Redes de señalización implicadas en la regulación del metabolismo de las ectoínas en la bacteria halófila Chromohalobacter salexigens y su potencial terapéutico como agente neuroprotector

Abstract

Chromohalobacter salexigens is a halophilic bacterium which naturally produces ectoine and hydroxyectoine, two biotechnologically important compatible solutes that are accumulated in response to osmotic and heat stress, respectively. The exploitation of C. salexigens in biotechnology as a producer of ectoines requires an integral understanding of the regulatory pathways controlling ectoines metabolism. The availability of its genomic sequence, physiological, biochemical, genetic, and high performance transcriptomics data, previously obtained from our Research Group, together with regulatory studies (this work), will allow us to obtain a more global knowledge of the metabolism of the ectoines in C. salexigens. The aim of this PhD was two-fold. On the one hand, to gain insight on the regulatory circuits controlling the metabolism of ectoines in C. salexigens. On the other, to progress in the biomedical use of ectoine for the prevention and treatment of neurodegenerative diseases.

The findings presented in this work provide clues for a better understanding of the complex regulation of ectoine metabolism and osmoadaptation in this moderately halophilic bacterium, and its contribution to adaptation to extreme environments. Our results will be overlayed on the existing high-quality genome-based metabolic model and global analyzes, in order to facilitate the rational design of new strains for Systems metabolic engineering. Additionally, we have investigated the in vitro and in vivo application of ectoine as neuroprotective- and inflammation-preventive- treatment in an Aβ murine model of Alzheimer's disease. Thus, we have got more insight in its potential applications in biomedicine, especially in neurodegenerative disorders as Alzheimer’s disease (AD).

EupR is the first-described response regulator of a two-component system involved in bacterial osmoregulation (Rodríguez-Moya et al., 2010). Through in silico studies, EupK was selected as its putative cognate histidine kinase. Phenotypic analyses of eupR and eupK mutants showed that both, EupR and EupK, participate in the control of the three processes within the metabolism of ectoines, synthesis, degradation/recycling and uptake, as a key system that allows the fine adjustment of the intracellular concentration of these solutes. Nevertheless, the association between EupK and EupR could not be confirmed by these analyses, as eupK did not always reproduce the eupR phenotype. This finding pointed out to the involvement of additional HKs and/or RR that would participate in the control of ectoines metabolism. Additional in vivo protein-protein interaction and phosphotransfer experiments, demonstrated not only the interaction between EupK and EupR, but also the functionality of the two component system, as EupK was able to

phosphorylate EupR. As EupK is a hybrid HK that lacks the HPt domain involved in phosphotransfer during phosphorelay events, three hybrid HKs, Csal_1062, Csal_1635 and Csal_2667, were selected as the most suitable candidates to form part of the EupK/EupR signalling network by providing this phosphotransfer domain. Our results indicate that EupK and EupR constitute a two-component system, which form part of a "many to one"-type complex signaling network with the other hybrid HKs, where EupR is phosphorylated by all of them. These cross-talk events within the response regulator could be developed either by a direct phosphorylation of the REC domain of EupR by the HisKA domains of the hybrid histidine kinases, or through a phosphorelay mechanism involving additional HPt domains, and could depend on the environmental conditions. Besides, this branched and/or crossed regulation circuit would be able to detect and respond to both salinity and extracellular ectoine stimuli.

Furthermore, transcriptomic experiments revealed that the elimination of eupR affected more than 18 and 22% of C. salexigens genes at low and high salinity respectively, distributed throughout the different functional categories described by COG. This result suggested that EupR has a global impact on C. salexigens physiology. Among them, 20 transcriptional regulators were differentially expressed in the mutant, strongly suggesting that EupR is a regulator of regulators, and explaining the large number of genes affected by its deletion. The importance of this regulator should be highlighted in the so-called "nitrogen channeling" that occurs in C. salexigens (Pastor et al., 2013), by controlling the balance between protein synthesis, nitrogen metabolism and ectoine synthesis, possibly through the global nitrogen regulator NtrC, among others. Another of the processes controlled by this regulator would be chemotaxis, which should be directly related to osmodetection in C. salexigens, as flagellum motility is Na+-dependent (Salvador et al., 2018). This regulatory mechanism might be exerted through the control of other two-component systems such as CheA and CheY.

On the other hand, the implication of EupR in central metabolism was remarkable, with 212 and 306 genes differentially expressed at low and high salinity, respectively. Among them, we should highlight those involved in carbon metabolism, by influencing the different metabolic flows distribution balance: (i) glucose assimilation by regulating the Entner-Doudoroff (ED) periplasmic and cytoplasmic route; (ii) nitrogen metabolism and (iii) the energetic metabolism through the control of oxidative phosphorylation, especially at high salinity. Finally, the results revealed a strong influence of EupR on C. salexigens osmoadaptation, where EupR could have a role in regulating the hierarchical accumulation of compatible solutes, in order to ensure the appropriate intracellular concentration of the

different solutes according to the environmental constraints, the carbon source, or the presence of osmoprotectants in the external medium, among others.

A final conclusion is that EupR would meet all the criteria to be postulated as a global regulator involved in the control of the metabolism of compatible solutes, especially that of ectoines, as well as the energy producing processes and central metabolism of C. salexigens. Hence, it would influence the C. salexigens metabolic and physiological adaptations to the external osmolarity. The results arising from this work will contribute to elucidate the complex regulatory network involved in the control of ectoine synthesis, which in turn will allow the construction of a signaling and transcriptional regulatory model for C. salexigens.

On the other hand, the in vitro and in vivo neuroprotective capacity of ectoine was evaluated in this work. Our results demonstrate the ectoine’s protective capacity against the oxidative damage, its antioxidant activity in human neuronal cells, as well as its capacity to prevent the formation of ADDL oligomers responsible for inflammation. In addition, ectoine was able to cross the blood-brain barrier and reach cerebral areas where Alzheimer diseased is developed, as cortex or hippocampus, and to remain with pharmacologically active concentrations during 48h. Intragastric administration of ectoine during 3 months in young transgenic APP/PS1 mice (which develop AD), improved their memory and diminished Aβ concentration in brain tissue. A decrease in plaques size accompanied by an increased recruitment of the microglia was also observed. Additionally, ectoine intragastric inoculation led to similar concentrations in brains that those achieved upon intraperitoneal administration, within the range of activity observed in vitro, suggesting that oral administration of ectoine would be feasible. All these results pointed to ectoine as an interesting candidate to be used as a drug for the prevention and treatment of neurodegenerative diseases.