Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants.

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

Anti­glycolipid disorder effect of epigallocatechin­3­gallate on high­fat diet and STZ­induced T2DM in mice.

Epigallocatechin-3-gallate (EGCG) is beneficial for inhibiting dyslipidemia and reducing hyperlipidemic risk. The purpose of the present study was to investigate the glycolipid regulatory effects and potential mechanisms of EGCG in a high­fat diet and streptozotocin­induced type 2 diabetes mellitus (T2DM) mouse model. The results demonstrated that EGCG can decrease blood glucose levels and increase insulin resistance in T2DM mice. In addition, EGCG can regulate serum lipid levels, including those of total cholesterol, triglyceride and low­density lipoprotein receptor (LDL­r), and reduce lipid deposition in vascular endothelial cells in a dose­dependent manner. In addition, the gene and protein expression of related scavenger receptors, including cluster of differentiation 36, sterol regulatory element binding protein 2 (SREBP), SREBP cleavage­activating protein and LDL­r, were downregulated in a dose­dependent manner. The present study noted that EGCG possesses potential as a natural product for preventing and treating metabolic hyperlipidemia syndrome, probably by reducing the blood lipid levels, alleviating vascular endothelial cell damage, maintaining normal lipid metabolism in blood vessels and ameliorating glycolipid disorders.

Absence of lactobacilli containing glycolipids with the α-galactose epitope and the enhanced fucosylation of a receptor glycolipid GA1 in the digestive tracts of immune-deficient scid mice.

The Lactobacillus species in the digestive tracts of immune-deficient scid mice was distinct from that in control mice, i.e. Lactobacillus murinus in scid and L. johnsonii in control mice, according to their 16S-rRNA, indicating that a symbiotic relationship between lactobacilli and a host is established under pressure from the immune system. The caecal and colonal contents rich in L. murinus of scid mice were loose with a strong sour smell, resulting in diarrhoea, and those with L. johnsonii in control mice included abundant solid materials. Lactobacillus glycolipids were revealed to be recognized by the immune system, and by TLC-immunostaining, LacTetH-DG (Galα1-6Galα1-6Galα1-2Glcα1-3'DG) of L. johnsonii was detected in the stomach, caecum and colon of control mice, but not in those of scid ones, in which fucosylation of a receptor GA1 for L. johnsonii was enhanced more than 4-fold compared with in the control mice. Thus, structural modification of receptor glycolipids was revealed to occur in the process of establishment of a symbiotic relationship between lactobacilli and a host. LacTetH-DG was also immunogenic to human, because of the presence of natural antibodies against it, and the antibody binding to it was comparable to that of blood group- and species-related glycosphingolipids.

A new class of plant lipid is essential for protection against phosphorus depletion.

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.

Milk fat globule-epidermal growth factor-factor 8 attenuates neutrophil infiltration in acute lung injury via modulation of CXCR2.

Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factor-factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8(-/-) mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8(-/-) mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-α, MIP-2, and myeloperoxidase. An increased number of apoptotic cells was trapped into the lungs of Mfge8(-/-) mice compared with WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2-mediated chemotaxis revealed higher migration of neutrophils of Mfge8(-/-) mice than those of WT mice via increased surface exposures to CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-α(v) integrin Abs. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.

A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

Abnormal morphology of Bacillus subtilis ugtP mutant cells lacking glucolipids.

Bacillus subtilis Marburg 168 cells with disrupted ugtP, which encodes UDP-glucosyltransferase involved in glucolipid synthesis, were bent and distended. In the ugtP mutant cells, the extracytoplasmic function sigmas SigM, SigV and SigX, were found to be activated. Introduction of a disrupted allele of sigM into the ugtP strain caused even more abnormal morphology, with cells taking on a balloon-like shape; growth of these cells in LB medium was hampered by addition of 1.5% NaCl. Addition of MgSO4 or MnCl2 suppressed the abnormal morphology. In ugtP mutant cells the transcription of the mreB operon from an upstream promoter in maf (designated Pupstream mreB) and PmreBH was 4.3- and 2.3-fold higher, respectively, and localization of GFP-MreB was not in discrete dots (in an apparently helical pattern), but faint and in irregular clusters. GFP-MreB protein was reduced in the ugtP mutant cells. We suggest that glucolipids are important for MreB isoforms to take on the configuration that appears as discrete dots and plays a role in shaping cells into straight rods.

