Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.     

Nitrogen Stress Affects the Turnover and Size of Nitrogen Pools Supplying Leaf Growth in a Grass

The effect of nitrogen (N) stress on the pool system supplying currently assimilated and (re)mobilized N for leaf growth of a grass was explored by dynamic ¹⁵N labeling, assessment of total and labeled N import into leaf growth zones, and compartmental analysis of the label import data. Perennial ryegrass (Lolium perenne) plants, grown with low or high levels of N fertilization, were labeled with ¹⁵NO₃⁻/¹⁴NO₃⁻ from 2 h to more than 20 d. In both treatments, the tracer time course in N imported into the growth zones fitted a two-pool model (r² > 0.99). This consisted of a “substrate pool,” which received N from current uptake and supplied the growth zone, and a recycling/mobilizing “store,” which exchanged with the substrate pool. N deficiency halved the leaf elongation rate, decreased N import into the growth zone, lengthened the delay between tracer uptake and its arrival in the growth zone (2.2 h versus 0.9 h), slowed the turnover of the substrate pool (half-life of 3.2 h versus 0.6 h), and increased its size (12.4 μg versus 5.9 μg). The store contained the equivalent of approximately 10 times (low N) and approximately five times (high N) the total daily N import into the growth zone. Its turnover agreed with that of protein turnover. Remarkably, the relative contribution of mobilization to leaf growth was large and similar (approximately 45%) in both treatments. We conclude that turnover and size of the substrate pool are related to the sink strength of the growth zone, whereas the contribution of the store is influenced by partitioning between sinks.This article examines the nitrogen (N) supply system of growing grass leaves, and it investigates how functional and kinetic properties of this system are affected by N stress. The N supply of growing leaves is a dominant target of whole-plant N metabolism. This is primarily related to the high N demand of the photosynthetic apparatus and the related metabolic machinery of new leaves (Evans, 1989; Makino and Osmond, 1991; Grindlay, 1997; Lemaire, 1997; Wright et al., 2004; Johnson et al., 2010; Maire et al., 2012). The N supply system, as defined here, is an integral part of the whole plant: it includes all N compounds that supply leaf growth. Hence, it integrates all events between the uptake of N from the environment (source), intermediate uses in other processes of plant N metabolism, and the eventual delivery to the leaf growth zone (sink; Fig. 1). N that does not ultimately serve leaf growth is not included in this system; all N that serves leaf growth is included, irrespective of its localization in the plant. Conceptually, two distinct sources supply N for leaf growth: N from current uptake and assimilation that is directly transferred to the growing leaf (“directly transferred N”) and N from turnover/redistribution of organic compounds (“mobilized N”).Open in a separate windowFigure 1.Schematic representation of N fluxes in the leaf growth zone and in the N supply system of leaf growth in a grass plant. A, Scheme of a growing leaf, with its growth zone (including zones of cell division, expansion, and maturation) and recently produced tissue (RPT). N import (I; μg h⁻¹) into the growth zone is mostly in the form of amino acids. Inside the growth zone, the nitrogenous substrate is used in new tissue construction. Then, N export (E; μg h⁻¹) is in the form of newly formed, fully expanded nitrogenous tissue (tissue-bound export with RPT) and is calculated as leaf elongation rate (L_ER; mm h⁻¹) times the lineal density of N in RPT (ρ; μg mm⁻¹): E = L_ER × ρ (Lattanzi et al., 2004). In a physiological steady state, import equals export (I = E) and the N content of the growth zone (G; μg [not shown]) is constant. Labeled N import into the growth zone (I_lab) commences shortly after labeling of the nutrient solution with ¹⁵N. The labeled N content of the growth zone (G_lab; μg) increases over time (dG_lab/dt) until it eventually reaches isotopic saturation (Fig. 2B). Similarly, the lineal density of labeled N in RPT (ρ_lab) increases until it approaches ρ. At any time, the export of labeled N in RPT (E_lab) equals the concurrent ρ_lab × L_ER. The import of labeled N is obtained as I_lab = E_lab + dG_lab/dt (Lattanzi et al., 2005) and considers the increasing label content in the growth zone during labeling. The fraction of labeled N in the import flux (f_{lab I}) is calculated as f_{lab I} = I_lab/I. The time course of f_{lab I} (Fig. 3) reflects the kinetic properties of the N supply system of leaf growth (C). B, Scheme of a vegetative grass plant (reduced to a rooted tiller with three leaves) with leaf growth zone. N import into the growth zone (I) originates from (1) N taken up from the nutrient solution that is transferred directly to the growth zone following assimilation (directly transferred N) and (2) N derived from turnover/redistribution of stores (mobilized N). The store potentially includes proteins in all mature and senescing tissue in the shoot and root of the entire plant. As xylem, phloem, and associated transfer cells/tissue provide for a vascular network that connects all parts of the plant, the mobilized N may principally originate from any plant tissue that exhibits N turnover/mobilization. The fraction of total N uptake that is allocated to the N supply system of the growth zone equals U (see model in C). The fraction of total mobilized N allocated to the growth zone equals M (see model in C). C, Compartmental model of the source-sink system supplying N to the leaf growth zone, as shown by Lattanzi et al. (2005) and used here. Newly absorbed N (U; μg h⁻¹) enters a substrate pool (Q₁); from there, the N is either imported directly into the growth zone (I) or exchanged with a store (Q₂). Q₁ integrates the steps of transport and assimilation that precede the translocation to the growth zone. Q₂ includes all proteins that supply N for leaf growth during their turnover and mobilization. The parameters of the model, including the (relative) size and turnover of pools Q₁ and Q₂, the deposition into the store (D; μg h⁻¹), and the mobilization from the store (M; μg h⁻¹), and the contribution of direct transfer relative to mobilization to the N supply of the growth zone are obtained by fitting the compartmental model to the f_{lab I} data (A) obtained in dynamic ¹⁵N labeling experiments (for details, see “Materials and Methods”). During physiological steady state, the sizes of Q₁ and Q₂ are constant, I = U, and M = D. [See online article for color version of this figure.]Amino acids are the predominant form in which N is supplied for leaf growth in grasses, and incorporation in new leaf tissue occurs mainly in the leaf growth zone (Gastal and Nelson, 1994; Amiard et al., 2004). This is a heterotrophic