Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor.

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models.

Hybrid polymeric nanoparticles: potential candidate for ophthalmic delivery.

Nanotechnology that deals with submicron particles has the potential to deeply impact ophthalmic delivery owing to its ability to transport incorporated active ingredient/drugs to the different compartments of the eye while protecting them from any external assault. Polymeric nanoparticles, in particular hybrid nanoparticles made up of different biodegradable and biocompatible polymers, has the potential to revolutionize ocular delivery carrier systems by taking advantage of their highly tunable physicochemical properties. Here we report a synthesis method of hybrid nanoparticles made up of chitosan-poly(lactide-co-glycolide) (PLGA, as a building block of the nanocarrier) as a carrier for ophthalmic delivery. The synthesis method, characterization, and uptake study in retinal epithelial cells by confocal imaging is described herein.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Molecular characterization of AfuFleA, an L-fucose-specific lectin from Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. We have found that extracts of mycelial mats of A. fumigatus contain a potent hemagglutinin. To clarify the characteristics of this factor, the hemagglutinin was purified from late-stage cultures and characterized at the molecular level. The hemagglutinin is a 32-kilodalton protein that shows activity as an L-fucose lectin. The gene encoding this protein, AfufleA, was identified from a genomic DNA library utilizing consensus primers designed for amino acid sequences obtained from peptides following limited trypsin proteolysis. An open reading frame was found that consists of 942 nucleotides encoding 314 amino acids with a deduced molecular mass of 34,498 and contains all seven of trypsin-digested peptide sequences; four short introns, 49-63 bp, were also identified. AfufleA shares homology with a fucose-specific lectin produced by the orange peel mushroom, Aleuria aurantia. The role of AfufleA fucose-specific lectin is not clear, but this lectin may enhance attachment of fungal spores to mammalian cell membranes and contribute to the pathogenicity of A. fumigatus.

Pseudomonas fluorescens SBW25 produces furanomycin, a non-proteinogenic amino acid with selective antimicrobial properties.

BACKGROUND: Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors' laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid. RESULTS: P. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2'R,5'S)-2-amino-2-(5'methyl-2',5'-dihydrofuran-2'-yl)acetic acid]. CONCLUSIONS: The identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.

Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis.

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

Infections in patients with hematologic neoplasms and hematopoietic stem cell transplantation: neutropenia, humoral, and splenic defects.

Infections are common in patients with hematologic neoplasms and following allogeneic hematopoietic transplantation. Neutropenia and defects in adaptive B-cell-mediated immunity and/or lack of splenic function predispose patients to a host of diverse and often serious infections. It is important to recognize that patients who undergo treatment for hematologic neoplasms may have mixed immune defects, and their vulnerability to infection may continue to change, in part as a reflection of the dynamic developments in the practice of oncology. The main obstacle in providing targeted, evidence-based antimicrobial treatment is the unpredictable results of even the new generation of diagnostic assays. A definite diagnosis for most end-organ opportunistic diseases requires tissue samples that are seldom available. Because immune defects may coexist, empirical therapy is directed toward a wide spectrum of pathogens. Real-time information about innate and adaptive immune functions and the role of acute and chronic phase molecules may improve target-specific therapy.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6.

BACKGROUND: Pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. RESULTS: We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system. CONCLUSIONS: The improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

4-Formylaminooxyvinylglycine, an herbicidal germination-arrest factor from Pseudomonas rhizosphere bacteria.

A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over 20 other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The germination-arrest factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine on the basis of NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally active rhizosphere bacteria in our collection.

A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection.

The purpose of this study was to develop and characterize a rat model of choroidal neovascularization (CNV) as occurs in age-related macular degeneration. The lipid hydroperoxide 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) is found in submacular Bruch's membrane in aged humans and has been reported to generate neovascularization in a rabbit model. Three weeks after a single subretinal injection of 30 microg of HpODE, eyes of Sprague-Dawley rats were harvested. Follow-up fluorescein angiography was done on other animals until 5 weeks postinjection. Histological studies, immunohistochemical staining, and flatmount choroids for CNV measurements were performed. In addition, we used murine neuronal, bovine endothelial, and human ARPE19 cells for testing the in vitro effects of HpODE. CNV developed in 85.7% of HpODE-injected eyes. The neovascular areas were significantly greater in HpODE-injected eyes compared with those in control eyes (P = 0.023). The CNV had maximum dye leakage at 3 weeks, which subsided by the 5th week. Histologically, CNV extended from the choriocapillaris into the subretinal space. ED1-positive macrophages were recruited to the site. In vitro assays demonstrated that only 30 ng/ml HpODE induced cell proliferation and migration of endothelial cells. HpODE-induced CNV was highly reproducible, and its natural course seems to be ideal for evaluating therapeutic modalities. Because HpODE has been isolated from aged humans, the HpODE-induced rat model seems to be a relevant experimental model for CNV in age-related macular degeneration.