Biological roles of sulfoglycolipids and pathophysiology of their deficiency.

Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is expressed on the spermatogenic cells. Cerebroside sulfotransferase (CST)-deficient mice generated by gene targeting completely lack sulfatide and seminolipid all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis, indicating that sulfation of glycolipids is essential for myelin formation and spermatogenesis. Moreover, CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction, an experimental model of renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms by which sulfoglycolipids participate in these biological processes are ongoing.

Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria.

Sulfoquinovosyl diacylglycerol (SQDG) is involved in the maintenance of photosystem II (PSII) activity in Chlamydomonas reinhardtii[Minoda, A., Sato, N., Nozaki, H., Okada, K., Takahashi, H., Sonoike, K. & Tsuzuki, M. et al. (2002) Eur. J. Biochem.269, 2353-2358]. To understand the spread of the taxa in which PSII interacts with SQDG, especially in cyanobacteria, we produced a mutant defective in the putative sqdB gene responsible for SQDG synthesis from two cyanobacteria, Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942. The mutant of PCC6803, designated SD1, lacked SQDG synthetic ability and required SQDG supplementation for its growth. After transfer from SQDG-supplemented to SQDG-free conditions, SD1 showed decreased net photosynthetic and PSII activities on a chlorophyll (Chl) basis with a decrease in the SQDG content. Moreover, the sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine was increased in SD1. However, SD1 maintained normal amounts of cytochrome b559 and D1 protein (the subunits comprising the PSII complex) on a Chl basis, indicating that the PSII complex content changed little, irrespective of a decrease in the SQDG content. These results suggest that the role of SQDG is the conservation of the PSII properties in PCC6803, consistent with the results obtained with C. reinhardtii. In contrast, the SQDG-null mutant of PCC7942 showed the normal level of PSII activity with little effect on its sensitivity to PSII herbicides. Therefore, the difference in the SQDG requirement for PSII is species-specific in cyanobacteria; this could be of use when investigating the molecular evolution of the PSII complex.

Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues.

Myelin-associated glycoprotein and myelin galactolipids stabilize developing axo-glial interactions.

We have analyzed mice that lack both the myelin-associated glycoprotein (MAG) and the myelin galactolipids, two glial components implicated in mediating axo-glial interactions during the myelination process. The single-mutant mice produce abnormal myelin containing similar ultrastructural abnormalities, suggesting that these molecules may play an overlapping role in myelin formation. Furthermore, the absence of the galactolipids results in a disruption in paranodal axo-glial interactions, and we show here that similar, albeit less severe, abnormalities exist in the developing MAG mutant. In the double-mutant mice, maintenance of axo-glial adhesion is significantly more affected than in the single mutants, supporting the overlapping function hypothesis. We also show that independently of MAG, galactolipids, and paranodal junctional components, immature nodes of Ranvier form normally, but rapidly destabilize in their absence. These data indicate that distinct molecular mechanisms are responsible for the formation and maintenance of axo-glial interactions.

Effects of galactolipid elimination on oligodendrocyte development and myelination.

The galactolipids galactocerebroside and sulfatide, which require the enzyme UDP-galactose:ceramide galactosyltransferase (CGT) for their synthesis, are among the most prevalent molecules in the myelin sheath. Numerous studies, mainly using antibody perturbation methods in vitro, have suggested that these molecules are crucial mediators of oligodendrocyte differentiation and myelin formation. Although we have previously demonstrated that myelin formation occurs in CGT null mutant mice, which are incapable of synthesizing the myelin galactolipids, here we show that there are developmental alterations in the CNS of these animals. There is a significant decrease in the number of myelinated axon segments in the mutant spinal cord despite normal levels of myelin gene-specific mRNAs and proteins. Also, there is an increased cellularity in the mature mutant spinal cord and the distinctive morphology of the additional cells suggests that they are actively myelinating oligodendrocytes. Using in situ hybridization techniques, we show that there is a 50% increase in the number of oligodendrocytes in the mutant spinal cord. The data suggest that galactolipids play an important developmental role in regulating the maturation program and final number of oligodendrocytes.

