Novobiocin inhibits membrane synthesis and vacuole formation of Enterococcus faecalis protoplasts

We demonstrate that plasma membrane biosynthesis and vacuole formation require DNA replication in Enterococcus faecalis protoplasts. The replication inhibitor novobiocin inhibited not only DNA replication but also cell enlargement (plasma membrane biosynthesis) and vacuole formation during the enlargement of the E. faecalis protoplasts. After novobiocin treatment prior to vacuole formation, the cell size of E. faecalis protoplasts was limited to 6 μm in diameter and the cells lacked vacuoles. When novobiocin was added after vacuole formation, E. faecalis protoplasts grew with vacuole enlargement; after novobiocin removal, protoplasts were enlarged again. Although cell size distribution of the protoplasts was similar following the 24 h and 48 h novobiocin treatments, after 72 h of novobiocin treatment there was a greater number of smaller sized protoplasts, suggesting that extended novobiocin treatment may inhibit the re-enlargement of E. faecalis protoplasts after novobiocin removal. Our findings demonstrate that novobiocin can control the enlargement of E. faecalis protoplasts due to inhibition of DNA replication.

Shared and Distinctive Ultrastructural Abnormalities Expressed By Megakaryocytes in Bone Marrow and Spleen from Patients with Primary Myelofibrosis

Several evidences indicate that abnormal megakaryocyte (MK) maturation drives the development of Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative neoplasms: a) presence in bone marrow (BM) of numerous clusters of MK with ultrastructural abnormalities indicating retarded maturation was the first cell hallmark reported for MF (Zucker-Franklin, Dahlem Workshop on Myelofibrosis-Osteosclerosis Syndrome Germany-West: Pergammon Press; 1975); b) studies in mouse models with MK-restricted JAK2V617F expression demonstrated that abnormal MK are necessary and sufficient to induce MF (Zhan, Leukemia 2016;30:23324) and c) both in patients and mouse models, the driver mutations induce a RSP14 ribosomopathy that reduces expression of GATA1 in MK, leading to retarded maturation (Vannucchi, AmJPathol 2005;167:849; Gilles, JCI 2017;127:1316). Due to fibrosis and hematopoietic failure in BM, MF patients develop hematopoiesis in spleen which also is rich of MK. Whether in MF MK in BM and spleen express similar ultrastructural abnormalities has not been investigated as yet. By transmission electron microscopy, we compared the ultrastructural features of MK from BM of 5 MF patients and spleen of 11 MF patients (9 JAK2V617Fhomo, 1 JAK2V617Fhomo/ASXl1R1068X, 2 JAK2V617Fhetero, 1 JAK2V617F hetero/ASXl1R693X, 1 CARLtyp1/TET2R1214W and 2 triple negative; 12 primary and 4 post-PV, IPSS score: low-intermedium 2, DIPSS score: intermedium 1-2). MF underwent splenectomy to reduce discomfort of large spleen size (>20 cm from the left costal margin). BM from 2 healthy controls (HC) and post-trauma spleen from 1 HC were analyzed as control. MK were numerous both in BM (43±5 vs 30±2/area in MF vs HC, p=0.027, Anova) and spleen (47±18 in MF vs below detection in HC). The majority (98%) of MK from MF BM had immature ultrastructural morphology while 59% of MK from HC BM were mature (p=0.00003). To a surprise, the majority (69%) of MK from MF spleen had also mature morphology with developed platelet territories. However, by contrast with the heavy electron dense granules found in the cytoplasm of HC MK, the cytoplasm of MK from both BM (56%) and spleen (32%) contained granules with light electron density, an indication of reduced protein content. MK from MF BM and spleen consistently expressed distinctive ultrastructural abnormalities: 100% of BM MK presented dilated nuclear membranes with abnormally large nuclear pores which in 33% of the cases were associated with thickening of the plasma membrane with extrusion of pseudopods and presence of glycogen droplets. By contrast, these abnormalities were not presented by spleen MK, 49% of which presented instead mitochondria with reduced crests. These results suggest that MK from BM and spleen express distinctive metabolic abnormalities: those from BM express insufficient lipid metabolism impairing membrane biosynthesis while those from spleen have impaired aerobic metabolism. As predicted by the impaired RSP14 signature reported in MF (Gilles, JCI 2017;127:1316), the cytoplasm of MK from MF BM contained reduced number of ribosomes (455±125 vs 1059±41 in MF vs HC, p=0.004) but the percent of ribosomes engaging with the rough endoplasmic reticulum (RER) was the same in both cases (41±2 vs 46±10%, p=0.80). The number of ribosomes was lower also in MK from spleen (157±85, p=0.0003 ) but in addition, MK from the spleen were poorly engaged with the RER (33±22%, p=0.008). These results indicate that in addition to low number of ribosomes resulting in defective mRNA translation, MK from spleen express ribosomes that engage poorly with RER leading to defective folding check and post-translational modification of nascent proteins. These results predict that partially different mechanisms drive the alterations of platelet territories observed in MK from BM and spleen in MF: defective membrane biosynthesis drives the paucity of the granules and the retarded maturation of the platelet territories observed in MK from BM. Insufficient proteins synthesis drives the poor protein content of granules found in MK both from BM and spleen but in addition the function of the proteins in the granules of MK from spleen may be impaired by improper protein folding check/post-translational modification. These results suggests lipid metabolism and RER functions as two potential novel therapeutic targets to restore MK function in MF. Disclosures Vannucchi: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

