Genes required for phosphosphingolipid formation in Caulobacter crescentus contribute to bacterial virulence.

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.

Spectrum of SPTLC1-related disorders: a novel case of 'Ser331 syndrome' that expand the phenotype of hereditary sensory and autonomic neuropathy type 1A and motor neuron diseases.

We report a patient with early-onset hereditary sensory and autonomic neuropathy type 1A (HSAN-1A) who developed a distinct phenotype, with tongue fasciculation and atrophy, due to a mutation at serine 331 in the SPTLC1 gene. HSAN-1A manifestation causing tongue fasciculation and atrophy have been rarely found. Our report adds to the growing evidence of the existence of an overlap between hereditary neuropathy and motor neuron disease caused by pathogenic p.S331Y variant in SPTLC1 gene.

Rare mutations in ATL3, SPTLC2 and SCN9A explaining hereditary sensory neuropathy and congenital insensitivity to pain in a Brazilian cohort.

Hereditary sensory neuropathies (HSN) are a group of rare neurological disorders with heterogeneous clinical and genetic characteristics. Although at least 17 different genes have already been associated with HSN, the epidemiology of the disorder in Brazil is still unknown. Performing whole genome sequencing (WGS) in 23 unrelated Brazilian families diagnosed with HSN, we detected pathogenic variants in ATL3, SPTLC2, and SCN9A in 12 patients belonging to five unrelated families. Clinical features associated with heterozygous mutations in ATL3 (c.575A > G; p.(Tyr192Cys)) and SPTLC2 (c.529A > G; p.(Asn177Asp)) were sensory deficits, neuropathic pain, and recurrent ulcerations. Presenting as congenital insensitivity to pain, three unrelated probands carried biallelic loss-of-function mutations in SCN9A. The so far undescribed stop mutation c.2106G > A (p.(Trp702Ter)) and the likewise novel splicing variant c.3319-1G > A were found in compound-heterozygosity with, respectively, the known pathogenic variants c.2908G > T (p.Trp970Ter) and c.2690G > A (p.Glu897Ter). In total, we identified pathogenic mutations in 21.7% of our families, which suggests that most of the cases could be explained by yet to be discovered genes or unusual alleles. Our study represents the first mutational screen in a Brazilian HSN cohort, enabling additional insights for genotype-phenotype correlations, reducing misdiagnoses, and providing early treatment considerations.

Five structural genes required for ceramide synthesis in Caulobacter and for bacterial survival.

Sphingolipids are essential and common membrane components in eukaryotic organisms, participating in many important cellular functions. Only a few bacteria are thought to harbour sphingolipids in their membranes, among them the well-studied α-proteobacterium Caulobacter crescentus, a model organism for asymmetric cell division and cellular differentiation. Here, we report that C. crescentus wild type produces several molecular species of dihydroceramides, which are not produced in a mutant lacking the structural gene for serine palmitoyltransferase (spt). Whereas growth of a spt-deficient mutant and wild type are indistinguishable during the exponential phase of growth, survival of the spt-deficient mutant is much reduced, in comparison with wild type, during stationary phase of growth, especially at elevated temperatures. The structural gene for spt is located within a genomic cluster, comprising another 16 genes and which, like spt, are important for fitness of C. crescentus. Mutants deficient in genes linked to spt by high cofitness were unable to produce dihydroceramide or to survive in stationary phase of growth at elevated temperatures. At least five structural genes are required for dihydroceramide biosynthesis in C. crescentus and sphingolipid biosynthesis is needed for survival of this bacterium and the integrity of its outer membrane.

Myriocin, a serine palmitoyltransferase inhibitor, blocks cytokinesis in Leishmania (Viannia) braziliensis promastigotes.

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 µM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 µM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 µM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 µM myriocin, respectively). Transmission electron microscopy following treatment with 1 µM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici.

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.

Amino acid-containing membrane lipids in bacteria.

In the bacterial model organism Escherichia coli only the three major membrane lipids phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin occur, all of which belong to the glycerophospholipids. The amino acid-containing phosphatidylserine is a major lipid in eukaryotic membranes but in most bacteria it occurs only as a minor biosynthetic intermediate. In some bacteria, the anionic glycerophospholipids phosphatidylglycerol and cardiolipin can be decorated with aminoacyl residues. For example, phosphatidylglycerol can be decorated with lysine, alanine, or arginine whereas in the case of cardiolipin, lysine or d-alanine modifications are known. In few bacteria, diacylglycerol-derived lipids can be substituted with lysine or homoserine. Acyl-oxyacyl lipids in which the lipidic part is amide-linked to the alpha-amino group of an amino acid are widely distributed among bacteria and ornithine-containing lipids are the most common version of this lipid type. Only few bacterial groups form glycine-containing lipids, serineglycine-containing lipids, sphingolipids, or sulfonolipids. Although many of these amino acid-containing bacterial membrane lipids are produced in response to certain stress conditions, little is known about the specific molecular functions of these lipids.

