Intrinsically High Capacity of Animal Cells From a Symbiotic Cnidarian to Deal With Pro-Oxidative Conditions

The cnidarian-dinoflagellate symbiosis is a mutualistic intracellular association based on the photosynthetic activity of the endosymbiont. This relationship involves significant constraints and requires co-evolution processes, such as an extensive capacity of the holobiont to counteract pro-oxidative conditions induced by hyperoxia generated during photosynthesis. In this study, we analyzed the capacity of Anemonia viridis cells to deal with pro-oxidative conditions by in vivo and in vitro approaches. Whole specimens and animal primary cell cultures were submitted to 200 and 500 mu M of H2O2 during 7 days. Then, we monitored global health parameters (symbiotic state, viability, and cell growth) and stress biomarkers (global antioxidant capacity, oxidative protein damages, and protein ubiquitination). In animal primary cell cultures, the intracellular reactive oxygen species (ROS) levels were also evaluated under H2O2 treatments. At the whole organism scale, both H2O2 concentrations didn't affect the survival and animal tissues exhibited a high resistance to H2O2 treatments. Moreover, no bleaching has been observed, even at high H2O2 concentration and after long exposure (7 days). Although, the community has suggested the role of ROS as the cause of bleaching, our results indicating the absence of bleaching under high H2O2 concentration may exculpate this specific ROS from being involved in the molecular processes inducing bleaching. However, counterintuitively, the symbiont compartment appeared sensitive to an H2O2 burst as it displayed oxidative protein damages, despite an enhancement of antioxidant capacity. The in vitro assays allowed highlighting an intrinsic high capacity of isolated animal cells to deal with pro-oxidative conditions, although we observed differences on tolerance between H2O2 treatments. The 200 mu M H2O2 concentration appeared to correspond to the tolerance threshold of animal cells. Indeed, no disequilibrium on redox state was observed and only a cell growth decrease was measured. Contrarily, the 500 mu M H2O2 concentration induced a stress state, characterized by a cell viability decrease from 1 day and a drastic cell growth arrest after 7 days leading to an uncomplete recovery after treatment. In conclusion, this study highlights the overall high capacity of cnidarian cells to cope with H2O2 and opens new perspective to investigate the molecular mechanisms involved in this peculiar resistance.

Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Francesca Zagari

Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.

EPFL2012

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

The functioning of coral reef systems, as biodiversity hotspots, is largely dependent on the symbiotic association between dinoflagellate symbionts (Symbiodinium spp.) and scleractinian coral hosts. The breakdown of this symbiosis (coral bleaching), as a result of global warming and other stressors, therefore has profound implications for the tropical marine environment. Corals associate with a variety of Symbiodinium genotypes, and it is this mosaic nature that contributes to the variable stress thresholds of corals. Research over the past 25 years has established that the generation and scavenging of reactive oxygen species (ROS) in both partners, under light and thermal stress, is a fundamental element of the bleaching response. However, while the existence of more thermally susceptible and tolerant symbiont types has been recognized, the differences in the antioxidant systems that may accompany these properties have received less attention. The purpose of this thesis was to explore the role of the antioxidant network in explaining the different thermal susceptibilities of various symbiont types and how the activity of key antioxidants in both partners under thermal stress relates to bleaching patterns in different corals. Thus, the specific objectives were to: (1) assess the antioxidant network response in different Symbiodinium types; (2) investigate the activity and structural diversity of key enzymatic antioxidants in different Symbiodinium types; (3) examine the regulation of these antioxidants at the transcriptomic and proteomic levels; and (4) contrast the symbiont’s and host’s antioxidant responses under bleaching conditions. Symbiodinium types in culture were found to differ significantly with regards to the concentration and activity of specific antioxidants, exhibiting magnitude scale differences in some of them. However, the response of the main removal pathway, involving superoxide dismutase (SOD) and ascorbate peroxidase (APX), under lethal thermal stress was fairly similar. Instead, the typespecific differences were found to lie in more downstream systems, and particularly in those associated with the maintenance of the glutathione redox state. A declining glutathione redox state was the common feature of the three thermally susceptible Symbiodinium types: B1, C1, and E. Indeed, in comparison to the most sensitive type (B1), the tolerant type F1 exhibited stronger antioxidant up-regulation and the successful preservation of the highly reduced glutathione pool. Comparing antioxidant gene orthologues from members of different Symbiodinium clades (A-E) revealed a higher degree of sequence variation at the amino acid level for peroxidases, which reflected the genetic radiation of the genus. In contrast, primary defences in the form of SOD isoforms were highly conserved. Sequence variations between Symbiodinium types involved residues that constitute binding sites of substrates and co-factors, and therefore likely affect the catalytic properties of these enzymes. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between transcriptomic expression and proteomic activity due to high variability in expression between replicates, and little response in their enzymatic activity over three days. In contrast to previous findings, up-regulation of antioxidant defences was not evident in Symbiodinium cells inside the host (i.e. in hospite). In fact, oxidative stress in the thermally sensitive corals Acropora millepora and Pocillopora damicornis was only apparent from increased host catalase activity, which interestingly preceded photosynthetic dysfunction of their symbionts. Baseline antioxidant activities of thermally tolerant and susceptible host species showed no differences, though the scavenging activities of the hosts were considerably higher than those of the symbionts. Baseline activities for the symbionts were different, however, with Symbiodinium C15 from the thermally tolerant coral Montipora digitata exhibiting the lowest activities for SOD and catalase peroxidase. This thesis provides significant findings with respect to the variability in antioxidant activity, structure, and network response in different Symbiodinium types in culture, and how these relate to thermal tolerance. What effect these differences have on the response in the intact symbiosis remains unclear, however, as the findings contradict the classic bleaching model of photoinhibition and symbiont-derived ROS. I argue, using previously published data, that heating rates might profoundly affect the way we perceive the antioxidant response of both partners to thermal stress, and that host antioxidant defences might not be as easily overwhelmed by symbiont ROS as suggested previously. This thesis reports important findings on the antioxidant system in different Symbiodinium types, but also raises new questions about the antioxidant response of the intact coral.

Victoria University of Wellington2014

Differential coral bleaching—Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress

Thomas Krüger

Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28 °C and 33 °C. A. millepora at 33 °C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33 °C. Increased host catalase activity in the susceptible coral after 5 days at 33 °C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

Elsevier2015

Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Francesca Zagari

EPFL2012

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

Victoria University of Wellington2014