piece of tissue that includes the zones of cell division and elongation, is located at the base of the leaf, and is encircled by the sheath of the next older leaf (Volenec and Nelson, 1981; MacAdam et al., 1989; Schnyder et al., 1990; Kavanová et al., 2008). As most N is taken up in the form of nitrate but supplied to the growth zone in the form of amino acids, the path of directly transferred N includes a series of metabolic and transport steps. These include transfer to and loading into the xylem, xylem transport and unloading, reduction and ammonium assimilation, cycling through photorespiratory N pools, amino acid synthesis, loading into the phloem, and transport to the growth zone (Hirel and Lea, 2001; Novitskaya et al., 2002; Stitt et al., 2002; Lalonde et al., 2003; Dechorgnat et al., 2011). The time taken to pass through this sequence is unknown at present, as is the effect of N deficiency on that time. Also, it is not known how much N is contained in, and moving through, the different compartments that supply leaf growth with currently assimilated N.At the level of mature organs, mainly leaves, there is considerable knowledge about N turnover and redistribution. Much less is known about the fate of the mobilized N and its actual use in sink tissues like the leaf growth zone. The processes in mature organs are associated with the maintenance metabolism of proteins, organ senescence, and adjustments in leaf protein levels to decreasing irradiance inside growing canopies when leaves become shaded by overtopping newer ones (Evans, 1993; Vierstra, 1993; Hikosaka et al., 1994; Anten et al., 1995; Hirel et al., 2007; Jansson and Thomas, 2008; Moreau et al., 2012). N mobilization in shaded leaves supports the optimization of photosynthetic N use efficiency at plant and canopy scale (Field, 1983; Evans, 1993; Anten et al., 1995), it reduces the respiratory burden of protein maintenance costs (Dewar et al., 1998; Amthor, 2000; Cannell and Thornley, 2000), and it provides a mechanism for the conservation of the most frequently growth-limiting nutrient (Aerts, 1996). Mobilization of N involves protein turnover and net degradation (Huffaker and Peterson, 1974), redistribution in the form of amino acids (Simpson and Dalling, 1981; Simpson et al., 1983; Hörtensteiner and Feller, 2002), and (at least) some of the mobilized N is supplied to new leaf growth (Lattanzi et al., 2005).N fertilizer supply has multiple direct and indirect effects on plant N metabolism (Stitt et al., 2002; Schlüter et al., 2012). In particular, it modifies the N content of newly produced leaves, leaf longevity/senescence, and the dynamics of light distribution inside expanding canopies (Evans, 1983, 1989; Lötscher et al., 2003; Moreau et al., 2012). Thus, N fertilization influences the availability of recyclable N. At the same time, it augments the availability of directly transferable N to leaf growth. The net effect of these factors on the importance of mobilized versus directly transferred N substrate for leaf growth is not known. Also, it is unknown how N fertilization influences the functional characteristics of the N supply system, such as the size and turnover of its component pools.The assessment of the importance of directly transferred versus mobilized N for leaf growth requires studies at the sink end of the system (i.e. investigations of the N import flux into the leaf growth zone). Directly transferred N and mobilized N can be distinguished on the basis of their residence time in the plant, the time between uptake from the environment and import into the leaf growth zone: direct transfer involves a short residence time (fast transfer), whereas mobilized N resides much longer in the plant before it is delivered to the growth zone (slow transfer; De Visser et al., 1997; Lattanzi et al., 2005). Such studies require dynamic labeling of the N taken up by the plant (Schnyder and de Visser, 1999) and monitoring of the rate and isotopic composition/label content of N import into the leaf growth zone (Lattanzi et al., 2005). For grass plants in a physiological steady state, N import and the isotopic composition of the imported N are calculated from the leaf elongation rate and the lineal density of N in newly formed tissue (Fig. 1A; Lattanzi et al., 2004) and the change of tracer content in the leaf growth zone and recently produced leaf tissue over time (Lattanzi et al., 2005). Such data reveal the temporal change of the fraction of labeled N in the N import flux (f_{lab I}), which then can be used to characterize the N supply system of leaf growth via compartmental modeling. So far, there is only one study that has partially characterized this system (Lattanzi et al., 2005): this work was conducted with a C₃ grass, perennial ryegrass (Lolium perenne), and a C₄ grass, Paspalum dilatatum, growing in mixed stands and indicated that two interconnected N pools supplied the leaf growth zone in both species: a “substrate pool” (Q₁), which provided a direct route for newly absorbed and assimilated N import into the leaf growth zone (directly transferred N), and a mobilizing “store” (Q₂), which supplied N to the leaf growth zone via the substrate pool (Fig. 1C). The relative contribution of mobilization from the store was least important in the fast-growing, dominant individuals and most important in subordinate, shaded individuals. That work did not address the role of N deficiency, and the limited short-term resolution of the study (labeling intervals of 24 h or greater) precluded an analysis of the fast-moving parts of the system.Accordingly, this work addresses the following questions. How does N deficiency influence the substrate supply system of the leaf growth sink in terms of the number, size, and turnover (half-life) of its kinetically distinct pools? How does N deficiency affect the relationship between directly transferred and mobilized N for leaf growth? And what additional insight on the compartmental structure of the supply system is obtained when the short-term resolution of the analysis is increased by 1 order of magnitude? The work was performed with vegetative plants of perennial ryegrass grown in constant conditions with either a low (1.0 mm; termed low N) or high (7.5 mm; high N) nitrate concentration in the nutrient solution. In both treatments, a large number of plants were dynamically labeled with ¹⁵N over a wide range of time intervals (2 h to more than 20 d). The import of total N and ¹⁵N tracer into growth zones was estimated at the end of each labeling interval. Tracer data were analyzed with compartmental models following principles detailed by Lattanzi et al. (2005, 2012) and Lehmeier et al. (2008) to address the specific questions. Previous articles reported on root and shoot respiration (Lehmeier et al., 2010) and cell division and expansion in leaf growth zones (Kavanová et al., 2008) in the same experiment.     