1827. Genetic Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates Associated with the Development of Reduced Susceptibility to Vancomycin from Latin America

Background Vancomycin (VAN) is a first-line therapeutic option for severe MRSA infections, especially in Latin America where other options are limited. However, reduced susceptibility to VAN may lead to therapeutic failures. The molecular mechanisms leading the development of VAN-intermediate S. aureus (VISA) and heterogeneous-VISA (hVISA) phenotypes are still unclear. We explored genetic signatures associated with hVISA phenotype in MRSA isolates recovered from bacteremic patients in 9 Latin American countries (2011–2014) in order to develop a genomic platform to identify these isolates. Methods From 538 VAN-susceptible MRSA (MIC90 = 1 µg/mL) we identified 30 hVISA isolates using GRD and macromethod E-tests; from these, 3 were confirmed by PAP-AUC. Whole-genome sequencing was performed in all 30 isolates using Illumina platform. Based on previous studies, we selected 46 genes involved in hVISA development. Multiple Blast alignments were performed using genomes of ATCC29213 and N315 (VAN-susceptible), Mu3 (hVISA) and Mu50 (VISA) as references. Results A total of 130 changes in 46 predicted proteins belonging to 8 functional categories were determined: 48 changes related to cell wall biogenesis, 22 to DNA/RNA processing, 17 to regulatory systems, 12 to cofactors and enzymes, 11 to membrane biosynthesis, 9 to virulence, 6 to amino acid metabolism, and 5 to transport of nitrogen and putrescine/spermidine. The most common changes identified in all the hVISA were: Y38H in Atl, N16S in PBP4, S160A in RpoB, L14I in WalK and E156G in YvqF, compared with VSSA strains. The proteins with the highest number of changes detected in the isolates confirmed by PAP-AUC were: CapP, DltA, Pbp4, TcaA, LytM (Cell wall biogenesis); MutL, RpoB (DNA/RNA processing); GraS (Regulatory systems); and PgsA (Membrane biosynthesis). Conclusion Changes in genes associated with cell wall biogenesis, DNA/RNA processing, regulatory systems, and membrane biosynthesis were the most prevalent in Latin American hVISA strains. Genetic signatures in genes encoding GraR (N197S), RpoB (H481Y, H481N), VraS (I5N), WalK (L14F, R222K) and MrsR (E146K) are potentially associated with this phenotype. These changes could be used to develop a platform for possible identification of hVISA isolates. Disclosures All Authors: No reported Disclosures.

