Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria.

The outer membrane (OM) of Gram-negative bacteria provides the cell with a formidable barrier that excludes external threats. The two major constituents of this asymmetric barrier are lipopolysaccharide (LPS) found in the outer leaflet, and glycerophospholipids (GPLs) in the inner leaflet. Maintaining the asymmetric nature and balance of LPS to GPLs in the OM is critical for bacterial viability. The biosynthetic pathways of LPS and GPLs are well characterized, but unlike LPS transport, how GPLs are translocated to the OM remains enigmatic. Understanding this aspect of cell envelope biology could provide a foundation for new antibacterial therapies. Here, we report that YhdP and its homologues, TamB and YdbH, members of the "AsmA-like" family, are critical for OM integrity and necessary for proper GPL transport to the OM. The absence of the two largest AsmA-like proteins (YhdP and TamB) leads to cell lysis and antibiotic sensitivity, phenotypes that are rescued by reducing LPS synthesis. We also find that yhdP, tamB double mutants shed excess LPS through outer membrane vesicles, presumably to maintain OM homeostasis when normal anterograde GPL transport is disrupted. Moreover, a yhdP, tamB, ydbH triple mutant is synthetically lethal, but if GPL transport is partially restored by overexpression of YhdP, the cell shape adjusts to accommodate increased membrane content as the cell accumulates GPLs in the IM. Our results therefore suggest a model in which "AsmA-like" proteins transport GPLs to the OM, and when hindered, changes in cell shape and shedding of excess LPS aids in maintaining OM asymmetry.

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis.

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

A Whole-Cell Screen Identifies Small Bioactives That Synergize with Polymyxin and Exhibit Antimicrobial Activities against Multidrug-Resistant Bacteria.

The threat of diminished antibiotic discovery has global health care in crisis. In the United States, it is estimated each year that over 2 million bacterial infections are resistant to first-line antibiotic treatments and cost in excess of 20 billion dollars. Many of these cases result from infection with the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are multidrug-resistant bacteria that often cause community- and hospital-acquired infections in both healthy and immunocompromised patients. Physicians have turned to last-resort antibiotics like polymyxins to tackle these pathogens, and as a consequence, polymyxin resistance has emerged and is spreading. Barring the discovery of new antibiotics, another route to successfully mitigate polymyxin resistance is to identify compounds that can complement the existing arsenal of antibiotics. We recently designed and performed a large-scale robotic screen to identify 43 bioactive compounds that act synergistically with polymyxin B to inhibit the growth of polymyxin-resistant Escherichia coli Of these 43 compounds, 5 lead compounds were identified and characterized using various Gram-negative bacterial organisms to better assess their synergistic activity with polymyxin. Several of these compounds reduce polymyxin to an MIC of <2 µg/ml against polymyxin-resistant and polymyxin-heteroresistant Gram-negative pathogens. Likewise, four of these compounds exhibit antimicrobial activity against Gram-positive bacteria, one of which rapidly eradicated methicillin-resistant Staphylococcus aureus We present multiple first-generation (i.e., not yet optimized) compounds that warrant further investigation and optimization, since they can act both synergistically with polymyxin and also as lone antimicrobials for combating ESKAPE pathogens.

Briareolate esters from the gorgonian Briareum asbestinum.

Two new briarane diterpenoids briareolate esters J (1) and K (2) were isolated from the methanolic extract of the octocoral Briareum asbestinum collected off the coast of Boca Raton, Florida. The structures of briaranes 1 and 2 were elucidated by interpretation of spectroscopic data. Briareolate ester K (2) showed weak growth inhibition activity against human embryonic stem cells (BG02).

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Bicyclic C21 terpenoids from the marine sponge Clathria compressa.

Three new bicyclic C(21) terpenoids, clathric acid (1) and two N-acyl taurine derivatives, clathrimides A (2) and B (3), were isolated from the marine sponge Clathria compressa. The structures of these compounds were elucidated by interpretation of spectroscopic data. Clathric acid showed mild antibacterial activity against several Gram-positive bacteria.

Bioactive diterpenoid containing a reversible "spring-loaded" (E,Z)-dieneone Michael acceptor.

Three new briarane diterpenoids, briareolate esters L-N (1-3), have been isolated from a gorgonian Briareum asbestinum. Briareolate esters L (1) and M (2) are the first natural products possessing a 10-membered macrocyclic ring with a (E,Z)-dieneone and exhibit growth inhibition activity against both human embryonic stem cells (BG02) and a pancreatic cancer cell line (BxPC-3). Briareolate ester L (1) was found to contain a "spring-loaded" (E,Z)-dieneone Michael acceptor group that can form a reversible covalent bond to model sulfur-based nucleophiles.

Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor beta family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, approximately 70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions.