Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of Penicillium Rot of Apples

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Most compounds were highly active against F. oxysporum (MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in F. oxysporum microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against P. expansum of a 125-member peptide library with the general structure R-X¹KLFKKILKX¹⁰L-NH₂, where X¹ and X¹⁰ corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X¹, and X¹⁰ positions and where Ac is an acetyl group. The peptides had sporicidal activity against P. expansum conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of P. expansum infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH₂ (BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.The discovery of antimicrobial compounds to treat plant diseases of economical importance in agriculture remains a major scientific challenge (1). Antimicrobial peptides are being considered as a good alternative to current fungicides and a great deal of scientific effort has been invested in studying their application in plant disease control (29, 34, 35).Antimicrobial peptides have been reported to display interesting activities against pathogenic microbes that are resistant to conventional antibiotics and to exhibit a broad spectrum of activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (7-10, 19, 20, 40, 49). The mechanism of action of these peptides against fungi consists of cell lysis by binding to the membrane surface and disrupting its structure, interference with the synthesis of essential cell wall components, or interaction with specific internal targets (12, 13, 15, 23, 29).Despite their good lytic activity, major concerns about the use of antimicrobial peptides as pesticides in plant protection are the high production cost associated with synthetic procedures and their low stability toward protease degradation. Several design strategies have been devised in order to find shorter and more stable peptides, while maintaining or increasing the activity with a low cytotoxicity. These strategies include the juxtaposition of fragments of natural antimicrobial peptides, the modification of natural peptides, and the de novo design of sequences maintaining the crucial features of native antimicrobial peptides (2, 3, 11, 24, 32, 38, 42). However, the process involved in the development of lead candidates is time consuming and limited by the number of individual compounds that can be synthesized. Combinatorial chemistry has allowed the rapid preparation of synthetic libraries and their screening has led to the identification of peptides with high activity against selected phytopathogenic bacteria and fungi (4, 26, 27, 33).During our current research oriented to the development of new antimicrobial agents for use in plant protection, we designed linear undecapeptides (CECMEL11) derived from the cecropin A-melittin hybrid peptide WKLFKKILKVL-NH₂ (Pep3) (5, 17). Using a combinatorial approach, we identified peptides with high activity against plant pathogenic bacteria, such as Erwinia amylovora, Xanthomonas vesicatoria, and Pseudomonas syringae, and with low susceptibility to protease degradation (4, 5).In order to broaden the study, we decided to test the CECMEL11 peptides against the plant pathogenic fungi Fusarium oxysporum, Aspergillus niger, Rhizopus stolonifer, and Penicillium expansum. The fungus F. oxysporum causes Fusarium wilt in more than a hundred species of plants, and it is an important pathogen in horticultural crops (44). Several Rhizopus and Penicillium species cause soft rot and blue mold rot, respectively, which are important postharvest diseases in stone and pome fruits (6, 18, 22, 39). Apart from the economic losses, Aspergillus and Penicillium species are also of interest from a public health point of view due to the production of mycotoxins (45, 47). The importance of Penicillium species in the postharvest of fruits emphasizes the interest to develop antimicrobial peptides to control this fungus.Taking into account the relevance of these pathogens, the aim of the present study was the analysis of the antifungal activity profile of the CECMEL11 peptides in order to identify sporicidal sequences against the above fungi. As a proof of concept, the feasibility of using such peptides to protect fruits from fungal spoilage was evaluated using a P. expansum/apple model.     

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression

Background

Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish.

Methodology/Principal Findings

Individual zebrafish (n = 10) were able to swim at a critical swimming speed (U_crit) of 0.548±0.007 m s⁻¹ or 18.0 standard body lengths (BL) s⁻¹. The optimal swimming speed (U_opt) at which energetic efficiency is highest was 0.396±0.019 m s⁻¹ (13.0 BL s⁻¹) corresponding to 72.26±0.29% of U_crit. The cost of transport at optimal swimming speed (COT_opt) was 25.23±4.03 µmol g⁻¹ m⁻¹. A group-wise experiment was conducted with zebrafish (n = 83) swimming at U_opt for 6 h day⁻¹ for 5 days week⁻¹ for 4 weeks vs. zebrafish (n = 84) that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb), insulin-like growth factor 1 receptor a (igf1ra), troponin C (stnnc), slow myosin heavy chain 1 (smyhc1), troponin I2 (tnni2), myosin heavy polypeptide 2 (myhz2) and myostatin (mstnb).

