A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

AIMS: Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. METHODS AND RESULTS: The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. CONCLUSIONS: The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. SIGNIFICANCE AND IMPACT OF THE STUDY: D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started.

The interaction of DIAP1 with dOmi/HtrA2 regulates cell death in Drosophila.

Mitochondrial proteins such as cytochrome c, Smac/DIABLO and Omi/HtrA2 play important roles in the cell death pathways of mammalian cells. In Drosophila, the role of mitochondria in cell death is less clear. Here, we report the identification and characterization of the Drosophila ortholog of human Omi/HtrA2. We show that Drosophila Omi/HtrA2 is imported into the mitochondria where it undergoes proteolytic maturation to yield two isoforms, dOmi-L and dOmi-S. dOmi-L contains a canonical N-terminal IAP-binding motif (AVVS), whereas dOmi-S contains a distinct N-terminal motif (SKMT). DIAP1 was able to bind to both isoforms via its BIR1 and BIR2 domains. This resulted in cleavage of the linker region of DIAP1 between the BIR1 and BIR2 domains and further degradation of the BIR1 domain by the proteolytic activity of dOmi. The binding of DIAP1 to dOmi also resulted in DIAP1-mediated polyubiquitination of dOmi, suggesting that DIAP1 could target dOmi for proteasomal degradation. Consistent with this, expression of DIAP1 in Drosophila eye discs protected them from dOmi-induced eye ablation, indicating that DIAP1 plays an important role in protecting cells from the potentially lethal effects of dOmi. The ability of IAPs to bind to and ubiquitinate mitochondrial proteins such as dOmi may be a key conserved function to counterbalance the lethal effects of these proteins if accidentally released into the cytosol.

In vitro microbicidal activities of cecropin peptides D2A21 and D4E1 and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis.

Topically applied microbicides that eradicate pathogens at the time of initial exposure represent a powerful strategy for the prevention of sexually transmitted infections. To aid in the further development of an effective topical microbicide, we assessed the minimum cidal concentration (MCC) of two cecropin peptides, D2A21 and D4E1, and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis in vitro. The MCC of peptide D2A21 was 5 microM (18.32 microg/ml), and that of peptide D4E1 was 7.5 microM (21.69 microg/ml). The MCC of gel formulations containing 2% D2A21 was 0.2 mM (0.7 mg/ml), and that of gel formulations containing 0.5% D2A21 was 0.2 mM (0.7 mg/ml). There was no significant variation in the results when two different C. trachomatis strains were tested, and the addition of 10% human blood did not significantly alter the MCCs. pH values above and below 7 reduced the activity of the D2A21 peptide alone, but the MCC of the 2% D2A21 gel formulation was only slightly altered at the various pHs tested. Ultrastructural studies indicated that C. trachomatis membranes were disrupted after D2A21 exposure, resulting in leakage of the cytoplasmic contents. These in vitro results suggest that these cecropin peptides may be an effective topical microbicide against C. trachomatis and support the need for further evaluation.

Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity.

Bicoid directs anterior development in Drosophila embryos by activating different genes along the anterior-posterior axis. However, its activity is down-regulated at the anterior tip of the embryo, in a process known as retraction. Retraction is under the control of the terminal polarity system, and results in localized repression of Bicoid target genes. Here, we describe a Drosophila homolog of human SAP18, a member of the Sin3A/Rpd3 histone deacetylase complex. dSAP18 interacts with Bicoid in yeast and in vitro, and is expressed early in development coincident with Bicoid. In tissue culture cells, dSAP18 inhibits the ability of Bicoid to activate reporter genes. These results suggest a model in which dSAP18 interacts with Bicoid to silence expression of Bicoid target genes in the anterior tip of the embryo.

Efficacy of a synthetic lytic peptide in the treatment of prostate cancer.

In the last several years, significant effort has been applied to identifying novel agents with effectiveness against prostate cancer. These studies were designed to determine the efficacy of one of these novel compounds, D2A21, in the treatment of an animal model of prostate cancer. Using the Mat-Ly-Lu(MLL) line of the Dunning R-3327 rat prostate adenocarcinoma model, the optimal dose, schedule and route of administration of D2A21 were established. A study involving the G line was used to further support these findings. In addition, hemotoxylin and eosin stained tissue samples were examined to investigate the extent of inhibition of lung metastases in animals injected with MLL cells. When D2A21 was injected intraperitoneally or subcutaneously, MLL and G cell tumor growth was inhibited 50-72% as demonstrated by both tumor volumes and weights. The optimal dosage of 0.179 mg/injection was established and it was determined to be most efficacious when administered five times per week. At this concentration, D2A21 appears to have no significant toxicity. Additionally, D2A21 increased the survival rate from only 25% to 70-75% in animals that were challenged with a large number of tumor cells. The peptide D2A21 is able to significantly inhibit tumor growth in rat models of prostate cancer. In addition, it can inhibit metastases and decrease deaths resulting from metastases in these animals.