Zymographical techniques for detection of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases associated with extracellular matrix degradation, cellular migration, tissue remodeling, and angiogenesis. The activity of MMPs is regulated by the tissue inhibitors of metalloproteinases (TIMPs). Zymography and reverse zymography are useful to detect MMPs and TIMPs activities from various samples, for example vitreous, retina, plasma, and so on. Sample proteins are separated in substrate containing polyacrylamide gel by electrophoresis. The gel is incubated and then stained with Coomassie Blue. MMPs' activities are detected as clear bands.

From nutraceuticals to pharmaceuticals to nanopharmaceuticals: a case study in angiogenesis modulation during oxidative stress.

This report reviews the potential applications of nanotechnology in various therapeutics and diagnostics areas with special emphasis on key frontiers in angiogenesis modulation using naturally driven drug targets including compounds that modulate oxidative stress and inflammatory pathways for the potential treatment of vascular, cancer, inflammatory, and ocular disorders. Recent advances of the nanotechnology mediated gene delivery are also described in this paper.

Effect of a dihydrobenzofuran derivative on lipid hydroperoxide-induced rabbit corneal neovascularization.

The aim of this study was to investigate the effect of A-3922, a dihydrobenzofuran derivative, on linoleic acid hydroperoxide (LHP)-induced corneal neovascularization (NV) in a rabbit model. Male New Zealand rabbits received intraperitoneal (i.p.) injections of 10 or 30 mg/kg per day A-3922 or its vehicle as control for 3 days. One day after i.p. injections, LHP was injected with a 30-gauge needle into the corneal stroma of the superior quadrant 4.5-mm below the limbus. Photographs of the vessels were taken for digital analysis with a surgical microscope. Vascular endothelial growth factor (VEGF) was measured using an immunoassay kit, and matrix metalloproteinase (MMP)-9 was measured by gelatin zymography in corneal samples. At 7 days post-LHP injection, the total vessel length was 26.7 +/- 3.8 mm in the control animals (n = 8), 16.1 +/- 0.8 mm in the A-3922 (10 mg/kg)-treated group (n = 5), and 11.4 +/- 2.1 mm in the 30 mg/kg group (n = 8, P<0.01 vs control), respectively. After LHP injection, the content of VEGF and MMP-9 activity were increased in the superior cornea, but these were not influenced by A-3922 treatments. These results indicate that LHP-induced corneal NV is inhibited by treatment with A-3922 and therefore may represent a potential pharmacological intervention for ocular neovascularization disorders.

Effect of lipid-hydroperoxide-induced oxidative stress on vitamin E, ascorbate and glutathione in the rabbit retina.

BACKGROUND: It is possible that oxidative stress causes several retinal diseases. However, the natural biogenic role of antioxidants in the retina is not clear. PURPOSE: This study investigates the change in concentration of vitamin E (VE), ascorbate and glutathione (GSH) in the retina following vitreous injection of 600 mug 18:2 linoleic acid hydroperoxide (LHP) in male New Zealand rabbits. METHOD: LHP was injected above the retinal surface. The animals were sacrificed and the eyes enucleated before LHP injection, 1, 3, 6, 12, 24 h and 4 and 7 days after LHP injection. Retinas were removed, VE and ascorbate measured by HPLC, and GSH determined by a fluorometric method. RESULTS: The concentration of VE in the retina decreased from pretreatment levels of 154.6 +/- 29.7 nmol/g wet weight (n = 7) and was lowest at 6 h (61.1 +/- 18.1 nmol/g wet weight, n = 4, p < 0.05), then increased gradually, returning slowly to pre-LHP levels by 7 days. The concentration of ascorbate in control retinas decreased at 6 h from pretreatment levels of 7.33 +/- 0.93 micromol/g wet weight (n = 7) to 2.74 +/- 0.16 micromol/g wet weight (n = 4, p < 0.05) and returned to pretreatment levels rapidly by 24 h after injection. The concentration of GSH in retinas decreased from baseline levels of 109.53 +/- 8.19 microg/g wet weight (n = 9), was lowest at 12 h (72.40 +/- 11.17 microg/g wet weight, n = 5, p < 0.05) and returned to pretreatment levels by 7 days. CONCLUSION: The results suggest that intravitreous LHP injection is a contributor to oxidative stress in the rabbit retina by causing a reduction in antioxidant capacity.

Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide.

PURPOSE: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. METHODS: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by electrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. RESULTS: AP-1 binding activity was increased at 1-3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. CONCLUSIONS: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.