Inquisition of Microcystis aeruginosa and Synechocystis nanowires: characterization and modelling

Identification of extracellular conductive pilus-like structures (PLS) i.e. microbial nanowires has spurred great interest among scientists due to their potential applications in the fields of biogeochemistry, bioelectronics, bioremediation etc. Using conductive atomic force microscopy, we identified microbial nanowires in Microcystis aeruginosa PCC 7806 which is an aerobic, photosynthetic microorganism. We also confirmed the earlier finding that Synechocystis sp. PCC 6803 produces microbial nanowires. In contrast to the use of highly instrumented continuous flow reactors for Synechocystis reported earlier, we identified simple and optimum culture conditions which allow increased production of nanowires in both test cyanobacteria. Production of these nanowires in Synechocystis and Microcystis were found to be sensitive to the availability of carbon source and light intensity. These structures seem to be proteinaceous in nature and their diameter was found to be 4.5-7 and 8.5-11 nm in Synechocystis and M. aeruginosa, respectively. Characterization of Synechocystis nanowires by transmission electron microscopy and biochemical techniques confirmed that they are type IV pili (TFP) while nanowires in M. aeruginosa were found to be similar to an unnamed protein (GenBank : CAO90693.1). Modelling studies of the Synechocystis TFP subunit i.e. PilA1 indicated that strategically placed aromatic amino acids may be involved in electron transfer through these nanowires. This study identifies PLS from Microcystis which can act as nanowires and supports the earlier hypothesis that microbial nanowires are widespread in nature and play diverse roles.

No outbreeding depression at a regional scale for a habitat-forming intertidal alga with limited dispersal

Hormosira banksii is distributed throughout southern Australasia, but dispersal of propagules is thought to be limited. In the present study, the hypothesis that outbreeding depression occurs in H. banksii was tested by assessing fertilisation success and early development of embryos in crosses between populations at local to regional spatial scales. Hierarchical experiments were conducted at three spatial scales with nesting present within each scale: small scale (within a rocky shore population), intermediate scale (regions separated by 70 km) and large scale (450-km separation between two states: Victoria and Tasmania). In each experiment, eggs and sperm were crossed within and between each population located in the spatial scale of interest. There were no consistent patterns of variable fertilisation success and subsequent development within a population or at different spatial scales. It was concluded that outbreeding depression is not detected in analyses of fertilisation success or early development processes in H. banksii. The results suggest one of the following to be likely: (1) H. banksii is capable of longer distance dispersal than previously considered, thus maintaining gene flow between distant populations, (2) gene flow is restricted by limited dispersal, but populations have not been isolated for a sufficient length of time to cause genetic divergence or (3) outbreeding depression is manifested as effects on later life-history stages.

Small-scale temporal variation in propagule supply of an intertidal red alga

Information on the variability in supply of algal propagules is scarce, hindered by the difficulty in identifying propagules, but this variability may affect the distribution and abundance of algal assemblages. This study examined the small-scale (½ hourly to hourly) temporal variation in propagule supply of Chondrus verrucosus (Gigartinaceae, Rhodophyta) over a dense, isolated bed in south-eastern Japan in summer and winter of 1999. Either 0.5 litre scoop samples or 5 litre pump samples were collected ½ hourly to hourly over 13, 22.5, and 30 h on three occasions in summer (June & July) and 32 h on one occasion in winter (December). Sampling was conducted around either the new moon (two occasions in summer) or full moon (one occasion in both summer and winter) and incorporated full tidal sequences including daytime (summer) and nighttime (winter) low-low (LL) tides. Chondrus verrucosus was the only red alga with spores within the size range of 15–20 μm that was fertile in the study area and surrounds at the time of sampling facilitating identification of spores. Spores in scoop samples were settled onto Petri dishes and identified on the basis of cell shape, colour and size. Pump samples were filtered onto transparent membrane filters and identified using epifluorescence microscopy: C. verrucosus spores fluoresced bright yellow and were easily distinguished from other micro-organisms of similar size, which fluoresced red or green. Results showed that while propagules could be found in the water column at most times, propagule supply of C. verrucosus was greatest during the 1–2 h period following LL tides. Variability in propagule supply was less than in previous studies examining surface or offshore waters. Spore release is thought to be stimulated by either desiccation or salinity changes associated with periods of emersion at low tide followed by re-immersion on incoming tides.