Choline: Exploring the Growing Science on Its Benefits for Moms and Babies

The importance of ensuring adequate choline intakes during pregnancy is increasingly recognized. Choline is critical for a number of physiological processes during the prenatal period with roles in membrane biosynthesis and tissue expansion, neurotransmission and brain development, and methyl group donation and gene expression. Studies in animals and humans have shown that supplementing the maternal diet with additional choline improves several pregnancy outcomes and protects against certain neural and metabolic insults. Most pregnant women in the U.S. are not achieving choline intake recommendations of 450 mg/day and would likely benefit from boosting their choline intakes through dietary and/or supplemental approaches.

Membrane Remodeling by a Bacterial Phospholipid-Methylating Enzyme

ABSTRACT Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a membrane-deforming activity of the phospholipid N-methyltransferase PmtA from the plant-pathogenic bacterium Agrobacterium tumefaciens. PmtA catalyzes the successive three-step N-methylation of phosphatidylethanolamine to phosphatidylcholine. Here, we defined the lipid and protein requirements for the membrane-remodeling activity of PmtA by a combination of transmission electron microscopy and liposome interaction studies. Dependent on the lipid composition, PmtA changes the shape of spherical liposomes either into filaments or small vesicles. Upon overproduction of PmtA in A. tumefaciens, vesicle-like structures occur in the cytoplasm, dependent on the presence of the anionic lipid cardiolipin. The N-terminal lipid-binding α-helix (αA) is involved in membrane deformation by PmtA. Two functionally distinct and spatially separated regions in αA can be distinguished. Anionic interactions by positively charged amino acids on one face of the helix are responsible for membrane recruitment of the enzyme. The opposite hydrophobic face of the helix is required for membrane remodeling, presumably by shallow insertion into the lipid bilayer. IMPORTANCE The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid N-methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of in vivo and in vitro methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes. IMPORTANCE The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid N-methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of in vivo and in vitro methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes.

The Fascio Effect: Biotin and Sleep

<p class="1Body">An Italian maxim says “I bambini che dormono tanto crescono bene”, and the English maxim also says that “All children need to sleep in order to grow and develop”. In this review, we would like to demonstrate the truth of this proverb about sleep together with biotin (vitamin H) and lipoic acid (thioctic acid). Recently, we have found that D-biotin regulates the membrane biosynthesis in the cells. Previously, we have found that purified biotin-recycling enzyme biotinidase is also able to hydrolyze enkephalin and neuro-peptides. Thus, biotin state and sleep should be strongly linked to each other. In this report, we have investigated the expression of hormone receptors in some diseases using the new proteomics of protein-direct-microsequencing-deciphering (PDMD) method, and have indicated that having adequate sleep and nutrition is important to for healthy immunological and mental conditions in humans. It is also summarized that human breast milk is superior to bovine milk for human healthy babies to sleep and grow well.</p>

Reduced expression of CDP-DAG synthase changes lipid composition and leads to male sterility in Drosophila

Drosophila spermatogenesis is an ideal system to study the effects of changes in lipid composition, because spermatid elongation and individualization requires extensive membrane biosynthesis and remodelling. The bulk of transcriptional activity is completed with the entry of cysts into meiotic division, which makes post-meiotic stages of spermatogenesis very sensitive to even a small reduction in gene products. In this study, we describe the effect of changes in lipid composition during spermatogenesis using a hypomorphic male sterile allele of the Drosophila CDP-DAG synthase ( CdsA ) gene. We find that the CdsA mutant shows defects in spermatid individualization and enlargement of mitochondria and the axonemal sheath of the spermatids. Furthermore, we could genetically rescue the male sterile phenotype by overexpressing Phosphatidylinositol synthase (dPIS) in a CdsA mutant background. The results of lipidomic and genetic analyses of the CdsA mutant highlight the importance of correct lipid composition during sperm development and show that phosphatidic acid levels are crucial in late stages of spermatogenesis.