Controlled release of chlorhexidine from a mesoporous silica-containing macroporous titanium dental implant prevents microbial biofilm formation.

Roughened surfaces are increasingly being used for dental implant applications as the enlarged contact area improves bone cell anchorage, thereby facilitating osseointegration. However, the additional surface area also entails a higher risk for the development of biofilm associated infections, an etiologic factor for many dental ailments, including peri-implantitis. To overcome this problem, we designed a dental implant composed of a porous titanium-silica (Ti/SiO2) composite material and containing an internal reservoir that can be loaded with antimicrobial compounds. The composite material consists of a sol-gel derived mesoporous SiO2 diffusion barrier integrated in a macroporous Ti load-bearing structure obtained by powder metallurgical processing. The antimicrobial compounds can diffuse through the porous implant walls, thereby reducing microbial biofilm formation on the implant surface. A continuous release of µM concentrations of chlorhexidine through the Ti/SiO2 composite material was measured, without initial burst effect, over at least 10 days and using a 5 mM chlorhexidine solution in the implant reservoir. Metabolic staining, CFU counting and visualisation by scanning electron microscopy confirmed that Streptococcus mutans biofilm formation on the implant surface was almost completely prevented due to chlorhexidine release (preventive setup). Moreover, we demonstrated efficacy of released chlorhexidine against mature Streptococcus mutans biofilms (curative setup). In conclusion, we provide a proof of concept of the sustained release of chlorhexidine, one of the most widely used oral antiseptics, through the Ti/SiO2 material thereby preventing and eradicating biofilm formation on the surface of the dental implant. In principle, our flexible design allows for the use of any bioactive compound, as discussed.

Digital microfluidics for time-resolved cytotoxicity studies on single non-adherent yeast cells.

Single cell analysis (SCA) has gained increased popularity for elucidating cellular heterogeneity at genomic, proteomic and cellular levels. Flow cytometry is considered as one of the most widely used techniques to characterize single cell responses; however, its inability to analyse cells with spatio-temporal resolution poses a major drawback. Here, we introduce a digital microfluidic (DMF) platform as a useful tool for conducting studies on isolated yeast cells in a high-throughput fashion. The reported system exhibits (i) a microwell array for trapping single non-adherent cells by shuttling a cell-containing droplet over the array, and allows (ii) implementation of high-throughput cytotoxicity assays with enhanced spatio-temporal resolution. The system was tested for five different concentrations of the antifungal drug Amphotericin B, and the cell responses were monitored over time by time lapse fluorescence microscopy. The DMF platform was validated by bulk experiments, which mimicked the DMF experimental design. A correlation analysis revealed that the results obtained on the DMF platform are not significantly different from those obtained in bulk; hence, the DMF platform can be used as a tool to perform SCA on non-adherent cells, with spatio-temporal resolution. In addition, no external forces, other than the physical forces generated by moving the droplet, were used to capture single cells, thereby avoiding cell damage. As such, the information on cellular behaviour during treatment could be obtained for every single cell over time making this platform noteworthy in the field of SCA.

Ydc1p ceramidase triggers organelle fragmentation, apoptosis and accelerated ageing in yeast.

Saccharomyces cerevisiae dihydroceramidase Ydc1p hydrolyzes ceramide, resulting in accumulation of free long-chain bases and their phosphates. Yeast mutants lacking YDC1 are characterized by increased chronological lifespan. Moreover, we found YDC1 up-regulated in a yeast mutant displaying reduced chronological lifespan. These data suggest an important role for Ydc1p in chronological lifespan determination in yeast. Mitochondria are known to play an important role in chronological lifespan and apoptosis. In this study we demonstrated that overexpression of YDC1 results in reduced chronological lifespan and increased apoptotic cell death. We found YDC1 overexpression to result in mitochondrial fragmentation and dysfunction. Interestingly, vacuoles also appeared to be fragmented and dysfunctional upon YDC1 overexpressing. Exogenous addition of ceramide to YDC1-overexpressing cultures increased chronological lifespan and restored organelle function. In conclusion, this study describes a direct link between ceramide metabolism in yeast and mitochondrial and vacuolar fragmentation and function, with consequences for chronological lifespan in yeast.

The mode of antifungal action of plant, insect and human defensins.

Defensins are small (~5 kDa), basic, cysteine-rich antimicrobial peptides that fulfill an important role in the innate immunity of their host by combating pathogenic invading micro-organisms. Defensins can inhibit the growth or virulence of microorganisms directly or can do so indirectly by enhancing the host's immune system. Because of their wide distribution in nature, defensins are believed to be ancient molecules with a common ancestor that arose more than a billion years ago. This review summarizes current knowledge concerning the mode of antifungal action of plant, insect and human defensins.

