Esencialidad de la tiorredoxina TrxA en cianobacterias. Estudio de un mutante condicional en Synechocystis sp. PCC 6803

Introducción: el cáncer de cabeza y cuello (CCC) constituye la novena neoplasia maligna más común en el mundo, con una importante morbilidad y mortalidad, sobre todo en los países en desarrollo. El cáncer de laringe es la segunda neoplasia maligna más común de este grupo; tiene una incidencia mundial (ASIRw) de 2,9 por 100 000 habitantes según datos de GLOBOCAN en 2018 y constituye el 1,5 % del total de tumores en España. Históricamente, el carcinoma laríngeo localmente avanzado se ha tratado con cirugía combinada y RT posoperatoria. Sin embargo, el abordaje quirúrgico primario mediante una laringectomía total implica la pérdida de la voz. Durante las últimas décadas, se han explorado nuevas modalidades de tratamientos no quirúrgicos para lograr la cura de la enfermedad localmente avanzada y preservar la laringe para mantener la función del órgano. Estos enfoques terapéuticos han consistido en RT aislada o combinada con quimioterapia. No obstante, el tratamiento de elección para los carcinomas laríngeos T3 y T4 sigue siendo controvertido debido a la heterogeneidad de los resultados de estudios tanto poblacionales como de ensayos clínicos. Objetivos: se analizó la efectividad de la preservación de órgano mediante quimio y radioterapia vs. tratamiento quirúrgico recibidos por los pacientes diagnosticados de cáncer de laringe avanzado y su impacto en la supervivencia. Materiales y métodos: se diseñó un estudio analítico observacional longitudinal de tipo cohortes prospectivo y retrospectivo con la finalidad de comparar la efectividad de los tratamientos de preservación de órgano vs. cirugía desde el punto de vista de resultados oncológicos y funcionales. Se trató de una muestra constituida por 640 pacientes con diagnóstico de cáncer de laringe avanzado, en estadios III y IV, tratados en los Servicios de Otorrinolaringología del Hospital Universitario Virgen del Rocío en el periodo comprendido entre 1980 y 2016. Resultados: se observó un riesgo aumentado en el análisis bivariado de mortalidad global (p <0,05; RR 2,05), mortalidad específica (p <0,05; RR 1,87), mortalidad competitiva (p <0,05; RR 2,47) y recidiva o progresión de la enfermedad (p <0,05; RR 2,12) para los pacientes tratados con protocolo de preservación de órgano con respecto a los tratados quirúrgicamente. Se consiguió en cifras absolutas una preservación del 60 % de los casos, sin embargo, si tomamos en cuenta los vivos con laringe preservada esta cifra es del 27 %. El 18 % de pacientes que reciben el protocolo de preservación de órgano consiguieron estar vivos con laringe preservada y funcionante. La laringectomía de rescate se asocia a un riesgo aumentado de mortalidad (p <0,05; RR 2,07) y de incidencia de fístulas faringocutáneas (FFC) (p <0,05; RR 1,65). Asimismo, las FFC tras cirugía de rescate suelen tardar más en cerrar con tratamiento conservador (p <0,05; 2,037 vs. 1,084) y necesitan con mayor frecuencia reconstrucción mediante colgajos microvasculares (p <0,05; RR 13,39) que aquellas que aparecen tras tratamiento quirúrgico primario. En el análisis de supervivencia observamos una menor supervivencia global (p <0,05; HR 1,79), supervivencia específica (p <0,05; HR 1,62), supervivencia libre de enfermedad (p <0,05; HR 2,59) y supervivencia tras laringectomía (p <0,05; HR 1,945) en los pacientes tratados con protocolo de preservación de órgano con respecto a los tratados quirúrgicamente. En los pacientes tratados con QTRT concomitante se observó que a los 10 años el 30,3 % mantuvo preservada su laringe y el 16,1 % tenía su laringe preservada y funcionante. Discusión: el tratamiento con preservación de órgano se asocia a una mayor mortalidad global, mortalidad específica, mortalidad competitiva, recidiva o progresión de la enfermedad que la LT primaria en nuestros resultados. Del mismo modo, se asocia a menor supervivencia global, supervivencia específica, supervivencia tras laringectomía y supervivencia libre de enfermedad. Tras el tratamiento con QTRT concomitante se logró preservar de manera global el 60 %. No obstante, si se tienen en cuenta cuántos pacientes viven con su laringe preservada esta cifra cae al 27 %. Además, las supervivencias de laringe preservada y funcionante difieren considerablemente del porcentaje de laringe preservada que suelen describir en la mayoría de los estudios. Si tomamos en cuenta el tiempo, estos tienden también a disminuir. Hacen falta más estudios en nuestro medio que comparen directamente ambos esquemas de tratamiento para poder observar con mayor potencia estadística la efectividad de la aplicación de estos protocolos y la identificación de factores intervinientes. La LT de rescate se asocia a una mayor mortalidad, menor supervivencia y mayor incidencia de complicaciones como la FFC. La FFC tras una LT de rescate suele tener un mayor impacto en la vida de los pacientes que la sufren al tardar en remitir con tratamiento conservador y tener mayor necesidad de reconstrucción con técnicas de colgajo microvascular. Conclusiones: se recomienda que se debe informar a los pacientes de la significativa desventaja en la supervivencia y control de la enfermedad sobre todo a largo plazo asociadas con la terapia no quirúrgica, como parte del proceso de toma de decisiones compartida durante la selección del tratamiento