Axo-glial interactions regulate the localization of axonal paranodal proteins.

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.

Myelination in the absence of galactolipids and proteolipid proteins.

The galactolipids galactocerebroside and sulfatide and the proteolipid protein (PLP) and its splice variant DM20 are the most abundant lipid and protein components of central nervous system myelin. Recent studies have found that mice lacking either the galactolipids or PLP are able to form myelin sheaths with apparently normal periodicity and near normal compaction. Here, we have generated galactolipid/proteolipid double mutants to examine the possibility that these molecules have overlapping functions. We show that the absence of the galactolipids and PLP has pleotropic effects on myelin formation. While oligodendrocytes in the postnatal day 20 galactolipid/proteolipid-deficient mouse are able to elaborate myelin with close to normal intraperiod lines, there is an increased frequency of uncompacted myelin sheaths as well as unmyelinated axons. Moreover, the double mutants display extensive white matter vacuolization of the cerebellum that initiates around postnatal day 16, which correlates with the onset of a severe ataxic phenotype and an increased percentage of apoptotic nuclei in the cerebellar internal granule cell layer. These data indicate that the galactolipids and PLP/DM20 are not required for intraperiod line formation, but they suggest a role for these molecules in mediating myelin compaction and in maintaining the integrity of the cerebellum.

Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana.

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.

Effects of rumpshaker mutation on CNS myelin composition and structure.

Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X-ray diffraction measurements on unfixed optic nerves showed that the jprsh period is 5-10 A larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.

Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes.

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.

Biosynthesis and function of LFA-3 in human mutant cells deficient in phosphatidylinositol-anchored proteins.

Mutants that lack expression of phosphatidylinositol (PI)-anchored proteins were derived from the human B lymphoblastoid JY cell line. It was demonstrated that unlike wild-type cells, which normally express both a transmembrane and a PI-linked form of LFA-3 glycoprotein, the mutant cells expressed only the transmembrane form of LFA-3. [3H]Ethanolamine was not incorporated into LFA-3 of mutant cells, indicating that the anchor moiety was entirely missing. Blockade of normal biosynthesis of the PI-anchored form led to accumulation of two intermediates that may have intact and truncated polypeptide chains. The truncated LFA-3, which was not attached to the cell membrane, was secreted by mutant cells into culture supernatants. A possible division of adhesion function between the two forms of LFA-3 was studied by using the JY cell lines as targets for CTL. Wild-type and mutant JY cells formed conjugates with CTL and were subsequently lysed to a similar extent. In addition, wild-type and mutant JY cells stimulated CTL proliferation to the same extent. Antibody-blocking experiments demonstrated a predominant role for the CD2/LFA-3 pathway in interaction of both wild-type and mutant cells with CTL. Because E exclusively express only the PI-linked LFA-3 form, and this form is known to mediate cell adhesion, the present results indicate that the two distinct membrane-anchored LFA-3 forms are each capable of mediating adhesion. A possible division of signaling functions between the two forms of LFA-3 is under investigation.

Failure of expression of alpha-3-L-fucosyltransferase in human serum is coincident with the absence of the X (or Le(x)) antigen in the kidney but not on leucocytes.

The X antigen, beta Gal(1----4)[alpha Fuc(1----3)]beta GlcNAc-R, is mostly found in epithelial cells of the digestive tract, proximal convoluted tubules of the kidney, and granulocytes. The alpha-3-L-fucosyltransferase responsible for synthesis of this antigen is normally present in the serum, but we found 2 individuals, a mother and her daughter, who lack more than 90% of the serum fucosyltransferase. They are of African origin and are both Le(a-b-). The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, her mother and herself normally possess the X antigen on granulocytes and its sialylated form on monocytes. Anephric patients showed reduced serum alpha-3-L-fucosyltransferase activity, suggesting that the kidney contributes to an important fraction of serum enzymic activity. It, therefore, appears that there are distinct genetic controls governing expression of the X antigen in different body compartments. Possibly, different alpha-3-L-fucosyltransferases could be at work in kidney and leucocytes.