Fungal sphingolipids as targets for the development of selective antifungal therapeutics.

Sphingolipids are essential membrane components, present in all eukaryotic cells, but structurally distinct in mammalian and fungal cells. Therefore, they represent an attractive new target for the development of novel antimycotics. This review will briefly highlight sphingolipid biosynthesis and functions in the yeast Saccharomyces cerevisiae. In addition, naturally occurring antifungal compounds that interact with fungal-specific sphingolipids, resulting in fungal growth arrest, will be discussed regarding their mode of action, and therapeutic value. These compounds include plant and insect defensins, syringomycin E and antifungal antibodies to sphingolipids.

Root and Foot Rot on Tomato Caused by Phytophthora infestans Detected in Belgium.

In January 2003, a severe root and foot rot was observed on 2-month-old wilted tomato (Lycopersicon esculentum Mill.) plants in a large-scale (2.5 ha) commercial greenhouse setting in Belgium. Tomato plants (10%) produced from healthy nursery-grown seedlings and planted to new, clean rockwool and drip irrigation with UV-disinfected water developed symptoms. Symptom development was restricted to lower plant parts with severe rotting of the entire root system and dark lesions girdling the stem base. No symptoms of disease were observed on the foliage or upper stems. Cross sections of the stem base revealed brown discoloration of internal tissue, including the vascular tissue and pith. Dark brown lesions also occurred on the roots. Sections of the stem base, the upper roots (sampled near to the stem base), and the lower roots (sampled on roots deeper in the rockwool) were plated separately on corn meal agar. The oomycete pathogen Phytophthora infestans (Mont.) de Bary was identified in each sample on the basis of morphological characteristics observed directly with light microscopy. Branched sporangiophores with slight swellings and characteristic lemon-shaped sporangia (35 × 20 µm and ratio length/width of 1.75 µm) at their tips were obvious after incubation in darkness at 24°C. Oospores and chlamydospores were not observed. After multiple soil treatment with oomycete-specific fungicides, the plants recovered. Since the occurrence of P. infestans on roots is unusual, the identity of the pathogen on the diseased plant tissues was confirmed with three techniques, DNA array identification, internal transcribed spacer (ITS) sequencing, and a polymerase chain reaction (PCR) amplification using P. infestans-specific primers. DNA was directly processed from separate samples of upper and lower root and stem base tissue. The DNA array used was originally developed to detect and identify the key fungal pathogens of tomato (2). Among detector probes for other tomato pathogens, this array contains oligonucleotide detector probes for P. infestans (PIN1: 5'-GGT TGT GGA CGC TGC TAT T and PIN2: 5'-AAT GGA GAA ATG CTC GAT TC). These probes are based on ITS sequences (ITS I and ITS II). Using conserved ribosomal primers OOMUP18Sc (5'-TGC GGA AGG ATC ATT ACC ACA C) and ITS4, oomycete DNA was amplified by PCR and simultaneously labeled with alkaline-labile digoxigenin (2). All generated amplicons strongly hybridized to the oligonucleotide detector probes for P. infestans and not to any other pathogen-specific detector probe present on the array. The pathogen could not be detected in roots and stem bases of symptomless plants. In addition, the ITS-region was sequenced and showed 100% homology with multiple GenBank accessions of P. infestans sequences. As a third confirmatory test, a PCR was performed on DNA extracts from infected root and stem base tissues using a primer set specific to P. infestans (O8-3/O8-4 [1]). A band of the expected size was produced for the infected stem base and root samples. Until now, this pathogen was known worldwide to cause late blight on potatoes and tomatoes. To our knowledge, this is the first report of root and foot rot of tomato caused by P. infestans. References: (1) H. S. Judelson and P. W. Tooley. Phytopathology 90:1112, 2000. (2) B. Lievens et al. FEMS Microbiol. Lett. 223:113, 2003.

Rapid detection and identification of tomato vascular wilt pathogens using a DNA array.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or V. dahliae, are devastating diseases of tomato (Lycopersicon esculentum Mill.) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We demonstrate that by using this array these pathogens can be detected within 24 h from complex substrates like soil, plant material, and samples as they are collected by tomato growers in their greenhouses.

Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens.

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

The complexity of disease signaling in Arabidopsis.

