Antimicrobial peptides from chilli pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chilli pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H⁺-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.     

Isolation and characterization of luffacylin,a ribosome inactivating peptide with anti-fungal activity from sponge gourd (Luffa cylindrica) seeds

A purification scheme involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on CM-Sepharose and Mono S was employed to isolate a peptide with a molecular weight of 7.8kDa from sponge gourd seeds. The peptide, which was designated luffacylin, exhibited an N-terminal sequence with pronounced resemblance to that of the 6.5kDa arginine-glutamate rich polypeptide previously isolated from sponge gourd seeds. Luffacylin inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 140pM and reacted positively in the N-glycosidase assay for ribosome inactivating proteins. Luffacylin exerted anti-fungal activity against Mycosphaerella arachidicola and Fusarium oxysporum.     

Antimicrobial peptides from Adenanthera pavonina L. seeds: characterization and antifungal activity

In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Peptides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently inhibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography, resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22 strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S. cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, revealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.     

Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides

Pre-exposure of the larvae of Galleria mellonella to Candida albicans or Saccharomyces cerevisiae protects against a subsequent infection with 10(6) C. albicans cells. This protection can also be induced by exposing larvae to glucan or laminarin prior to the administration of the potentially lethal inoculum. Analysis of the genes coding for galiomicin, a defensin in G. mellonella, a cysteine-rich antifungal peptide gallerimycin, an iron-binding protein transferrin and an inducible metalloproteinase inhibitor (IMPI) from G. mellonella demonstrated increased expression, which is at its highest after 24 h of the initial inoculum. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increased expression of a number of proteins associated with the insect immune response to infection 24 h after the initial exposure. This study demonstrates that the larvae of G. mellonella can withstand a lethal inoculum of C. albicans if pre-exposed to a non-lethal dose of yeast or polysaccharide 24 h previously which is mediated by increased expression of a number of antimicrobial peptides and the appearance of a number of peptides in the challenged larvae.     

A 2S albumin-homologous protein from passion fruit seeds inhibits the fungal growth and acidification of the medium by Fusarium oxysporum

Antimicrobial proteins have been isolated from a wide range of plant species. More recently, it has become increasingly clear that these types of proteins play an important role in the protection of plants. In this study, we investigate the presence of defense-related proteins from passion fruit (Passiflora edulis f. flavicarpa) seeds. Initially, seed flour was extracted for 2h (at 4 degrees C) with phosphate buffer, pH 5.5. The precipitate obtained between 0 and 70% relative ammonium sulfate saturation was re-dissolved in distilled water and heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant (F/0-70) was extensively dialyzed. A Sephadex G-50 size exclusion column was employed for further separation of proteins. The fraction with antifungal activity was pooled and submitted to CM-Sepharose cation exchange. Two proteins, named Pf1 and Pf2, were eluted in 0.1 and 0.2M of salt, respectively, and submitted to reverse-phase chromatography in HPLC. This fraction inhibited the growth, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae and strongly inhibited glucose-stimulated acidification of the medium by F. oxysporum in a dose-dependent manner. The molecular masses of these proteins, referred to now as Pf1-RP and Pf2-RP, were obtained by MALDI-TOF spectrometry and corresponded to 12,088 Da for Pf1-RP and 11,930 Da for Pf2-RP. These proteins were also subjected to automated N-terminal amino acid sequencing. Sequence comparisons for the heavy subunit of Pf2-RP showed the presence of a protein with a high degree of homology to storage 2S albumins.     

Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans.

We have used the two PFK genes of Saccharomyces cerevisiae encoding the alpha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose-negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero-oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Peptide enrichment and protein fractionation using selective electrophoresis

In recent times, the analysis of the peptidome has become increasingly valuable to gain a better understanding of the critical roles native peptides play in biological processes. Here, we show a technique using a novel electrophoretic device named MF10, for the fractionation of proteins and peptides based on size and also pH in low volume liquid phase under an electric field. A 1 microM, 7-protein and peptide standard mix ranging from 1 to 25 kDa has been used to show peptide migration into a fraction contained by 1-5 kDa membranes. Simultaneous fractionation of the higher mass protein standards to the correct fraction also occurred. To assess the MF10's ability to fractionate more complex samples, human plasma was used to enrich for the peptidome below 5 kDa in the presence of the proteome. Peptide enrichment was achieved while simultaneously fractionating higher mass proteins to three other mass restricted fractions. The utility of this approach is demonstrated with the identification (with at least 2 ppm mass accuracy) of 76 unique peptides, equating to 22 proteins enriched to the 1-5 kDa fraction of the MF10.     

