Ceramide synthase 2 knockdown suppresses trophozoite growth, migration, in vitro encystment and excystment of Entamoeba invadens.

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.

The metabolic pathway of sphingolipids biosynthesis and signaling in Entamoeba histolytica.

Sphingolipids (SLs) synthesis involves a complex metabolic pathway occurring between the endoplasmic reticulum (ER) and Golgi apparatus, generating ceramide synthesis and complex lipids, respectively. Here we show that E. histolytica, apparently lacking cellular organelles (ER and Golgi apparatus), synthesizes a wide variety of sphingolipid subspecies, being particularly abundant those of long-chain fatty acids. In silico analysis showed five putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the TLC domain, a region conserved in CerS of multiple organisms. These genes are abundantly expressed in different growth phases. Silencing and overexpression of CerS C4M4U4 (the closest homolog of human CerS 2 and 3) demonstrated its involvement in the synthesis of ceramide. Additionally, we identify C4M4U4, SMS2 and PKC (α, ßII) proteins and their subcellular localization of E. histolytica, suggesting that these subcellular compartments might be involved in the biosynthesis and signaling pathway of sphingolipids, and evidencing different sphingolipid synthesis pathways in Entamoeba.

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens.

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.

Srebf1a is a key regulator of transcriptional control for adipogenesis.

Adipogenesis is regulated by a complex cascade of transcriptional factors, but little is known about the early events that regulate the adipogenic program. Here, we report the role of the srebf1a gene in the differentiation of fibroblastic 3T3-F442A cells. We found that expression of srebf1a depended on GSK3ß activity and that GSK3ß activity was necessary for C/EBPß phosphorylation at Thr188. Knockdown of srebf1a inhibited the adipogenic program because it blocked the expression of genes encoding PPARγ2, C/EBPα, SREBP1c and even FABP4, demonstrating that SREBP1a activation is upstream of these three essential adipogenic transcription factors. Kinetic analysis during differentiation illustrated that the order of expression of adipogenic genes was the following: cebpb, srebf1a, pparg2, cebpa, srebp1c and fabp4. Our data suggest that srebf1a acts as an essential link between the GSK3ß-C/EBPß signaling axis and the beginning of the adipogenic transcriptional cascade.