Phosphatidylethanolamine Deficiency Impairs Escherichia coli Adhesion by Downregulating Lipopolysaccharide Synthesis, Which is Reversible by High Galactose/Lactose Cultivation.

As the initiation step of bacterial infection or biofouling, bacterial adhesion on cells or substrates is generally an optimal target for antibacterial design. Phosphatidylethanolamine (PE) is the principal phospholipid in bacteria, and its function in bacterial adhesion remains unclear. In this study, four E. coli strains including two PE-deficient mutants (PE-PC- and PE-PC+ strains) and two PE-containing wild-type controls (PE + PC- strains) were recruited to investigate the influence of PE deficiency on bacterial adhesion. We found that PE deficiency could impair E. coli adhesion on macrophages (human THP-1-derived and mouse RAW264.7 macrophages) or glass coverslips by downregulating lipopolysaccharide (LPS) biosynthesis, which could be reversible by high galactose/lactose but not glucose cultivation. The data imply that PE play important role in bacterial adhesion probably via affecting LPS biosynthesis and suggest that targeting PE biosynthesis is also a potential antibacterial strategy.

Phosphatidylethanolamine deficiency in Mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology.

Mitochondrial dysfunction is implicated in neurodegenerative, cardiovascular, and metabolic disorders, but the role of phospholipids, particularly the nonbilayer-forming lipid phosphatidylethanolamine (PE), in mitochondrial function is poorly understood. Elimination of mitochondrial PE (mtPE) synthesis via phosphatidylserine decarboxylase in mice profoundly alters mitochondrial morphology and is embryonic lethal (Steenbergen, R., Nanowski, T. S., Beigneux, A., Kulinski, A., Young, S. G., and Vance, J. E. (2005) J. Biol. Chem. 280, 40032-40040). We now report that moderate <30% depletion of mtPE alters mitochondrial morphology and function and impairs cell growth. Acute reduction of mtPE by RNAi silencing of phosphatidylserine decarboxylase and chronic reduction of mtPE in PSB-2 cells that have only 5% of normal phosphatidylserine synthesis decreased respiratory capacity, ATP production, and activities of electron transport chain complexes (C) I and CIV but not CV. Blue native-PAGE analysis revealed defects in the organization of CI and CIV into supercomplexes in PE-deficient mitochondria, correlated with reduced amounts of CI and CIV proteins. Thus, mtPE deficiency impairs formation and/or membrane integration of respiratory supercomplexes. Despite normal or increased levels of mitochondrial fusion proteins in mtPE-deficient cells, and no reduction in mitochondrial membrane potential, mitochondria were extensively fragmented, and mitochondrial ultrastructure was grossly aberrant. In general, chronic reduction of mtPE caused more pronounced mitochondrial defects than did acute mtPE depletion. The functional and morphological changes in PSB-2 cells were largely reversed by normalization of mtPE content by supplementation with lyso-PE, a mtPE precursor. These studies demonstrate that even a modest reduction of mtPE in mammalian cells profoundly alters mitochondrial functions.

Depletion of phosphatidylethanolamine--the major membrane phospholipid of Escherichia coli--depresses posttranslocational modification of alkaline phosphatase in the periplasm.

Unbalanced phospholipid composition due to depletion of the major phospholipid of Escherichia coli, phosphatidylethanolamine, was shown previously to significantly decrease the secretion and transcriptional expression of periplasmic alkaline phosphatase of this bacterium. The current work studies the effect of this unbalance on posttranslocational proteolytic modification of the enzyme, proceeding in the periplasm with the formation of its isoforms. It has been revealed that under phosphatidylethanolamine depletion the content of incompletely modified (intermediate) isoforms I and II increases. This increase does not depend on the level of enzyme synthesis and the mechanism of its regulation (expression of the chromosomal gene or the gene cloned in plasmid under the control of the endogenous promoter P(PHO) or exogenous promoter P(BAD)). Depression of posttranslocational modification of alkaline phosphatase in phosphatidylethanolamine-depleted cells is supposed to be a result of the lower efficiency of secretion of modifying proteinase (product of the iap gene) localized, like alkaline phosphatase, in the periplasm of Escherichia coli.