Exploring the Diversity of the Thioredoxin Systems in Cyanobacteria

Cyanobacteria evolved the ability to perform oxygenic photosynthesis using light energy to reduce CO2 from electrons extracted from water and form nutrients. These organisms also devel-oped light-dependent redox regulation through the Trx system, formed by thioredoxins (Trxs) and thioredoxin reductases (TRs). Trxs are thiol-disulfide oxidoreductases that serve as reducing sub-strates for target enzymes involved in numerous processes such as photosynthetic CO2 fixation and stress responses. We focus on the evolutionary diversity of Trx systems in cyanobacteria and discuss their phylogenetic relationships. The study shows that most cyanobacteria contain at least one copy of each identified Trx, and TrxA is the only one present in all genomes analyzed. Ferredoxin thiore-doxin reductase (FTR) is present in all groups except Gloeobacter and Prochlorococcus, where there is a ferredoxin flavin-thioredoxin reductase (FFTR). Our data suggest that both TRs may have coex-isted in ancestral cyanobacteria together with other evolutionarily related proteins such as NTRC or DDOR, probably used against oxidative stress. Phylogenetic studies indicate that they have different evolutionary histories. As cyanobacteria diversified to occupy new habitats, some of these proteins were gradually lost in some groups. Finally, we also review the physiological relevance of redox regulation in cyanobacteria through the study of target enzymes.Junta de Andalucía BIO-28

Deciphering the function and evolution of the TOR signaling pathway in microalgae

Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen in the planet and a major biotechnological target for sustainable biofuel production and CO2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as central regulator of cell growth and nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed the high conservation of central components of the TOR pathway likely present in the last eukaryotic common ancestor and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress on the study of this signaling pathway in these organisms.Ministerio de Ciencia y Tecnología PGC2018-099048-B-I00, PID2019-110080GB-I0

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.España, Ministerio de Economía y Competitividad (MINECO) (grant BFU2013-41712-P and BIO2016-75634-P)España, Junta de Andalucía (P12-BIO-1119

Dark side of cyanobacteria: searching for strategies to control blooms

Cyanobacteria are ecologically one of the most prolific groups of photosynthetic prokaryotes in marine and freshwater habitats. They are primary producer microorganisms and are involved in the production of important secondary metabolites, including toxic compounds such as cyanotoxins. Environmental conditions promote massive growth of these microbes, causing blooms that can have critical ecological and public health implications. In this highlight, we discuss some of the approaches being addressed to prevent these blooms, such as control of nutrient loading, treatments to minimize growth or monitoring interactions with other speciesPeer reviewe

