AbstractSphingoid bases, including sphingosine, are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria.Pseudomonas aeruginosais a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here, we determined that deletion of thesphBCDoperon reduced growth in the presence of sphingosine. Using deletion mutants, complementation, and growth assays inP. aeruginosaPAO1, we determined that thesphCandsphBgenes, encoding a periplasmic oxidase and periplasmic cytochrome c, respectively, were important for growth on sphingosine, whilesphDwas dispensable under these conditions. Deletion ofsphBCDinP. aeruginosaPA14,P. protegensPf-5, andP. fluorescensPf01 also showed reduced growth in the presence of sphingosine. TheP. aeruginosa sphBCgenes were also important for growth in the presence of two other sphingoid bases, phytosphingosine and sphinganine. In wild-typeP. aeruginosa, sphingosine is metabolized to an unknown non-inhibitory product, as sphingosine concentrations drop in the culture. However, in the absence ofsphBC, sphingosine accumulates, pointing to SphC and SphB as having a role in sphingosine metabolism. Finally, metabolism of sphingosine by wild-typeP. aeruginosaprotected susceptible cells from full growth inhibition by sphingosine, pointing to a role for sphingosine metabolism as a public good. This work shows that metabolism of sphingosine byP. aeruginosapresents a novel pathway by which bacteria can alter host-derived sphingolipids, but it remains an open question whether SphB and SphC act directly on sphingosine.

A common feature among nearly all Gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate. Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg 2+ as a cofactor. Mn 2+ , but not other divalent cations, could substitute for Mg 2+ . Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD-C6-ceramide (K m,app =19.2 ± 5.5 μM; V max,app =2586.29 ± 231.99 pmol/min/mg enzyme) and ATP (K m,app =0.29 ± 0.07 mM; V max,app =10067.57 ± 996.85 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

Summary Lipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se , underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Sphingolipids synthesis was thought to be rare in Gram-negative bacteria, previously only found in a handful of taxa. We recently discovered ceramides in Caulobacter crescentus and demonstrated that these lipids play an important role in antibiotic and phage sensitivity. However, the mechanism by which ceramides affect resistance to antimicrobials, as well as their effects on the integrity of the cell membrane are not yet clear. In this study, a coarse-grained molecular dynamics simulation of a prototypical bacterial outer membrane is used to observe changes in the conformation of the outer membrane lipids in the presence of ceramides. The outer membrane of a Gram-negative bacteria is asymmetric with an outer leaflet dominated by lipopolysaccharide (LPS). LPS is composed of three domains that extend from the outer membrane: a membrane-embedded lipid A, the attached core polysaccharide chain, and the O-antigen polysaccharide chain. Rough LPS consists of lipid A and core oligosaccharides only, while smooth LPS contains lipid A, core sugars, and O-antigen chains. Membranes were simulated with a one to one ratio of rough to smooth LPS, with ceramide concentrations ranging from ten to forty percent of total lipids. In order to understand the role of ceramide in outer membrane structure and function, this study considers their effects on the flexibility of O-antigen as well as the clustering and packing of LPS and membrane lipids.

Bacteria have a variety of mechanisms for adapting to environmental perturbations. Changes in oxygen availability result in a switch between aerobic and anaerobic respiration, whereas iron limitation may lead to siderophore secretion. In addition to metabolic adaptations, many organisms respond by altering their cell shape. Caulobacter crescentus, when grown under phosphate-limiting conditions, dramatically elongates its polar stalk appendage. The stalk is hypothesized to facilitate phosphate uptake; however, the mechanistic details of stalk synthesis are not well characterized. We used a chemical mutagenesis approach to isolate and characterize stalk-deficient mutants, one of which had two mutations in the phosphomannose isomerase gene (manA) that were necessary and sufficient to inhibit stalk elongation. Transcription of the pho regulon was unaffected in the manA mutant; therefore, ManA plays a unique regulatory role in stalk synthesis. The mutant ManA had reduced enzymatic activity, resulting in a 5-fold increase in the intracellular fructose 6-phosphate/mannose 6-phosphate ratio. This metabolic imbalance impaired the synthesis of cellular envelope components derived from mannose 6-phosphate, namely, lipopolysaccharide O-antigen and exopolysaccharide. Furthermore, the manA mutations prevented C. crescentus cells from efficiently entering stationary phase. Deletion of the stationary-phase response regulator gene spdR inhibited stalk elongation in wild-type cells, while overproduction of the alarmone ppGpp, which triggers growth arrest and stationary-phase entry, increased stalk length in the manA mutant strain. These results demonstrate that sugar-phosphate metabolism regulates stalk elongation independently of phosphate starvation.IMPORTANCE Metabolic control of bacterial cell shape is an important mechanism for adapting to environmental perturbations. Caulobacter crescentus dramatically elongates its polar stalk appendage in response to phosphate starvation. To investigate the mechanism of this morphological adaptation, we isolated stalk-deficient mutants, one of which had mutations in the phosphomannose isomerase gene (manA) that blocked stalk elongation, despite normal activation of the phosphate starvation response. The mutant ManA resulted in an imbalance in sugar-phosphate concentrations, which had effects on the synthesis of cellular envelope components and entry into stationary phase. Due to the interconnectivity of metabolic pathways, our findings may suggest more generally that the modulation of bacterial cell shape involves the regulation of growth phase and the synthesis of cellular building blocks.

The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeletons, few MreB interacting proteins have been well characterized. Here we identify a small protein from Caulobacter crescentus , AimB, as an Assembly Inhibitor of MreB. AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft. We validate this model through functional analysis of point mutants in both AimB and MreB, photo-crosslinking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings indicate that AimB promotes MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor.

