Draft Genome of Proteus mirabilis Serogroup O18 Elaborating Phosphocholine-Decorated O Antigen

Proteus mirabilis is a pathogenic, Gram-negative, rod-shaped bacterium that causes ascending urinary tract infections. Swarming motility, urease production, biofilm formation, and the properties of its lipopolysaccharide (LPS) are all factors that contribute to the virulence of this bacterium. Uniquely, members of the O18 serogroup elaborate LPS molecules capped with O antigen polymers built of pentasaccharide repeats; these repeats are modified with a phosphocholine (ChoP) moiety attached to the proximal sugar of each O unit. Decoration of the LPS with ChoP is an important surface modification of many pathogenic and commensal bacteria. The presence of ChoP on the bacterial envelope is correlated with pathogenicity, as decoration with ChoP plays a role in bacterial adhesion to mucosal surfaces, resistance to antimicrobial peptides and sensitivity to complement-mediated killing in several species. The genome of P. mirabilis O18 is 3.98 Mb in size, containing 3,762 protein-coding sequences and an overall GC content of 38.7%. Annotation performed using the RAST Annotation Server revealed genes associated with choline phosphorylation, uptake and transfer. Moreover, amino acid sequence alignment of the translated licC gene revealed it to be homologous to LicC from Streptococcus pneumoniae encoding CTP:phosphocholine cytidylyltransferase. Recognized homologs are located in the O antigen gene clusters of Proteus species, near the wzx gene encoding the O antigen flippase, which translocates lipid-linked O units across the inner membrane. This study reveals the genes potentially engaged in LPS decoration with ChoP in P. mirabilis O18.

Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.

Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40–50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.

Differential dephosphorylation of CTP:phosphocholine cytidylyltransferase upon translocation to nuclear membranes and lipid droplets

The translocation of CCTα, the rate-limiting enzyme for phosphatidylcholine synthesis, to nuclear membranes and nuclear lipid droplets results in reversible dephosphorylation of S319 and sustained phosphorylation of Y359+S362. Independent regulation of these phosphosites in the P-domain of CCTα is required for activation on nuclear membranes.

Lipid-associated PML domains regulate CCTα, Lipin1 and lipid homeostasis

ABSTRACTNuclear LDs (nLDs) originate at the inner nuclear membrane by a mechanism that involves the promyelocytic leukemia (PML) protein. Here we demonstrate that nLDs in oleate-treated U2OS cells are associated with Lipid-Associated PML (LAP) domains that differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100 and DAXX. nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1 and diacylglycerol (DAG). High resolution imaging revealed that LAP domains and CCTα occupy distinct polarized regions on nLDs, and that loss of LAP domains in PML knockout U2OS cells reduced the recruitment of CCTα onto nLDs by its amphipathic α-helical M-domain. The association of Lipin1 and DAG with nLDs was also LAP domain-dependent. The disruption of CCTα and Lipin1 localization on nLDs in PML knockout cells resulted in the inhibition of phosphatidylcholine and triacylglycerol synthesis indicating that LAP domains are a unique PML subdomain involved in nLD assembly and regulation of lipid metabolism.