Regulatory mechanisms within the mazEF family of toxin-antitoxin modules.

Abstract

Chromosomal Toxin-Antitoxin (TA) modules, which are involved in bacterial stress response and persistence cell formation, are small operons that encode for two proteins: a "toxin" that interferes with translation or transcription and an "antitoxin" that neutralizes the toxin and protects the cell from its destructive activity. Although there are several unrelated families of TA modules in terms of amino acid sequence and activity, TA modules operate under a similar principle: a pronounced difference in life-time between the stable, long-lived toxin and the labile, short-lived antitoxin. Upon exposure to stress (e.g. antibiotics or nutritional stress), the modules are activated by proteolytic degradation of the antitoxin, releasing the toxin. The targets of TA toxins can vary and include mRNA, elongation factor Tu, the ribosome or DNA gyrase, all part of the transcription/translation machinery.
The mazEF family of TA modules is very common in both Gram positive and Gram negative bacteria, with the Escherichia coli MazEF being the best characterized system.
The PhD research focused on the structural and biophysical studies on the mazEF modules from Staphylococcus aureus (SamazEF) as gram positive and the mazEF from Escherichia coli (EcmazEF) as gram negative. The research aimed to elucidate regulatory mechanisms at the level of transcription and protein activities. Although there is quite a bit of in vivo data available on mazEF modules, very little is known about their mechanisms of action at the molecular level. No quantitative data are available on the MazE-MazF interaction as well as on the MazE-DNA interaction for the E. coli family. Little is not known about the enzymatic reaction mechanism of the MazF toxins towards mRNA. The staphylococcal MazEF system remained so far surprisingly unstudied.

The biochemical activities of TA toxins typically lead to cell death upon overexpression and wild-type TA toxins can only be expressed in presence of their cognate antitoxin. They are difficult to obtain in large quantities needed for structural studies. So far, production of MazF of suitable quality and quantity for structural studies required a mutation that abrogates its RNAse activity. To overcome this limitation, we designed a purification procedure that allows separating MazF from MazE without compromising protein quality and function. This resulted in the production of active (RNA cleaving) protein from both the E. coli and S. aureus modules.
For the E. coli mazEF module, I focused on transcription regulation: the EcMazE acts as transcription factor, binding and negatively auto-regulating its own mazEF promoter. This was studied using a combination of NMR, SAXS, ITC and EMSA experiments. I find that transcription is regulated though a mechanism of conditional co-operativity and provide its thermodynamic and structural basis.
For the less studied SaMazEF system, its RNAse activity was studied in detail. The structure of SaMazF in its free form was determined using a combination of X-ray crystallography, NMR spectroscopy and SAXS. This led to the identification of a conserved mode of loop dynamics in the MazF/CcdB superfamily that is of direct relevance to substrate and antitoxin binding. The crystal structure of SaMazF was also determined in complex with a substrate mimic deoxy-RNA fragment, leading to new insights into its mechanism of action and basis for substrate specificity. The SaMazF ribonuclease activity can be modulated by nanobodies (Nbs). Crystal structures of SaMazF in complex with several, such Nbs was determined, revealing partial overlap between the Nb-binding sites and the RNA-binding site. Thermodynamics of the SaMazF-RNA and SaMazF-Nbs interaction and their competition provides useful information for the future design of inhibitors of SaMazF that can be developed into anti-persistence drugs.

Date of Award	12 May 2015
Original language	English
Awarding Institution	Vrije Universiteit Brussel
Sponsors	Research Foundation Flanders