ABSTRACT To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, we generated an sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila. The homozygous fly appears similar to wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, we quantified an average of 1100, 6200 and 2500 receptors per follicle cell (FC) at stages 8/9, 10 and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGFα-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, we followed the endosomal localization of EGFR in the FCs. The EGFR colocalized with the late endosome, but no significant colocalization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource for studying cellular localization and regulation of EGFR in tissues.

Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1-positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well-characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.

Molecular motors play important roles in force generation, migration, and intracellular trafficking. Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with clinical outcomes. We report here a new role for the KIF20A kinesin motor protein in intracellular mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe cortical mechanics, we first confirm that bladder urothelial cells soften with an increasing cancer grade. We then show that inhibiting KIF20A makes the intracellular environment softer for both high- and low-grade bladder cancer cells. Upon inhibition of KIF20A, cortical stiffness also decreases in lower grade cells, while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer.

The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall including muropeptide composition, glycan strand length, degree of crosslinking, type of crosslinking and other peptidoglycan modifications can be quantified using this approach. Well-established protocols provide us with highly-resolved and quantitatively reproducible chromatographic data, which can be used to investigate bacterial cell wall composition under a variety of environmental or genetic perturbations. The method described here enables the purification of muropeptide samples, their quantification by HPLC, and fraction collection for peak identification by mass spectrometry. Although the methods for peptidoglycan purification and HPLC analysis have been previously published, our method includes important details on how to re-equilibrate the column between runs to allow for automated analysis of multiple samples.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3',5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The rise of antibiotic-resistant bacteria is a rapidly growing global health concern. According to the Center for Disease Control, approximately 2 million illnesses and 23,000 deaths per year occur in the USA due to antibiotic resistance. In recent years, there has been a surge in the use of metal nanoparticles as coatings for orthopedic implants, wound dressings, and food packaging, due to their antimicrobial properties. In this report, we demonstrate that the antibacterial efficacy of silver nanoparticles (AgNPs) is enhanced with exposure to light from the visible spectrum. We find that the increased toxicity is due to augmented silver ion release and bacterial uptake. Interestingly, silver ion toxicity does not appear to depend on the formation of reactive oxygen species. Our findings provide a novel paradigm for using light to regulate the toxicity of AgNPs which may have a significant impact in the development of new antimicrobial therapeutics.

Escherichia coli J96 (O4:K6) was isolated from a human pyelonephritis patient. Here, we report the draft genome sequence of E. coli J96, which contains virulence genes, including adhesion factors, alpha-hemolysins, and cytotoxic necrotizing factor. J96 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis.

The Gram-negative bacterium Caulobacter crescentus forms a thin polar stalk, which mediates its attachment to solid surfaces. Whereas stalks remain short (1 µm) in nutrient-rich conditions, they lengthen dramatically (up to 30 µm) upon phosphate starvation. A long-standing hypothesis is that the Caulobacter stalk functions as a nutrient scavenging “antenna” that facilitates phosphate uptake and transport to the cell body. The mechanistic details of this model must be revisited, given our recent identification of a protein-mediated diffusion barrier, which prevents the exchange of both membrane and soluble proteins between the stalk extension and the cell body. In this report, we discuss the potential of stalks to facilitate nutrient uptake and propose additional physiological roles for stalk elongation in Caulobacter cells.

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins.

Mechanical properties of cells and extracellular matrices are critical determinants of function in contexts including oncogenic transformation, neuronal synapse formation, hepatic fibrosis and stem cell differentiation. The size and heterogeneity of biological specimens and the importance of measuring their mechanical properties under conditions that resemble their environments in vivo present a challenge for quantitative measurement. Centimeter-scale tissue samples can be measured by commercial instruments, whereas properties at the subcellular (nm) scale are accessible by atomic force microscopy, optical trapping, or magnetic bead microrheometry; however many tissues are heterogeneous on a length scale between micrometers and millimeters which is not accessible to most current instrumentation. The device described here combines two commercially available technologies, a micronewton resolution force probe and a micromanipulator for probing soft biological samples at sub-millimeter spatial resolution. Several applications of the device are described. These include the first measurement of the stiffness of an intact, isolated mouse glomerulus, quantification of the inner wall stiffness of healthy and diseased mouse aortas, and evaluation of the lateral heterogeneity in the stiffness of mouse mammary glands and rat livers with correlation of this heterogeneity with malignant or fibrotic pathology as evaluated by histology.

Anchorage-independent growth (AIG) of cancer cells requires escape from integrin-mediated signals. A protein frequently downregulated in cancer, caveolin-1 (Cav1), mediates integrin control of several growth-regulatory pathways. We report that loss of Cav1 results in faster exit from quiescence and progress through the cell cycle, proliferation without anchorage to substrate, and absence of cyclin D1 downregulation upon serum deprivation or detachment. Surprisingly, this proliferative advantage is independent of Erk-mitogen-activated protein kinase signaling; instead, cyclin expression and cell cycle progression in the absence of Cav1 are driven by increased membrane order and Rac targeting. AIG was induced in Cav1-expressing cells by forced membrane targeting of Rac1 or by inhibiting Cav1-mediated internalization of plasma membrane ordered domains at which Rac1 accumulates. Restoring Rho activity, which is downregulated after loss of Cav1, antagonizes Rac1 and prevents cyclin D1 accumulation after serum starvation or loss of adhesion. Anchorage independence and increased proliferation in Cav1-deficient tumoral and null cells are thus due to an increased fraction of active Rac1 at membrane ordered domains. These results provide insight into the mechanisms regulating growth of cancer cells, which frequently lose Cav1 function.