Use of in vitro critical inhibitory concentration, a novel approach to predict in vivo synergistic bactericidal effect of combined amikacin and piperacillin against pseudomonas aeruginosa in a systemic rat infection model

PURPOSE: This study was undertaken to explore the use of in vitro critical inhibitory concentration (CIC) as a surrogate marker relating the pharmacokinetic (PK) parameters to in vivo bactericidal synergistic effect [pharmacodynamic (PD)] of amikacin + piperacillin combination against Pseudomonas aeruginosa in a systemic rat infection model. METHODS: The in vitro antibacterial activities of amikacin and piperacillin, alone and in combinations at various ratios of the concentrations, were tested against a standard [5 x 10(5) colony-forming units (CFU)/ml] and a large (1.5 x 10(8) CFU/ml) inoculum of P. aeruginosa ATCC 9027 using a modified survival-time method. The CIC of each individual antibiotic for the different combinations was determined using a cup-plate method. In vivo studies were performed on Sprague-Dawley rats using a systemic model of infection with P. aeruginosa ATCC 9027. PK profiles and in vivo killing effects of the combination at different dosing ratios were studied. RESULTS: An inoculum effect was observed with the antibiotics studied. Synergy was seen against both the inocula at the following concentration ratios: 70% C(ami) + 30% C(pip) and 75% C(ami) + 25% C(pip), where C(ami) and C(pip) are the concentrations of amikacin and piperacillin to produce a 1000-fold decrease in bacterial population over 5 h, respectively. The CIC values determined corroborated with the order of in vitro bacterial killing observed for the antibiotic combinations. The dosing ratio of 12.6 mg/kg amikacin + 36 mg/kg piperacillin (a 70:30 ratio of the individual doses) exhibited the greatest killing in vivo when compared to the other ratios. The PK-PD relationships were described by simple, linear regression equations using the area under the in vivo killing curve as a PD marker and the AUCIC(ami)/CIC(ami) + AUCIC(pip)/CIC(pip), AUC(ami)/CIC(ami) + AUC(pip)/CIC(pip), C(max,ami)/CIC(ami) + C(max,pip)/CIC(pip), and AUCIC(ami)/MIC(ami) + AUCIC(pip)/MIC(pip) as PK markers for the amikacin + piperacillin combination. CONCLUSION: The combination of amikacin and piperacillin exhibited synergistic killing effect on P. aeruginosa that could be modeled using CIC as a surrogate marker relating the PK parameters to in vivo bactericidal effect.

First isolation and structural determination of cyclic β-(1→2)-glucans from an alga, Chlorella pyrenoidosa

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

Restoration of the habitat-forming fucoid alga Hormosira banksii at effluent-affected sites : competitive exclusion by coralline turfs

Fucoid algae often dominate intertidal rocky shores, providing habitat and modifying ecosystem resources for other species, but are susceptible to discharge of sewage effluent. In this study we assessed the potential for competition from coralline turfs to inhibit restoration of the intertidal fucoid macroalga Hormosira banksii at sites associated with an ocean outfall a scenario of improving following water quality in the nearshore coastal environment. The percentage cover and number of individuals of H. banksii were negatively correlated with both the percentage cover and turf height of Corallina officinalis. In contrast, H. banksii was positively associated with rocky substrata and recruited well to rock-surface substrata. Importantly, there appears to be a threshold abundance where the percentage cover of H. banksii rarely reaches above 20% cover amongst coralline turfs with >40% cover. These data support a model of alternative community states: H. banksii dominated canopy on rocky substrata versus C. officinalis turf. In field and laboratory experiments, extensive coralline turfs (up to 4 cm thick) were shown to inhibit recruitment of H. banksii. This study shows competitive exclusion by coralline turfs may limit the successful restoration of habitat provided by H. banksii to shores that have been affected by sustained discharge of secondarily treated sewage effluent. We suggest potential strategies for management authorities to consider when seeking ways of restoring fucoid communities affected by anthropogenic disturbances such as wastewater disposal.

The pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl− ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including b-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

The ΔF508-CFTR mutation results in increased biofilm formation by Pseodomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

Effect of bronchoalveolar lavage-directed therapy on pseudomonas aeruginosa infection and structural lung injury in children with cystic fibrosis

Context Early pulmonary infection in children with cystic fibrosis leads to increased morbidity and mortality. Despite wide use of oropharyngeal cultures to identify pulmonary infection, concerns remain over their diagnostic accuracy. While bronchoalveolar lavage (BAL) is an alternative diagnostic tool, evidence for its clinical benefit is lacking.

Objective To determine if BAL-directed therapy for pulmonary exacerbations during the first 5 years of life provides better outcomes than current standard practice relying on clinical features and oropharyngeal cultures.

Design, Setting, and Participants The Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) randomized controlled trial, recruiting infants diagnosed with cystic fibrosis through newborn screening programs in 8 Australasian cystic fibrosis centers. Recruitment occurred between June 1, 1999, and April 30, 2005, with the study ending on December 31, 2009.

Interventions BAL-directed (n=84) or standard (n=86) therapy until age 5 years. The BAL-directed therapy group underwent BAL before age 6 months when well, when hospitalized for pulmonary exacerbations, if Pseudomonas aeruginosa was detected in oropharyngeal specimens, and after P aeruginosa eradication therapy. Treatment was prescribed according to BAL or oropharyngeal culture results.

Main Outcome Measures Primary outcomes at age 5 years were prevalence of P aeruginosa on BAL cultures and total cystic fibrosis computed tomography (CF-CT) score (as a percentage of the maximum score) on high-resolution chest CT scan.

Results Of 267 infants diagnosed with cystic fibrosis following newborn screening, 170 were enrolled and randomized, and 157 completed the study. At age 5 years, 8 of 79 children (10%) in the BAL-directed therapy group and 9 of 76 (12%) in the standard therapy group had P aeruginosa in final BAL cultures (risk difference, −1.7% [95% confidence interval, −11.6% to 8.1%]; P=.73). Mean total CF-CT scores for the BAL-directed therapy and standard therapy groups were 3.0% and 2.8%, respectively (mean difference, 0.19% [95% confidence interval, −0.94% to 1.33%]; P=.74).

Conclusion Among infants diagnosed with cystic fibrosis, BAL-directed therapy did not result in a lower prevalence of P aeruginosa infection or lower total CF-CT score when compared with standard therapy at age 5 years.

Trial Registration anzctr.org.au Identifier: ACTRN12605000665639

Structural characterisation and bioactivities of hybrid carrageenan-like sulphated galactan from red alga Furcellaria lumbricalis

The red alga, Furcellaria lumbricalis from the coast of the Prince Edward Island (PEI) in Atlantic Canada, was extracted with hot water and fractionated with 0.125. M KCl to obtain a carrageenan-like polysaccharide. The polysaccharide was further purified on ion-exchange and gel-permeation chromatography to yield a fraction (FB1) of uniform size and charge, with an average molecular weight of 428. kD. Oligosaccharides generated with acid hydrolysis of FB1 were sequenced using the electrospray ionisation collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique. On the basis of chemical and spectroscopic analysis, FB1 was characterised to be composed of 1,4-linked 3,6-anhydro-galactose (40%), 1,3-linked 4-sulphated-galactose (30%), 1,3-linked galactose (20%), 1,4-linked galactose (8%) and 1,4-linked 3-O-methyl-galactose (2%), which makes it be a novel sulphated galactan hybrid. The β-secretase (BACE) inhibition and immunomodulation activities of FB1-derived oligosaccharides were evaluated in vitro.