Dark side of cyanobacteria: searching for strategies to blooms control

Cyanobacteria are ecologically one of the most prolific groups of photosynthetic prokaryotes in marine and freshwater habitats. They are primary producer microorganisms and are involved in the production of important secondary metabolites, including toxic compounds such as cyanotoxins. Environmental conditions promote massive growth of these microbes, causing blooms that can have critical ecological and public health implications. In this highlight, we discuss some of the approaches being addressed to prevent these blooms, such as control of nutrient loading, treatments to minimize growth or monitoring interactions with other species

Photosynthetic assimilation of CO2 regulates TOR activity

The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin–Benson–Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii. Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.Peer reviewe

Depletion of m-type thioredoxin impairs photosynthesis, carbon fixation, and oxidative stress in cyanobacteria

Thioredoxins (Trxs) are disulfide oxidoreductases that regulate many biological processes. The m-type thioredoxin (TrxA) is the only Trx present in all oxygenic photosynthetic organisms. Extensive biochemical and proteomic analyses have identified many TrxA target proteins in different photosynthetic organisms. However, the precise function of this essential protein in vivo is still poorly known. In this study, we generated a conditional Synechocystis sp. PCC 6803 mutant strain (STXA2) using an on-off promoter that is able to survive with only 2% of the TrxA level of the wild-type (WT) strain. STXA2 characterization revealed that TrxA depletion results in growth arrest and pronounced impairment of photosynthesis and the Calvin–Benson–Bassham (CBB) cycle. Analysis of the in vivo redox state of the bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase showed higher levels of oxidation that affected enzyme activity in STXA2. This result implies that TrxA-mediated redox regulation of the CBB cycle is conserved in both cyanobacteria and chloroplasts, although the targets have different evolutionary origins. The STXA2 strain also accumulated more reactive oxygen species and was more sensitive to oxidative stress than the WT. Analysis of the in vivo redox state of 2-Cys peroxiredoxin revealed full oxidation, corresponding with TrxA depletion. Overall, these results indicate that depletion of TrxA in STXA2 greatly alters the cellular redox state, interfering with essential processes such as photosynthetic machinery operativity, carbon assimilation, and oxidative stress response. The TrxA regulatory role appears to be conserved along the evolution of oxygenic photosynthetic organisms.Agencia Estatal de Investigación PID2019-104513GB-I00/AEI/10.13039/501100011033Ministerio de Economía y Competitividad BIO2016-75634-PJunta de Andalucía, cofinanced by European Regional Funds (FEDER) BIO-28

Deciphering the role of autophagy in the extremophilic microalga Chlamydomonas urium

Extremophilic microalgae have the ability to grow and adapt their metabolism to extreme conditions including acidic pH, high or very low temperature, or the presence of high concentrations of heavy metals. In addition to their ecological importance as primary producers in extreme environments, these organisms also have biotechnological potential since they produce several beneficial compounds as metabolism side-products including lipids or carbohydrates. Autophagy is a degradative process by which eukaryotic cells eliminate toxic or superfluous cellular components in order to cope with stress or unfavorable conditions and maintain cellular homeostasis. We have demonstrated that autophagy is a pro-survival mechanism that is upregulated by a wide range of stress conditions such as nutrient starvation or metal toxicity in the model microalga Chlamydomonas reinhardtii. However, autophagy has never been investigated in extremophiles and it is currently unknown whether this catabolic process play a role in the adaptation of these organisms to adverse environments. To address this question, we have isolated a new microalga, Chlamydomonas urium, from the Tinto River (Nerva, Spain), a well-characterized extremely acidic river with a high content of heavy metals. Genome sequencing and annotation revealed that core ATG genes are conserved in C. urium, indicating that autophagy must be conserved in extremophilic organisms. Our studies by western blot analysis, ultrastructural microscopy and metabolomics showed that autophagy is a highly dynamic process in C. urium. Interestingly, the inhibition of autophagic flux results in a large increase of triacylglycerols and starch, the two main carbon storage molecules. Thus, our results pinpoint autophagy as a central process regulating carbon storage in the new extremophilic microalga C. urium.This work was supported in part by Junta de Andalucía (Grant P20_00057