Groucho augments the repression of multiple Even skipped target genes in establishing parasegment boundaries.

Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped has previously been shown to contain a Groucho-independent repression activity. In the Even skipped protein, outside the Groucho-independent repression domain, we have identified a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein. Even skipped interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Even skipped with a mutated Groucho interaction motif, which abolished binding to Groucho, showed a significantly reduced ability to rescue the even skipped null phenotype when driven by the complete even skipped regulatory region. Replacing this motif with a heterologous Groucho interaction motif restored the rescuing function of Even skipped in segmentation. Further functional assays demonstrated that the Even skipped C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain was active on two target genes that are normally repressed by Even skipped at different concentrations, paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Even skipped to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions.

even skipped is required to produce a trans-acting signal for larval neuroblast proliferation that can be mimicked by ecdysone.

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.

Use of intravaginal microbicides to prevent acquisition of Trichomonas vaginalis infection in Lactobacillus-pretreated, estrogenized young mice.

D2A21, a novel peptide antibiotic has in vitro activity against a wide spectrum of sexually transmitted diseases (STD). In this study we tested the hypothesis that intravaginal D2A21 would interfere with acquisition of Trichomonas vaginalis infection in a modified mouse model. T. vaginalis infections of estrogenized young mice pretreated with Lactobacillus vaginalis or Lactobacillus rhamnosus were more frequent and persistent than those in mice pre-treated with Lactobacillus gasseri or Lactobacillus acidophilus. One hundred percent T. vaginalis infection was achieved for 2-4 days post-challenge when intravaginal L. rhamnosus pre-treatments were given to estrogenized mice 48 hr prior to a single T. vaginalis challenge. Estrogenized mice pre-treated with L. rhamnosus were pre-medicated with intravaginal placebo gel, 0.5% or 2% D2A21 gel, or 500 microg/mL metronidazole gel prior to T. vaginalis challenge. Both 2% D2A21 and metronidazole gels were significantly more efficacious (10% or none infected) than placebo gel (53% infected) in preventing vaginal T. vaginalis infections in mice.

Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila.

During the development of the nervous system embryonic neurons are incorporated into neural networks that underlie behaviour. For example, during embryogenesis in Drosophila, motor neurons in every body segment are wired into the circuitry that drives the simple peristaltic locomotion of the larva. Very little is known about the way in which the necessary central synapses are formed in such a network or how their properties are controlled. One possibility is that presynaptic and postsynaptic elements form relatively independently of each other. Alternatively, there might be an interaction between presynaptic and postsynaptic neurons that allows for adjustment and plasticity in the embryonic network. Here we have addressed this issue by analysing the role of synaptic transmission in the formation of synaptic inputs onto identified motorneurons as the locomotor circuitry is assembled in the Drosophila embryo. We targeted the expression of tetanus toxin light chain (TeTxLC) to single identified neurons using the GAL4 system. TeTxLC prevents the evoked release of neurotransmitter by enzymatically cleaving the synaptic-vesicle-associated protein neuronal-Synaptobrevin (n-Syb) [1]. Unexpectedly, we found that the cells that expressed TeTxLC, which were themselves incapable of evoked release, showed a dramatic reduction in synaptic input. We detected this reduction both electrophysiologically and ultrastructurally.

In vitro activities of designed antimicrobial peptides against multidrug-resistant cystic fibrosis pathogens.

