Unravelling Crosstalk in Two-component Signalling Systems and Its Role in Mycobacterium Species

Abstract

A typical two-component signaling system (TCSs) is composed of two transcriptionally coupled proteins (cognate pair) where one is a signal-sensing histidine kinase (HK) and the other, an output-generating response regulator (RR). Typically, signal transmission in TCSs is restricted to cognate pairs, and crosstalk is considered deleterious. However, recent evidence suggests that crosstalk between TCS proteins could improve bacterial adaptive capacity living in predefined environments. M. tuberculosis, a human tuberculosis pathogen, encodes only 12 paired TCS and five orphan RRs, which play a very crucial role in the bacterium’s adaptation inside the host. During the course of infection, it is anticipated that M. tuberculosis pathogen will encounter a large number of stresses; however, to effectively respond to these stresses and take complex decisions such as proliferation, dormancy/latency, and reactivation, exclusively ‘specific’ TCS signaling may not be sufficient. Previous in vitro studies, focusing on potential cross-communication, have proposed an extensive crosstalk network among TCS proteins of M. tuberculosis. However, this network's in vivo existence and physiological relevance remain unclear. Making use of biochemical, biophysical, and genetic approaches, in the present study, I report the atypical crosstalk networks that operates between the MtrAB-NarSL, NarSL-SenX3RegX3, and NarSL-TcrYX TCS systems in M. tuberculosis. To establish this crosstalk network, a transcriptional response mapping was performed which revealed an extensive regulon overlap between the NarS-NarL system and the SenX3-RegX3, TcrX-TcrY, MtrB-MtrA TCSs. The study indicated a potential regulatory network between the NarS-NarL and SenX3-RegX3, TcrX-TcrY TCSs, and provides the possible physiological implications of crosstalk for the in vivo scenarios. The investigation of MtrB-NarL crosstalk node suggested that RR NarL acts as a strong binder for the HK MtrB, and able to tune the cognate MtrB to MtrA signaling output necessary for the bacterium’s adaptation. Further biochemical experiments unveiled a novel crosstalk node between the RR NarL and the non-cognate HK SenX3 as well as RR RegX3. The comprehensive investigation into the signaling of the NarS-NarL TCS suggested its implication in key cellular processes and pathogenicity of M. tuberculosis. Overall, through this study, I propose a master crosstalk node, the NarS-NarL TCS, which operates as a central hub, with MtrB-MtrA and SenX3-RegX3 TCSs serving as supporting arms. The MtrB feeds into this node through its higher binding affinity to RR NarL, while the HK SenX3 modulates this node by robust phosphorylation of RR NarL. Seeing the integrative capacity and implications of this master node in adaptation and pathogenicity of the TB bacterium, could serves as a promising target for pharmaceutical interventions.