Etude du système CovM-CovRS de régulation distale de la compétence chez Streptococcus salivarius

Details

Supervisors

Hols, Pascal

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

enCompetence for natural DNA transformation in Streptococcus salivarius is proximally regulated by the ComRS system, Induced by the presence of signalling peptides in the extracellular medium. This primary layer of regulation is itself modulated by a three-component system (distal regulation) composed of the CovRS two-component system and its associated modulator, the pseudo-histidine kinase CovM. To date, the functioning of the CovM-CovRS system remains poorly understood, particularly regarding its activity (kinase vs. phosphatase) and the interactions between its partners. The main objective of this master thesis was to investigate the interactions among CovS, CovM, and CovR, as well as their impact on the activation of competence in S. salivarius. Firstly, the existence of a tripartite system was demonstrated using the Split NanoLuc technique (α-complementation). The interaction between CovM and CovS had already been validated in a previous study. In this work, we showed that the response regulator CovR interacts with the histidine kinase CovS and also with the pseudo-histidine kinase CovM. Furthermore, this study demonstrated that the interaction between CovR and CovM is dependent on the presence of CovS, indicating that CovM and CovR interact via CovS. Collectively, these findings support the presence of a tripartite complex, though its stoichiometry remains to be further investigated. Secondly, point mutations were introduced to better understand the mechanism by which these proteins modulate the ComRS system via the control of comR expression. The first significant mutations aimed to destabilize the CovS-CovS dimer by disrupting π-sulfur interactions between phenylalanine 260 (F260) and three methionines (M231, M263, and M264) in the HAMP domain. The most intriguing results were obtained with the F260A mutation. Although this mutation did not disrupt the formation of the CovS-CovS dimer, it appeared to induce a conformational change that increased intrinsic phosphatase activity of CovS. Due to its location in the HAMP domain, this mutation may mimic the activation of CovS's receiver domain by a signal favouring phosphatase activity. Another mutation also led to increased CovS phosphatase activity: the mutation of asparagine 367 (N367A) in the magnesium-binding motif of CovM. Magnesium, an essential cofactor for classical histidine kinases, stabilizes ATP binding. This gain-of-function mutation was unexpected for a pseudo-kinase lacking a catalytic histidine. A plausible hypothesis is that CovM is dimeric and that the N367A mutation destabilizes the CovM dimer. This destabilization might increase the monomeric form of CovM, which could interact with CovS and positively modulate its phosphatase activity. The destabilization of the CovM dimer by the N367A mutation may mimic a conformational change that occurs only when CovM is activated by its specific stimuli. This thesis has thus demonstrated for the first time the existence of a tripartite complex between CovM, CovS, and CovR. Additionally, several point mutations revealed gain-of-function effects, opening new perspectives on the role of pseudo-histidine kinases as modulators of two-component systems in Gram-positive bacteria.