The emergence of multidrug-resistant pathogens renders antibiotics ineffective in the treatment of lung infections in patients with cystic fibrosis (CF). Designed antimicrobial peptides (DAPs) are laboratory-synthesized peptide antibiotics that demonstrate a wide spectrum of antibacterial activity. Optimal conditions for susceptibility testing of these peptides have not yet been established. Medium composition is clearly a major factor influencing the results and reproducibilities of susceptibility tests. Using time-kill assays, we tested the effects of different media and buffers on the bactericidal activities of the peptides D2A21 and D4E1 on Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853. Each peptide at 1 and 5 microM was incubated with bacteria in the different media and buffers. Both peptides were most active in Tris-HCl buffer against S. aureus and P. aeruginosa. Among the more complex media tested, modified RPMI medium was the medium in which the peptides demonstrated the highest activity, while it supported the growth of the bacteria. The broth microdilution technique was used to test the activities of D2A21 and D4E1 in modified RPMI medium against multidrug-resistant pathogens from patients with CF. The MICs of DAPs for methicillin-resistant S. aureus ranged from 0.25 to 4 microg/ml, those for multidrug-resistant P. aeruginosa ranged from 0.125 to 4 microg/ml, those for Stenotrophomonas maltophilia ranged from 0.5 to 32 microg/ml, and those for Burkholderia cepacia ranged from 32 to >/=64 microg/ml. When the activity of peptide D2A21 was compared with that of the tracheal antimicrobial peptide (TAP), D2A21 had greater potency than TAP against P. aeruginosa. In addition, no difference in the MICs of D2A21 was seen when it was tested in nutrient broth supplemented with NaCl at different concentrations. Thus, DAPs are a class of salt-insensitive antibiotics potentially useful in the treatment of CF patients harboring multidrug-resistant P. aeruginosa.

Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.

Evaluation of the effect of peptidyl membrane-interactive molecules on avian coccidia.

This study examined the lytic effect of seven different synthetic peptidyl membrane-interactive molecules (Peptidyl-MIMs) on sporozoites of five different species of Eimeria infecting chickens and merozoites of two different species that infect chickens. All Peptidyl-MIMs (pMIMs) demonstrated antiparasitic effects at concentrations of 1-50 microM during incubation periods varying from 1 to 20 min. In addition, electron microscopy showed that ultrastructural degeneration of the pellicle of sporozoite stages of the parasites occurred within 5-10 min of exposure to 5-microM concentrations of three different pMIMs. Pore-like openings were seen in the pellicle of the sporozoites at the ultrastructural level, which indicated that the pMIMs had the same mechanism of action on the parasites as that reported from studies done on bacteria. A reduction in lesion scores was seen in chickens treated orally with 10-, 50-, or 75-microM concentrations of two different proteolytic stabilized (methylated) pMIMs after challenge with three different species of avian coccidia in battery-cage trials. Collectively these data indicate that pMIMs may be useful in the control of coccidiosis in poultry.

Drosophila homeobox gene eve enhances trol, an activator of neuroblast proliferation in the larval CNS.

The regulation of stem cell division by developmental cues is critical for the assembly and function of multicellular organisms. Stem cell division in the Drosophila brain is controlled by trol, which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. We show that the transcriptional regulator eve is part of the trol activation pathway by identifying eve as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally, genetic and molecular analysis reveals that an independent mutation which acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction.

Peptidyl membrane-interactive molecules are cytotoxic to prostatic cancer cells in vitro.

Cytotoxic membrane disruption via lytic peptides is a well-recognized mechanism of immune surveillance for antifungal and antibacterial host protection. Naturally occurring lytic peptides were shown to exhibit antitumor activity as well. Peptidyl membrane-interactive molecules (MIMs) are synthetic lytic peptides specifically designed to maximize antitumor activity. We tested nine novel Peptidyl MIMs for activity against four androgen-insensitive prostate-cancer cell lines using a standard microculture tetrazolium (MTT) assay. Five Peptidyl MIMs known to form alpha-helical secondary structures were active against prostate carcinoma and were chosen for further study. Three peptides configured in beta-pleated sheets were noticeably less effective. Concentrations lethal to 50% of the prostate-cancer cell lines treated (D50 values) with the five chosen Peptidyl MIMs ranged from 0.6 to 1.8 microM. For comparison, two alpha-helically structured peptides, D2A21 and DP1E, were tested on several other cancer types: breast (n = 2), colon (n = 2). bladder, cervical and lung carcinomas (n = 1 each). Resulting LD50 values obtained in breast carcinoma cells were significantly higher (P < 0.05) than those observed in prostate cancer cells. LD50 values recorded for D2A21 and DP1E in cervical, colon, bladder, and lung cancer lines were similar to those obtained in prostate cancer cells. As compared with cisplatin, a standard chemotherapeutic drug, the LD50 values recorded for D2A21 were significantly lower (P < 0.04) in prostate-cancer cell lines, suggesting the therapeutic efficacy of Peptidyl MIMs. These data demonstrate for the first time the cytotoxic potential of Peptidyl MIMs against prostate cancer cells and suggest a dependence on a specific secondary alpha-helical structure of the peptide.

Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.

The fungicidal properties of the synthetic peptide D4E1 were studied with nongerminated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium moniliforme, and Fusarium oxysporum. The minimal lethal concentrations (MLC) needed to kill 100% of germinating conidia of A. fumigatus, A. flavus, and A. niger were 12.5, 12.5, and 25 microM, respectively. The MLC value for nongerminated and germinating conidia of both Fusarium spp. was 3.0 microM. Except for A. fumigatus, D4E1 was inactive against the nongerminated conidia of the Aspergillus spp. Physicochemical studies showed D4E1 complexed with ergosterol, a sterol present in conidial walls. Cholesterol, present in nongerminated conidia of F. moniliforme, had a greater affinity for D4E1 than did ergosterol. D4E1 was more resistant to fungal and plant protease degradation than the natural peptide, cecropin A. These in vitro results suggest D4E1 is a candidate for transgenic expression in plants to enhance host resistance to fungal infection.

Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes.

Active transcriptional repression has been characterized as a function of many regulatory factors. It facilitates combinatorial regulation of gene expression by allowing repressors to be dominant over activators under certain conditions. Here, we show that the Engrailed protein uses two distinct mechanisms to repress transcription. One activity is predominant under normal transient transfection assay conditions in cultured cells. A second activity is predominant in an in vivo active repression assay. The domain mediating the in vivo activity (eh1) is highly conserved throughout several classes of homeoproteins and interacts specifically with the Groucho corepressor. While eh1 shows only weak activity in transient transfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relative activities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of the assay to the Groucho interaction domain, albeit to a lesser extent. This suggests that it utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on integrated target genes.

Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus.

The addition of an antimicrobial that can be synthesized by the mammalian immune system at the point of challenge may enhance disease resistance. A possible group of agents are cecropins, broad-spectrum antimicrobial peptides, which have been described and characterized. They are relatively non-toxic to normal cells from multicellular organisms but are toxic to a wide range of bacteria, protozoa and fungi, as well as infected and abnormal cells. Twenty-six lines of transgenic mice were produced by pronuclear injection of DNA consisting of the 5'-flanking region from -593 to +110 of the mouse interleukin 2 (IL-2) gene, Shiva 1a (a synthetic cecropinclass lytic peptide), and the SV40 polyadenylation/splice signal. A reverse-transcription PCR assay determined that two lines of transgenic mice were produced whose spleen-derived lymphocytes could be induced to transcribe and mature mRNA for Shiva 1a by exposure to 3.25 mg ml-1 of Con A. Two lines were challenged with an inoculation of 5 x 10(4) Brucella abortus strain 2308. After four weeks, there were significantly fewer B. abortus organisms in the spleens of transgenic mice than in non-transgenic control mice of the same strain (p < 0.05). Since the controlling regions of the IL-2 enhancer and the amino acid sequence of the signal peptide are highly conserved among several species, it is likely that this recombinant gene will function in other mammals.

An amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus-1 (HSV-1)-induced cell fusion and virus spread.

The effects of hecate, a 23-amino acid synthetic peptide analogue of melittin, on HSV-1-induced cell fusion and virus multiplication was investigated. Hecate completely inhibited cell fusion induced by HSV-1 syncytial (syn) mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP) at a concentration of 5.0 microM. Metabolic labeling experiments indicated that hecate did not adversely affect cellular growth and protein synthesis. The synthesis of virus-specified glycoproteins B, C, D, and H was reduced in the presence of hecate; however, the transport of these glycoproteins to the surface of infected cells was not affected. Production of infectious virions for wild-type and syn mutants tsB5 and MP was reduced in the presence of hecate. The effect of hecate on virus titer was dependent on the multiplicity of infection. Virus titers were reduced 2-28-fold at an M.O.I. of 0.1, 3-6-fold at an M.O.I. of 0.5, and 0-2.5-fold at an M.O.I. of 2.5. Direct treatment of semipurified virions with hecate reduced titers by approximately 4-fold for KOS, 2-fold for tsB5, and over 30-fold for MP.