Not more than 10 years ago it was generally accepted that pathogen-inducible defense mechanisms in plants are triggered through a central signaling cascade that regulates a multicomponent defense response. Now we know that the plant defense system is regulated through a complex network of various signaling cascades.

A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii).

We have previously identified a Saccharomyces cerevisiae mutant that is markedly more resistant than wild-type to Dahlia merckii antimicrobial peptide 1 (DmAMP1), an antifungal plant defensin isolated from seeds of dahlia (Dahlia merckii). A complementation approach was followed that consisted of the introduction of a genomic library of DmAMP1-sensitive wild-type yeast into the DmAMP1-resistant yeast mutant and screening for restored sensitivity to DmAMP1. The gene determining sensitivity of S. cerevisiae to DmAMP1 was identified as IPT1, a gene encoding an enzyme involved in the last step of the synthesis of the sphingolipid mannose-(inositol-phosphate)(2)-ceramide. Strains with a nonfunctional IPT1 allele lacked mannose-(inositol-phosphate)(2)-ceramide in their plasma membranes, bound significantly less DmAMP1 compared with wild-type strains, and were highly resistant to DmAMP1-mediated membrane permeabilization. All of these phenotypic deviations could be restored by reintroduction of a functional IPT1 gene. Our data support a model in which membrane patches containing sphingolipids act as binding sites for DmAMP1 or, alternatively, are required to anchor membrane or cell wall-associated proteins, which themselves interact with DmAMP1.

The promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid.

The plant defensin PDF1.2 has previously been shown to accumulate systemically via a salicylic acid-independent pathway in leaves of Arabidopsis upon challenge by fungal pathogens. To further investigate the signalling and transcriptional processes underlying plant defensin induction, a DNA fragment containing 1184 bp and 1232 bp upstream of the transcriptional and translational start sites, respectively, was cloned by inverse PCR. To test for promoter activity this DNA fragment was linked to the beta-glucuronidase (GUS)-encoding region of the UidA gene as a translational fusion and introduced into Arabidopsis ecotype C-24. Challenge of the transgenic plants with the fungal pathogens Alternaria brassicicola and Botrytis cinerea resulted in both local and systemic induction of the reporter gene. Wounding of the transgenic plants had no effect on GUS activity. Treatment of the transgenic plants with either jasmonates or the active oxygen generating compound paraquat strongly induced the reporter gene. In contrast, neither salicylate nor its functional analogues 2,6-dichloroisonicotinic acid and 1,2,3-benzothiodiazole-7-carbothioic acid S-methyl ester resulted in reporter gene induction. These results are consistent with the existence of a salicylic acid-independent signalling pathway, possibly involving jasmonates as regulators, that is triggered by pathogen challenge but not by wounding. The transgenic plants containing the PDF1.2-based promoter-reporter construct will provide useful tools for future genetic dissection of this novel systemic signalling pathway.

Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens.

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2, 6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247-1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

A novel family of small cysteine-rich antimicrobial peptides from seed of Impatiens balsamina is derived from a single precursor protein.

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

Genetic transformation of banana and plantain (Musa spp.) via particle bombardment.

We have developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the beta-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

Expression of functional Raphanus sativus antifungal protein in yeast.

Rs-AFP2 is a 51 amino acid cysteine-rich peptide isolated from radish (Raphanus sativus) seeds that exhibits potent inhibitory activity against filamentous fungi. A cDNA clone encoding the Rs-AFP2 preprotein was modified by recombinant DNA methods to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N-terminus the prepro-sequences derived from the precursor of the yeast pheromone mating factor alpha 1. These sequences allow secretion of the biologically active peptide in a correctly processed form. Deletion of the mating factor alpha 1 pro-peptide drastically reduced the expression level of the peptide.

Gene-encoded antimicrobial peptides from plants.

On the basis of an extensive screening of seeds from various plant species, we have isolated and characterized several different antimicrobial peptides. They were all typified by having a broad antifungal activity spectrum, a relatively low molecular weight (3-14 kDa), a high cysteine content and a high isoelectric point (pI > 10). With respect to their amino acid sequence, these peptides can be classified into six structural classes. Synergistic enhancement (up to 73-fold) of antimicrobial activity was demonstrated in some combinations of peptides belonging to different classes. cDNA clones corresponding to different antifungal peptides were isolated and used to transform tobacco plants. Extracts of these transgenic plants showed higher (up to 16-fold) antifungal activity than untransformed control plants. Such antimicrobial peptides may find applications in molecular breeding of plants with increased disease resistance.