Different Pathways of Choline Metabolism in Two Choline-Independent Strains of Streptococcus pneumoniae and Their Impact on Virulence

ABSTRACT The two recently characterized Streptococcus pneumoniae strains—R6Chi and R6Cho−—that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho− strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho−. These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho− by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho−. The identification in R6Cho− of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a “backup” system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.

Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption

A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.

Early Embryonic Lethality in Mice with Targeted Deletion of the CTP:Phosphocholine Cytidylyltransferase α Gene (Pcyt1a)

ABSTRACT CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (PtdCho). Multiple CCT isoforms, CCTα, CCTβ2, and CCTβ3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTα in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a − / − zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTα transcripts are significantly more abundant than transcripts encoding CCTβ or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTα transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTβ and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTβ (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTα expression in adult liver and other tissues from Pcyt1a +/ − heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a +/ − mice. Liver PtdCho mass was the same in Pcyt1a +/+ and Pcyt1a + / − adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTα expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues.

Lysosomal-type PLA2and turnover of alveolar DPPC

This study evaluated the role of a lysosomal-type phospholipase A2(aiPLA2) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [3H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA2. Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by ∼40–50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by ∼65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA2is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.

Channelling of intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine in mammalian cells

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T½ fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus α-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to < 1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of α-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, α-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.