Candida albicans and Saccharomyces cerevisiae expressing ALA1/ALS5 adhere to accessible threonine,serine, or alanine patches

Saccharomyces cerevisiae transformed with Candida albicans ALA1/ALS5 exhibits adherence properties similar to C. albicans. Adherence of the fungi to immobilized proteins involves hydrogen bonds, is stable to shear forces, and is resistant to competition from various biological molecules. The specificity determinants of target recognition in Ala1/Als5p-mediated adherence are not known. To determine features of target recognition, proteins and small peptides were covalently coupled at the N-terminus to the surface of carboxylate-modified magnetic beads. C. albicans yeast cells, germ tubes and pseudohyphae and S. cerevisiae expressing the adhesin, Ala1/Als5p, adhered to beads coated with fibronectin, laminin, type IV collagen, bovine serum albumin, and casein. No adherence to beads was observed if a single amino acid was coupled to the beads. However, 10-mer homopolymers of threonine, serine, and alanine served as ligands for adherence. The presence of a minimum of four contiguous threonine residues in a peptide was required for maximal adherence. Coupling of 10-mer peptides from fibronectin and Ala1/Als5p each possessing 5-7 threonine or serine residues also initiated adherence. On the other hand, a collagen and a fibronectin 10-mer peptide with few threonine and serine residues and lysine at the C-terminus did not serve as adherence ligands. Both of them are converted to adherence ligands by adding threonine or serine residues at the C-terminus or removing the lysine residue and adding threonine residues anywhere in the peptide. The presence of lysine at the C-terminus may have resulted in coupling of the peptides at both the N- and C-termini, thus making the threonine residues inaccessible for adherence. Thus, Ala1/Als5p recognizes patches of certain amino acids, which must be accessible before adherence will occur.     

Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.     

Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.     

Purification,biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties

Background

A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection.

Methods

Peptides from Coffea canephora seeds were extracted in Tris–HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses.

Results

The purified peptide of 9 kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9 kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP₁ exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP₁ interfered in a dose-dependent manner with glucose-stimulated, H⁺-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP₁ family, represented here by Cc-LTP₁, was also able to inhibit mammalian α-amylase activity in vitro.

Conclusions and general significance

In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.     

Inner kinetochore of the pathogenic yeast Candida glabrata

The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure of the C. glabrata inner kinetochore, we isolated several C. glabrata homologs of S. cerevisiae inner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative to S. cerevisiae. CgNDC10, CgCEP3, and CgCTF13 are required for growth in C. glabrata and are specifically found at CgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception of CgCSE4, are species specific and cannot functionally substitute for the corresponding genes in S. cerevisiae deletion strains. Likewise, the S. cerevisiae CBF3 genes NDC10, CEP3, and CTF13 cannot functionally replace their homologs in C. glabrata CBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins of S. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of the C. glabrata inner kinetochore is similar to that in S. cerevisiae.     

A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane

A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.     

Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain

A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.     

NDV F48E9株与La Sota株F蛋白B细胞表位差异分析区段的鉴定和免疫原性比较

本文对预测的NDV F48E9强毒株和La Sota 疫苗株F蛋白优势表位进行比较分析。根据预测结果设计克隆了两个毒株F蛋白各4段表位区,分别将每个毒株的4段表位区连接到原核表达载体上进行了体外表达,鉴定得到4段多肽的大小分别是P1：8.0KD、P2:10.8KD、P5:13.5KD、P6:10.5KD。应用F48E9和LaSota特异性抗血清对表达各肽段进行了抗原性鉴定,P1、P2、P5、P6段均呈阳性反应,表明其中含有线性B细胞表位。其中F48E9 P5 (FP5)和La Sota P5(LP5)与血清反应时有明显差异。FP5和LP5与鸡IL-18成熟肽（m ChIL-18）蛋白以不同组合免疫SPF鸡,ELISA检测各组的免疫活性,结果发现FP5与m ChIL-18蛋白联合免疫组的抗体水平高于LP5与m ChIL-18蛋白联合免疫组、FP5、LP5单独免疫组;用NDV F48E9株攻毒结果显示FP5具有20%的保护率,说明P5段与F蛋白免疫原性相关,而LaSota P5不能抵抗强毒的攻击,证明该表位区域在两个毒株之间存在着免疫原性差异。     

Structure-activity relationships of the yeast alpha-factor

The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation. Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied. These peptides have been analyzed for their biological activities by various assays. Mutants of S. cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues. The mating system of S. cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.