Conclusions/Significance

From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.     

Accuracy of serum luteinizing hormone and serum testosterone measurements to assess the efficacy of medical castration in prostate cancer patients

Background

Luteinizing hormone-releasing hormone (LH-RH) agonists are the standard for androgen deprivation therapy (ADT) in prostate cancer (PCa) patients. Current guidelines recommend serum testosterone measurement to assess the efficacy of ADT and to define castration resistance. However, serum testosterone does not reflect the exclusive effect of castration due to its extratesticular production. The aim of this study is to analyze if serum LH reflects better than serum testosterone the activity of LH-RH agonists.

Methods

Serum LH and serum testosterone were measured with chemiluminescent immunoassay (CLIA) in a cohort study of 1091 participants: 488 PCa patients “on LH-RH agonists”, 303 “off LH-RH agonist” in whom LH-RH agonists were withdrawn, and 350 men with PCa suspicion “no LH-RH agonist” who never received LH-RH agonists. In a validation cohort of 147 PCa patients, 124 on “LH-RH agonists” and 19 “off LH-RH agonists”, serum testosterone was also measured with liquid chromatography and tandem mass spectrometry (LC MSMS).

Results

The area under the curve (AUC) to distinguish patients “on versus off LH-RH agonists” was 0.997 for serum LH and 0.740 for serum testosterone, P < 0.001. The 97.5 percentile of serum LH in patients “on LH-RH agonists” was 0.97 U/L, been the most efficient threshold 1.1 U/L. The AUCs for serum LH, testosterone measured with CLIA and with LC MSMS, in the validation cohort, were respectively 1.000, 0.646 and 0.814, P < 0.001. The efficacy to distinguish patients “on versus off LH-RH agonists” was 98.6%, 78.3%, and 89.5% respectively, using 1.1 U/L as threshold for serum LH and 50 ng/dL for serum testosterone regardless the method.

Conclusions

Serum LH is more accurate than serum testosterone regardless the method, to distinguish patients “on versus off LH-RH agonists”. The castrate level of serum LH is 1.1 U/l. These findings suggest that assessment of LH-RH agonist efficacy and castration resistance definition should be reviewed.

     

Structural basis for the enhanced activity of cyclic antimicrobial peptides: the case of BPC194

We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides.     

Improvement of the efficacy of linear undecapeptides against plant-pathogenic bacteria by incorporation of D-amino acids

A set of 31 undecapeptides, incorporating 1 to 11 d-amino acids and derived from the antimicrobial peptide BP100 (KKLFKKILKYL-NH(2)), was designed and synthesized. This set was evaluated for inhibition of growth of the plant-pathogenic bacteria Erwinia amylovora, Pseudomonas syringae pv. syringae, and Xanthomonas axonopodis pv. vesicatoria, hemolysis, and protease degradation. Two derivatives were as active as BP100, and 10 peptides displayed improved activity, with the all-d isomer being the most active. Twenty-six peptides were less hemolytic than BP100, and all peptides were more stable against protease degradation. Plant extracts inhibited the activity of BP100 as well as that of the d-isomers. Ten derivatives incorporating one d-amino acid each were tested in an infectivity inhibition assay with the three plant-pathogenic bacteria by using detached pear and pepper leaves and pear fruits. All 10 peptides studied were active against E. amylovora, 6 displayed activity against P. syringae pv. syringae, and 2 displayed activity against X. axonopodis pv. vesicatoria. Peptides BP143 (KKLFKKILKYL-NH(2)) and BP145 (KKLFKKILKYL-NH(2)), containing one d-amino acid at positions 4 and 2 (underlined), respectively, were evaluated in whole-plant assays for the control of bacterial blight of pepper and pear and fire blight of pear. Peptide BP143 was as effective as streptomycin in the three pathosystems, was more effective than BP100 against bacterial blight of pepper and pear, and equally effective against fire blight of pear.     