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells.

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line.

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM beta-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 muM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-(14)C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.

Hexachlorobenzene-induced alterations on neutral and acidic sphingomyelinases and serine palmitoyltransferase activities. A time course study in two strains of rats.

Hexachlorobenzene (HCB) induces porphyria both in humans and rodents, and hepatocarcinoma in rodents. In a previous work we observed that HCB produces a continuous decrease in hepatic sphingomyelin (SM) content in Wistar rats. A distinguishing characteristic of sphingolipids breakdown products is their participation in anti-proliferative and apoptotic processes and in the suppression of oncogenesis. As a first step to elucidate the role of SM decrease in the hepatotoxicity induced by HCB, the present study evaluates the metabolic causes of the continuous decrease in hepatic SM content observed in Wistar rats with HCB intoxication, and its relation with porphyria development. For this purpose, the time-course (3, 7, 15, 21 and 28 days) of the effects of HCB on hepatic SM levels and on some of the enzymes of SM synthesis (serine palmitoyltransferase, SPT) and catabolism (sphingomyelinases, SMases) was followed, using two strains of rats differing in their susceptibility to acquire porphyria: Chbb THOM (low) and Wistar (high). HCB (1 g kg(-1) b.w. per day) was administered by gastric intubation as an aqueous suspension. After 5 days of HCB treatment, animals were allowed a 2-day recovery period without HCB administration. Two phases in the HCB-induced damages to sphingolipid metabolism were observed. The first stage (7 days of treatment), common to both strains of rats, was characterized by a decrease in hepatic SM levels (17-25%) and in SPT activity (50-43%), while strain differences were found for the later stage. In Chbb THOM rats, hepatic SM content was restored to normal values concomitantly with an increase in SPT activity (44%, at day 28), and without any increase in SM catabolism. In addition, the level of the other phospholipids was not altered. In Wistar rats, hepatic SM levels decreased continuously throughout the experiment, accompanied by increases in SPT, acidic sphingomyelinase (A-SMase) and neutral sphingomyelinase (N-SMase) activities (86, 28.5 and 78% increase, respectively). A role for glutathione (GSH) in the interstrain differences or a direct effect of HCB on SM metabolism was not found. The present study: (a) demonstrates that N-SMase, A-SMase, and SPT are some of the enzymes that play a role in the HCB-induced decrease of hepatic SM content; (b) finds that HCB-induced alterations of SM metabolism do not correlate with HCB-induced accumulation of hepatic porphyrins; and (c) proposes a link between HCB-induced alterations in phospholipid pattern and in SM metabolism. The increased SM hydrolysis produced as a consequence of SMases induction could be regarded as a cellular response to liver injury elicited by HCB, perhaps acting through the activation of SM signal transduction pathway delaying the proliferative processes observed after long-term treatment with HCB in some rodent species. However, such protective mechanism appears to be strain-dependent.

Undernutrition decreases serine palmitoyltransferase activity in developing rat hypothalamus.

BACKGROUND/AIMS: Undernutrition reduces the hypothalamic ganglioside concentration. This may be attributed to some modifications in the contents of precursors of sphingolipid biosynthesis in undernourished rats. The present study evaluated the serine palmitoyl transferase activity (SPT; EC 2.3.1.50) during the development of the rat hypothalamus. This work also shows the L-[3-(14)C]serine metabolic labeling of hypothalamic sphingolipids in normal and undernourished rats at weaning. METHODS: The SPT activity was determined in microsomal fractions obtained from the hypothalamus of normal rats (diet: 25% protein) and pre- and postnatally undernourished rats (diet: 8% protein since pregnancy) at 21 days of gestational age and at 7, 14, and 21 days of postnatal life. RESULTS: The enzymatic activity was lower in the hypothalamus of undernourished than in the hypothalamus of control rats since the 7th postnatal day. Incorporation of the precursor L-[3-(14)C]serine into sphingolipid fraction was lower in the hypothalamus of undernourished rats than in the hypothalamus of control rats on the 21st postnatal day which coincided with the age of the highest difference in SPT activity between normal and undernourished rats. CONCLUSION: These results indicate that undernutrition reduces the biosynthesis of the main sphingolipids during the period of brain growth spurt.