Role of PdtaS-PdtaR two component signalling system in metabolic adaptation in Mycobacterium spp

includes bibliographical references

PhD;2024;Developmental Biology and Genetics

The ability to perceive and adapt to environmental fluctuations, both internal and external, is a fundamental characteristic of life across diverse organisms. Within the context of pathogenic bacteria such as Mycobacterium tuberculosis (Mtb), adaptation to the host environment is pivotal for its survival. Mtb encounters a range of stimuli, encompassing external cues like pH variations, hypoxia, and nutrient availability. To sense these stimuli, bacterial signal transduction relies on several key systems, with two-component systems (TCS) being one of the primary mechanisms. TCS typically consist of a sensor histidine kinase that auto-phosphorylates upon sensing a specific signal and subsequently transfers the phosphoryl group to the receiver domain of a response regulator. This phosphorylation event activates the effector domain of the protein, which then modulates signaling pathways via mechanisms like enhancing DNA/RNA-binding activity or modifying protein-protein interactions. The genome of Mtb encodes 12 genetically paired TCSs and 4 orphan response regulators (cognate sensor kinases unknown). Many of these TCS are also conserved in other non-pathogenic Mycobacterium species, indicating their importance in adapting to fundamental cues required by all mycobacterial species. This study specifically delves into the role of PdtaS-PdtaR TCS in Mycobacterium spp. and its role in sensing nutritional paucities through tandem GAF and PAS domains present in the PdtaS SK. The literature suggests that PAS/GAF domains often serve as versatile sensors and interaction modules in signal transduction proteins to regulate the activity of specific effector domains. This study primarily focuses on characterizing the role of the PdtaS-PdtaR TCS, with a particular emphasis on the role GAF-PAS domain and the possible targets of this TCS by employing M. smegmatis as our model organism. In this study, we identified c-di-GMP as one of the cues that are required by the GAF domain of PdtaS SK to further enhance its activity under conditions of nutrient scarcity. Through biochemical, biophysical, and genetic approaches, the study demonstrates that c-di-GMP interacts with the GAF domain, which is crucial for the signaling cascade from PdtaS to PdtaR and subsequently for PdtaR interaction with one of its targets, which is essential for its adaption in nutrient limiting conditions. Furthermore, through RNA immuno-precipitation, I also identified potential targets of PdtaR through which this regulatory effect is exerted. Additionally, this study delves into the implication of post-translational modifications, specifically acetylation, on PdtaS-PdtaR signaling. It is demonstrated that acetylation is necessary for the kinase activity of the sensor kinase, and a single acetylation mutation disrupts the entire signalling process and results in growth defects. In summary, utilizing non-pathogenic Mycobacteria as our model system, the study elucidates the functional significance of the GAF-PAS domain, identify the influence of phosphorylation status of PdtaR on its interactions with putative targets, and elucidate the role of post-translational modification on the signalling of SK (PdtaS) to RR (PdtaR).