Detection and discrimination between ochratoxin producer and non-producer strains of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> on a ham-based medium using an electronic nose

The aim of this work was to evaluate the potential use of qualitative volatile patterns produced by Penicillium nordicum to discriminate between ochratoxin A (OTA) producers and non-producer strains on a ham-based medium. Experiments were carried out on a 3% ham medium at two water activities (a_w ; 0.995, 0.95) inoculated with P. nordicum spores and incubated at 25°C for up to 14 days. Growing colonies were sampled after 1, 2, 3, 7 and 14 days, placed in 30-ml vials, sealed and the head space analysed using a hybrid sensor electronic nose device. The effect of environmental conditions on growth and OTA production was evaluated based on the qualitative response. However, after 7 days, it was possible to discriminate between strains grown at 0.995 a_w, and after 14 days, the OTA producer and non-producer strain and the controls could be discriminated at both a_w levels. This study suggests that volatile patterns produced by P. nordicum strains may differ and be used to predict the presence of toxigenic contaminants in ham. This approach could be utilised in ham production as part of a quality assurance system for preventing OTA contamination.     

Expression and characterization of a Mycoplasma genitalium glycosyltransferase in membrane glycolipid biosynthesis: potential target against mycoplasma infections

Mycoplasmas contain glycoglycerolipids in their plasma membrane as key structural components involved in bilayer properties and stability. A membrane-associated glycosyltransferase (GT), GT MG517, has been identified in Mycoplasma genitalium, which sequentially produces monoglycosyl- and diglycosyldiacylglycerols. When recombinantly expressed in Escherichia coli, the enzyme was functional in vivo and yielded membrane glycolipids from which Glcβ1,6GlcβDAG was identified as the main product. A chaperone co-expression system and extraction with CHAPS detergent afforded soluble protein that was purified by affinity chromatography. GT MG517 transfers glucosyl and galactosyl residues from UDP-Glc and UDP-Gal to dioleoylglycerol (DOG) acceptor to form the corresponding β-glycosyl-DOG, which then acts as acceptor to give β-diglycosyl-DOG products. The enzyme (GT2 family) follows Michaelis-Menten kinetics. k(cat) is about 5-fold higher for UDP-Gal with either DOG or monoglucosyldioleoylglycerol acceptors, but it shows better binding for UDP-Glc than UDP-Gal, as reflected by the lower K(m), which results in similar k(cat)/K(m) values for both donors. Although sequentially adding glycosyl residues with β-1,6 connectivity, the first glycosyltransferase activity (to DOG) is about 1 order of magnitude higher than the second (to monoglucosyldioleoylglycerol). Because the ratio between the non-bilayer-forming monoglycosyldiacylglycerols and the bilayer-prone diglycosyldiacylglycerols contributes to regulate the properties of the plasma membrane, both synthase activities are probably regulated. Dioleoylphosphatidylglycerol (anionic phospholipid) activates the enzyme, k(cat) linearly increasing with dioleoylphosphatidylglycerol concentration. GT MG517 is shown to be encoded by an essential gene, and the addition of GT inhibitors results in cell growth inhibition. It is proposed that glycolipid synthases are potential targets for drug discovery against infections by mycoplasmas.     

Mycobacterium hippocampi sp. nov., a Rapidly Growing Scotochromogenic Species Isolated from a Seahorse with Tail Rot

A Gram-positive, aerobic, non-motile, non-sporulating, acid-fast, and rod-shaped bacterium (BFLP-6^T), previously isolated from a seahorse (Hippocampus guttulatus) with tail rot, was studied using a polyphasic taxonomic approach. Growth occurred at 15–35 °C (optimum 25 °C), at pH 5.0–10.0 (optimum pH 7.0) and at NaCl concentrations between 0 and 6 % (w/v). The G+C content of DNA was 66.7 mol%. The predominant fatty acids were C_18:1 ω9c, C_16:0 and C_16:1 ω6c. A mycolic acid pattern of alpha-mycolates and keto-mycolates was detected. Analysis of concatenated sequences (16S rRNA, rpoB, ssrA and tuf genes), and chemotaxonomic and phenotypic features indicated that strain BFLP-6^T represents a novel species within the genus Mycobacterium, for which the name Mycobacterium hippocampi sp. nov. is proposed. The type strain is BFLP-6^T (=DSM 45391^T =LMG 25372^T).     

Fish Insulin, IGF-I and IGF-II Receptors: A Phylogenetic Approach

In fish, the structural and functional characteristics of insulinand IGF-I receptors have been well studied. Current evidenceindicates that all gnatostome animals, from fish to mammals,contain separate insulin and IGF-I molecules and specific receptorsfor insulin and IGF-I. However, qualitative differences in thefunctional aspects of insulin and IGF-I receptors among vertebratespecies can account for variations in the biological activityof insulin and IGF-I. In this paper we will focus on the functionalevolution of the insulin and IGF-I receptors in vertebratesand on the appearance of the unrelated IGF-II receptors.