is the best-suited nanobody for further research because it does not affect the toxin activity.
Datum prijs4 feb 2022
Originele taalEnglish
Prijsuitreikende instantie
  • Toegepaste Biologische Wetenschappen
  • Vrije Universiteit Brussel
BegeleiderRemy Loris (Promotor) & Marusa Prolic Kalinsek (Advisor)

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