develops the ability to survive during infection by resisting the host's immunological responses and previous work has suggested that activation of parasitic stress responses occurs during the invasive process 
Eukaryotic cells have evolved sophisticated mechanisms to sense stress in intracellular compartments such as the ER and respond appropriately by modulating nuclear gene expression. Within the ER, stress is induced by the presence of large amounts of unfolded or misfolded proteins 
. Cells can alleviate this shock by degrading or refolding the improperly folded proteins, one activity that needs an overproduction of chaperones.
As in a previous study 
, we identified an upregulation of hsp
genes. These were here newly annotated using a desktop application we developed for the retrieval of annotations of E. histolytica
's genome, thus providing the community with a genuine tool for rapid analysis of functional genomic data. We identified additional genes that were upregulated (hsp90
), while commonly identified genes (hsp20
) exhibited greater fold changes in our study. A more intense shock was induced in our experiments, due to the higher concentration of the NO donor used (1 mM versus 200 µM) and may explain the fact that only our dataset provide identification of a large panel of HSPs, whole pathways for DNA repair, metabolism or protein degradation.
The stress response triggers the induction of hsp
gene transcription upon exposure of cells to proteotoxic environments, which is a mechanism conserved from bacteria to mammals. In extreme stress, the protein quality control system can become saturated; this leads to accumulation and aggregation of misfolded proteins 
. These aggregates can potentially be eliminated by proteasome-mediated proteolysis in the cytosol. Under high thermal stress conditions (e.g.
>50°C), cells also induce a thermotolerance process capable of reactivating aggregated proteins via
the bi-chaperone Hsp101 (ClpB)-Hsp70 system, whose genes are seen to be the most highly over-expressed in E. histolytica
in the presence of NO.
In our experiments, hsp genes were overexpressed and massive cell death occurred. The protective function of hsp genes may be overwhelmed by misfolded proteins, resulting in cell death by loss of protein function or toxicity, however, one cannot exclude that prolonged hsp gene overexpression might be detrimental to cells.
Following SNP internalization and its activation, the NO generated can interact with a variety of molecules, especially aromatic residues, amines, thiols and metal ion-containing centers (such as heme or Fe-S clusters) and causes an inhibition of enzyme active sites. These assumptions suggest that increased turnover of Fe-S cluster-containing proteins and/or augmented Fe-S cluster synthesis or reparation is required to overcome the deleterious effects of NO treatment. Remarkably, we observed over-expression of genes encoding enzymes responsible for Fe-S cluster genesis and CS, the latter involved in the synthesis of Cys, the main antioxidant metabolite in amoeba and an essential aminoacid constituent of Fe-S clusters. The increased CS mRNA correlated with significant higher CS activity in the amebas exposed to SNP compared to control parasites. We also observed overexpression of Fe-S cluster-containing proteins such as PFOR, a metabolic enzyme that is essential in glucose metabolism in the parasite 
. Active PFOR requires intact Fe-S clusters and their transfer onto native PFOR apoproteins. The oxido-reductive properties of the iron atom make it highly susceptible to attack by ROS 
. The present results indicated that NO also inhibited the enzyme, but unlike to the reversible inactivation by ROS, NO produced a pronounced and irreversible inactivation. As fixation of Fe-S clusters by PFOR is mediated by cysteine residues, the inactivation we observed could be due to the formation of adducts between NO and the sulphur atoms of these amino acids. In the experiments we performed, either RNAs or proteins were purified directly after incubation with SNP; this implies that newly synthesized and active PFOR enzymes would be deactivated, as we have shown in vitro
by incubating active enzymes with SNP. Similar results were obtained for other glycolysis-related enzymes: a 4.3-fold up-regulation was observed for the gene that encodes the bifunctional aldehyde-alcohol dehydrogenase 2, the main NADH-dependent alcohol dehydrogenase activity in the parasite responsible of ethanol production and NADH reoxidation under anaerobic/microaerophilic conditions which was severely inhibited by SNP treatment and which has a prominent role in determining the glucose catabolism end-product profile in the parasites 
. Moreover, the NADPH-dependent ADH activity (alcohol dehydrogenase 3
gene) was also inhibited, although to a lesser extent; however, its contribution to ethanol synthesis is not well established yet. Malic enzyme activity was also inhibited by SNP treatment, which, together with malate dehydrogenase, is involved in reoxidation of the NADH produced during glycolysis under partial aerobic conditions where ADH2 is inhibited.
These results highlight that energy metabolism is highly impaired by NO, leading to a decrease in the levels of their end-products – ethanol and ATP 
– and maybe in the redox potential. These results also correlate with high glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and dihydroxyacetone-phosphate (DHAP) concentrations in NO-treated trophozoites 
; these accumulated metabolites are intermediates of reactions upstream of PFOR in the glycolytic pathway. Thus, our results indicate that the glycolysis-fermentation pathway is disrupted upon NO treatment and that a feedback response may lead to enhanced transcription of pfor
and other genes involved in glucose metabolism.
Cells will promote their survival by reducing misfolded protein levels during stress by activating UPR. Unexpectedly, the E. histolytica
genome does not contain gene paralogues encoding important UPR-related proteins such as PERK or ATF6 and indeed E. histolytica
does not seem to induce UPR upon NO treatment. The absence of UPR responses (or defects herein) has been found in other protozoan 
and has been noticed as well in important diseases such as in the Wolfram syndrome which includes diabetes at early stages, mutations in WFSI (Wolframine) leads to high levels of ER stress and cell apoptosis. WFS1 is a ER transmembrane protein acting in calcium homeostasis and it has recently been shown that WFS1 is a key feedback regulator of the ATF6 branch of the UPR 
. These evidences suggest that ER stress is a major player in the establishment of important human pathologies such as diabetes, characterized by high blood glucose levels, contributing to pancreatic ß-cell death and insulin resistance. Interestingly, in vitro
data suggest that the cytokines IL-1β and IFN-γ, putative mediators of ß -cell loss in type-1 diabetes, induce severe ER stress through NO-mediated depletion of ER calcium and inhibition of ER chaperones, respectively, thus hampering ß -cell defenses and amplifying the pro-apoptotic pathways. The physiopathology of ß -cells during type-1 diabetes thus share common features with E. histolytica's
response to NO.
One interesting aspect on ER morphology changes is that the ER components remains localized to the same compartments during the ER fission process. This fact indicates that the parasite has developed a way for the ER not to be obliterated upon contact with stress components. ER fragmentation and the subsequent potential and/or eventual fusion may be one of E. histolytica's strategies to rapidly recover ER function after removal of a stress, allowing its survival.
Reversible ER fission is possible and has been evidenced in neurons, depending on mechanisms other than energy depletion and cell death, whereas extracellular Ca2+
is obligatory for ER fragmentation 
. This other cellular system suggests that ER fission is not solely a passive mechanism due to a lack of energy, but may also be a way for the cell to respond to a stress, probably by compartmentalizing the continuous ER to avoid diffusion of toxic products within the organelle and towards the nucleus.
The cellular and molecular analysis presented here has highlighted a strong and complex response to NO linked to ER stress and glucose metabolism which is a topic of interest to understand the death of E. histolytica during amoebiasis and also the death of other cells during human metabolic diseases.