Cells store metabolic energy in the form of neutral lipids that are deposited within lipid droplets (LDs). In this study, we examine the biogenesis of LDs and the transport of integral membrane proteins from the endoplasmic reticulum (ER) to newly formed LDs. In cells that lack LDs, otherwise LD-localized membrane proteins are homogenously distributed in the ER membrane. Under these conditions, transcriptional induction of a diacylglycerol acyltransferase that catalyzes the formation of the storage lipid triacylglycerol (TAG), Lro1, is sufficient to drive LD formation. Newly formed LDs originate from the ER membrane where they become decorated by marker proteins. Induction of LDs by expression of the second TAG-synthesizing integral membrane protein, Dga1, reveals that Dga1 itself moves from the ER membrane to concentrate on LDs. Photobleaching experiments (FRAP) indicate that relocation of membrane proteins from the ER to LDs is independent of temperature and energy, and thus not mediated by classical vesicular transport routes. LD-localized membrane proteins are homogenously distributed at the perimeter of LDs, they are free to move over the LD surface and can even relocate back into the ER, indicating that they are not restricted to specialized sites on LDs. These observations indicate that LDs are functionally connected to the ER membrane and that this connection allows the efficient partitioning of membrane proteins between the two compartments.

The opportunistic human pathogen Pseudomonas aeruginosa is the causal agent of a wide variety of infections. This non-fermentative Gram-negative bacillus can colonize zones where the skin barrier is weakened, such as wounds or burns. It also causes infections of the urinary tract, respiratory system or bloodstream. P. aeruginosa infections are common in hospitalized patients for which multidrug-resistant, respectively extensively drug-resistant isolates can be a strong contributor to a high rate of in-hospital mortality. Moreover, chronic respiratory system infections of cystic fibrosis patients are especially concerning, since very tedious to treat. P. aeruginosa exploits diverse cell-associated and secreted virulence factors, which play essential roles in its pathogenesis. Those factors encompass carbohydrate-binding proteins, quorum sensing that monitor the production of extracellular products, genes conferring extensive drug resistance, and a secretion system to deliver effectors to kill competitors or subvert host essential functions. In this article, we highlight recent advances in the understanding of P. aeruginosa pathogenicity and virulence as well as efforts for the identification of new drug targets and the development of new therapeutic strategies against P. aeruginosa infections. These recent advances provide innovative and promising strategies to circumvent infection caused by this important human pathogen.

Most cells store neutral lipids in a dedicated compartment, the lipid droplet (LD). These LDs are structurally and functionally conserved across species. In higher eukaryotes, LDs are covered by abundant scaffolding proteins, such as the oleosins in plants and perilipins (PLINs) in animal cells. S. cerevisiae, however, has no homologues of these scaffolding proteins. To analyze a possible function of these proteins in the biogenesis of LDs, oleosin and perilipin family members (PLIN1, ADRP/PLIN2, and TIP47/PLIN3) were expressed in yeast cells and their targeting to LDs, membrane association and function in neutral lipid homeostasis and LD biogenesis were analyzed. When expressed in wild-type cells, these proteins were properly targeted to LDs. However, when expressed in cells lacking LDs, oleosin was localized to the ER bilayer and was rapidly degraded. PLINs, on the other hand, did not localize to the ER membrane in the absence of LDs and lost their membrane association. Photobleaching experiments revealed that PLIN2 and PLIN3 rapidly exchanged their LD association but PLINs did not move over the LD surface as quickly as did an integral membrane protein, such as oleosin. Interestingly, expression of these scaffolding LD proteins in mutant cells containing elevated levels of neutral lipids within the ER bilayer resulted in the formation of LDs. These results suggest that these LD scaffolding proteins promote the sequestration of neutral lipids from the ER bilayer and thereby induce LD formation. Consistent with this proposition, addition of a cell permeable diacylglycerol (DAG) was sufficient to promote LD formation in cells expressing the LD scaffolding proteins but lacking the capacity to synthesize storage lipids.

Members of the Chlamydiales order are major bacterial pathogens that divide at mid-cell, without a sequence homologue of the FtsZ cytokinetic tubulin and without a classical peptidoglycan cell wall. Moreover, the spatiotemporal mechanisms directing constriction in Chlamydia are not known. Here we show that the MreB actin homologue and its conserved regulator RodZ localize to the division furrow in Waddlia chondrophila, a member of the Chlamydiales order implicated in human miscarriage. RodZ is recruited to the septal site earlier than MreB and in a manner that depends on biosynthesis of the peptidoglycan precursor lipid II by the MurA enzyme. By contrast, crosslinking of lipid II peptides by the Pbp3 transpeptidase disperses RodZ from the septum. Altogether, these findings provide a cytological framework for understanding chlamydial cytokinesis driven by septal cell wall synthesis.

Chlamydiales are obligate intracellular bacteria including some important pathogens causing trachoma, genital tract infections and pneumonia, among others. They share an atypical division mechanism, which is independent of an FtsZ homologue. However, they divide by binary fission, in a process inhibited by penicillin derivatives, causing the formation of an aberrant form of the bacteria, which is able to survive in the presence of the antibiotic. The paradox of penicillin sensitivity of chlamydial cells in the absence of detectable peptidoglycan (PG) was dubbed the chlamydial anomaly, since no PG modified by enzymes (Pbps) that are the usual target of penicillin could be detected in Chlamydiales. We review here the recent advances in this field with the first direct and indirect evidences of PG-like material in both Chlamydiaceae and Chlamydia-related bacteria. Moreover, PG biosynthesis is required for proper localization of the newly described septal proteins RodZ and NlpD. Taken together, these new results set the stage for a better understanding of the role of PG and septal proteins in the division mechanism of Chlamydiales and illuminate the long-standing chlamydial anomaly. Moreover, understanding the chlamydial division mechanism is critical for the development of new antibiotics for the treatment of chlamydial chronic infections.