Keratoconjunctivitis sicca associated with meibomian secretion polar lipid abnormality.

OBJECTIVE: To determine whether an association between keratoconjunctivitis sicca (KCS) and meibomian gland lipids exists in patients with chronic blepharitis. METHODS: Meibomian gland lipids were collected from normal patients and those with chronic blepharitis. Some of the chronic blepharitis patients had an ocular surface abnormality with apparent aqueous deficiency similar to KCS. Lipids were separated by thin-layer chromatography and polar lipids were further separated by high-pressure liquid chromatography with detection by UV absorbance. Lipids were identified by retention time with comparison with standards and by gas chromatography and mass spectroscopy. RESULTS: A strong association between specific lipids and KCS signs was observed only with the polar lipids. Low levels of 2 phospholipids, identified as phosphatidylethanolamine and sphingomyelin, were significantly (P < .05) associated with ocular surface abnormalities that were consistent with KCS. CONCLUSIONS: Evaporative KCS syndrome (rather than tear insufficiency) in many individuals may be the result of polar lipid abnormalities. We believe that the 2 associated phospholipids identified in the patients with chronic blepharitis act as important structural components in the polar phase of the tear film lipid layer. We suggest that a deficiency in these lipids results in a poorly structured polar phase that in turn affects the nonpolar phase. Ultimately water transmission through the tear film lipid layer increases, thus resulting in evaporative KCS. These results should aid in development of tear film substitutes directed toward specific abnormalities.

Abnormal function of protein kinase C in cells having phosphatidylethanolamine-deficient and phosphatidylcholine-excess membranes.

When rat mammary carcinoma 64-24 cells are grown in the absence of ethanolamine, their membrane phospholipid composition changes significantly, becoming phosphatidylethanolamine-deficient and phosphatidylcholine-excess due to a reduced de novo rate of phosphatidylethanolamine synthesis, and growth stops. We have assumed that this membrane phospholipid environment is not suitable for membrane-associated functions. We have previously demonstrated that functions normally stimulated by tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, are not stimulated in ethanolamine-deprived cells, suggesting that function of protein kinase C may be abnormal under the altered membrane environment. In the present study, the behavior of protein kinase C in 64-24 cells grown in the presence and absence of ethanolamine (having normal and phosphatidylethanolamine-deficient/phosphatidylcholine-excess phospholipid) was compared by enzyme assay as well as Western blotting. The results show that the nature of association of protein kinase C to the membrane, which is induced by phorbol ester, is abnormal when cells have the altered membrane phospholipid, and thus argue that membrane phospholipid environment is important in the function of protein kinase C.

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.

Binding of epidermal growth factor to its receptor is affected by membrane phospholipid environment.

Cells of epithelial origin generally require ethanolamine to grow in culture; when these cells are grown without ethanolamine, the phosphatidylethanolamine content of their membrane phospholipid becomes 1/2 to 1/3 of the normal amount, and growth stops. We have hypothesized that growth ceases because the phospholipid environment becomes unsuitable for membrane-associated function. Using ethanolamine-requiring rat mammary cells, we have investigated the possible effect of phosphatidylethanolamine deficiency on the binding characteristics of epidermal growth factor. Apparent dissociation constant for the high-affinity sites in cells having normal membrane phospholipid was 1.7 X 10(-10) M, whereas that of phosphatidylethanolamine-deficient cells was 2.7 X 10(-10) M: the difference was small, but significant. Pretreatment with phorbol ester caused the loss of high-affinity sites in cells having normal membrane, whereas binding characteristics of epidermal growth factor became refractory to the pretreatment in phosphatidylethanolamine-deficient cells. In addition, the rate of internalization of bound epidermal growth factor in phosphatidylethanolamine-deficient cells was about 1/4 of normal cells. Further, whether cells had normal or phosphatidylethanolamine-deficient membranes seemed to affect the phosphorylation patterns of membrane proteins in response to epidermal growth factor or phorbol ester. These results suggest that membrane phospholipid environment affects the activity of the epidermal growth factor receptor.