Little evidence for niche partitioning among ectomycorrhizal fungi on spruce seedlings planted in decayed wood versus mineral soil microsites

Ectomycorrhizal fungal (EMF) communities vary among microhabitats, supporting a dominant role for deterministic processes in EMF community assemblage. EMF communities also differ between forest and clearcut environments, responding to this disturbance in a directional manner over time by returning to the species composition of the original forest. Accordingly, we examined EMF community composition on roots of spruce seedlings planted in three different microhabitats in forest and clearcut plots: decayed wood, mineral soil adjacent to downed wood, or control mineral soil, to determine the effect of retained downed wood on EMF communities over the medium and long term. If downed and decayed wood provide refuge habitat distinct from that of mineral soil, we would expect EMF communities on seedlings in woody habitats in clearcuts to be similar to those on seedlings planted in the adjacent forest. As expected, we found EMF species richness to be higher in forests than clearcuts (P ≤ 0.01), even though soil nutrient status did not differ greatly between the two plot types (P ≥ 0.05). Communities on forest seedlings were dominated by Tylospora spp., whereas those in clearcuts were dominated by Amphinema byssoides and Thelephora terrestris. Surprisingly, while substrate conditions varied among microsites (P ≤ 0.03), especially between decayed wood and mineral soil, EMF communities were not distinctly different among microhabitats. Our data suggest that niche partitioning by substrate does not occur among EMF species on very young seedlings in high elevation spruce-fir forests. Further, dispersal limitations shape EMF community assembly in clearcuts in these forests.     

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity

We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over‐expressing RCAN1 (RCAN1^ox), but not in wild‐type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK‐506, replicates this effect in WT cells but has no additional effect in RCAN1^ox cells. When we chronically expose WT cells to cyclosporine A and FK‐506 we find that catecholamine release per vesicle and pre‐spike foot (PSF) signal parameters are decreased, similar to that in RCAN1^ox cells. Inhibiting calcineurin activity in RCAN1^ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1^ox cells, the smaller vesicle and dense core size we observe in RCAN1^ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.     

Cardiomyopathy Mutations in the Tail of β-Cardiac Myosin Modify the Coiled-coil Structure and Affect Integration into Thick Filaments in Muscle Sarcomeres in Adult Cardiomyocytes

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301–1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.     

Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.     

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO₄ solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.