Antinematodal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Caenorhabditis elegans.

The antinematodal activity and mechanism of a 23-mer antimicrobial peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed a strong antinematodal activity against the eggs and worms of Caenorhabditis elegans. To investigate the antinematodal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. C. elegans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide (PI) staining than normal cells. Confocal microscopy showed that the peptide was localized in the egg's shell and cell membrane. The action of the peptide against C. elegans membranes was examined by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w). The result suggests that PMAP-23 may exert its antinematodal activity by disrupting the structure of the cell membrane via pore formation or via direct interaction with the lipid bilayers.     

Design of novel peptide analogs with potent fungicidal activity,based on PMAP-23 antimicrobial peptide isolated from porcine myeloid

PMAP-23 is a 23-mer peptide derived from porcine myeloid. To develop novel antifungal peptides useful as therapeutic drugs, it would require a strong fungicidal activity against pathogenic fungal cells. To this goal, several analogs, with amino acid substitutions, were designed to increase the net hydrophobicity by Trp (W)-substitution at positions 10, 13, or 14 at the hydrophilic face of PMAP-23 without changing the hydrophobic helical face. The Trp (W)-substitution (P6) showed an enhanced fungicidal and antitumor activities, with the fungicidal activity inhibited by salts and the respiratory inhibitor, NaN(3). The results suggested that the increase of hydrophobicity of the peptides correlated with fungicidal activity. The fungicidal effects of analog peptides were further investigated using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. In Candida albicans, the analog peptide (P6) exerted its fungicidal effect on the blastoconidia in 20% fetal bovine serum by disrupting the mycelial forms. Furthermore, P6 caused significant morphological changes, and these facts suggested that the fungicidal function of the novel analog peptide (P6) was by damaging the fungal cell membranes. Thus, this peptide may provide a useful template for designing novel antifungal peptides useful for the treatment of infectious diseases.     

Fungicidal effect of human lactoferrin against Candida albicans

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37 degrees C. Fe(3+)-saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37 degrees, 42 degrees C). The presence of 1 mM D-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the anti-fungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.     

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide,PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH₂) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K⁺ efflux were observed in Candida albicans cells treated with 5?µM PMAP-23. In contrast, 2.5?µM PMAP-23 caused membrane depolarisation and K⁺ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca²⁺ levels. Disruption of Ca²⁺ homeostasis altered the NAD⁺/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca²⁺ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca²⁺-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca²⁺-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca²⁺ and ROS in apoptosis regulation.     

猪源抗菌肽PMAP-36的序列设计及其结构优化的研究进展

抗生素的耐药性和动物源性食品中的药物残留问题严重威胁全球公共卫生系统.因此,开发出不易产生耐药性、抗菌活性高的新型抗菌药物迫在眉睫.抗菌肽因其分子量小、抗菌谱广、不易产生耐药性等优点受到科学家们的广泛关注,但天然抗菌肽具有抗菌活性低、溶血活性和细胞毒性等缺陷.随着抗菌肽序列和结构的不断优化,多种具有显著体内外抗菌活性且...     

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.     

Fungicidal effect of prenylated flavonol, papyriflavonol A, isolated from Broussonetia papyrifera (L.) vent. against Candida albicans

Papyriflavonol A (PapA), a prenylated flavonoid (5,7,3',4'-tetrahydroxy-6,5'-di-(r,r-dimethylallyl)-flavonol), was isolated from the root barks of Broussonetia papyriferra. Our previous study showed that PapA has a broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. In this study, the mode of action of PapA against Candida albicans was investigated to evaluate PapA as antifungal agent. The minimal inhibitory concentration (MIC) values were 10~25 microgram/ml for C. albicans and Saccharomyces cerevisiae, gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and gram-positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus). The kinetics of cell growth inhibition, scanning electron microscopy, and measurement of plasma membrane florescence anisotrophy revealed that the antifungal activity of PapA against C. albicans and S. cerevisiae is mediated by its ability to disrupt the cell membrane integrity. Compared with amphotericin B, a cell membrane disrupting polyene antibiotic, the hemolytic toxicity of PapA was negligible. At 10~25 microgram/ml of MIC levels for the tested strains, the hemolysis ratio of human erythrocytes was less than 5%. Our results suggest that PapA could be a therapeutic fungicidal agent having a broad spectrum antimicrobial agent.     

Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP1, from Impatiens balsamina against Candida albicans

The antifungal mechanism of a 20-mer peptide, Ib-AMP1, derived from Impatiens balsamina was investigated. The oxidized (disulfide bridged) Ib-AMP1 showed a 4-fold increase in antifungal activity against Aspergillus flavus and Candida albicans than reduced (non-disulfide bridged) Ib-AMP1. Ib-AMP1 had very low activity for phospholipid disruption when compared with cecropin A(1-8)-magainin 2(1-12), a -helical amphiphatic, antimicrobial peptide. Confocal microscopy showed that Ib-AMP1 binds on cell surface or penetrates into cell membranes. These results suggested that Ib-AMP1 may manifest its antifungal activity against Candida albicans by inhibiting a distinct cellular process rather than ion channel or pore formation in cell membrane.     

Fungicidal effect of antimicrobial peptide arenicin-1

Arenicin-1 is a 21-residue peptide which was derived from Arenicola marina. In this study, we investigated the antifungal effects and its mechanism of action towards human pathogenic fungi. Arenicin-1 exerted remarkable fungicidal activity with both energy-dependent and salt-insensitive manners. To investigate the fungicidal mechanisms of arenicin-1, the membrane interactions of arenicin-1 were examined. Flow cytometric analysis, using propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], as well as fluorescence analysis, regarding the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), were conducted against Candida albicans. The results demonstrated that arenicin-1 was associated with lipid bilayers and induced membrane permeabilization. Additionally, the membrane studies in regard to liposomes resembling the phospholipid bilayer of C. albicans confirmed the membrane-disruptive potency of arenicin-1. Therefore, the present study suggests that arenicin-1 exerts its fungicidal effect by disrupting fungal phospholipid membranes.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.     

A novel antimicrobial peptide derived from human BPIFA1 protein protects against Candida albicans infection

Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is an innate immunity defense protein. Our previous studies proved its antibacterial and antiviral effects, but its role in fungi remains unknown. The study aimed to identify antifungal peptides (AFP) derived from BPIFA1, and three antimicrobial peptides (AMP1–3) were designed. The antifungal effects were proved by growth inhibition assay. AMP3 activity was confirmed by germ tube growth experiment and XTT assay. Its effects on cell wall and membrane of Candida albicans were assessed by tannic acid and Annexin V-FITC/PI double staining, respectively. Additionally, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for morphological and ultrastructural observation. The expression of ALS1, EAP1, and SUN41 was tested by qPCR. Ultimately, three AMPs could fight against C. albicans in vitro, and AMP3 was highly effective. It functioned by destroying the integrity of cell wall and normal structure of cell membrane. It also inhibited biofilm formation of C. albicans. In addition, AMP3 down-regulated the expression of ALS1, EAP1, and SUN41, those are known to be involved in virulence of C. albicans. Altogether, the study reported successful development of a novel AFP, which could be used as a new strategy for antifungal therapy.     

家蝇抗菌肽AMP-17对白色念珠菌菌丝的抑制作用

【背景】AMP-17是从微生物诱导的家蝇转录组数据库筛选到的一条特异性高表达基因,采用原核表达体系获得其重组蛋白并证实了具有显著的抗菌效果,特别是对白色念珠菌具有较强的抗菌活性。【目的】研究抗菌肽AMP-17对白色念珠菌菌丝的抑制作用。【方法】采用微量液体稀释法测定AMP-17对11株白色念珠菌的最小抑菌浓度(minimal inhibitory concentration,MIC);根据对AMP-17的敏感程度选取3株绘制生长曲线;通过光学显微镜观察并计数经AMP-17作用后白色念珠菌芽生孢子生成率及芽管形成率;倒置荧光显微镜观察白色念珠菌酵母相向菌丝相转化及以菌丝相为起点AMP-17促进菌丝相转化为酵母相的情况。【结果】AMP-17对支气管肺泡灌洗液分离株16105的MIC为10μg/mL,对粪便分离株16214的MIC为40μg/mL,对其余9株白色念珠菌的MIC均为20μg/mL;白色念珠菌经不同浓度的AMP-17作用后,各时间点的芽生孢子生成率均显著低于对照组,尤其是40μg/mL的AMP-17组,芽生孢子生成率仅15%,显著低于阳性药物氟康唑;各实验组芽管形成率显著低于对照...     

Membrane perturbation by the antimicrobial peptide PMAP-23: A fluorescence and molecular dynamics study

Several bioactive peptides exert their biological function by interacting with cellular membranes. Structural data on their location inside lipid bilayers are thus essential for a detailed understanding of their mechanism of action. We propose here a combined approach in which fluorescence spectroscopy and molecular dynamics (MD) simulations were applied to investigate the mechanism of membrane perturbation by the antimicrobial peptide PMAP-23. Fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the peptide inside the membrane. MD simulations were performed starting from a random mixture of water, lipids and peptide, and following the spontaneous self-assembly of the bilayer. Both experimental and theoretical data indicated a peptide location just below the polar headgroups of the membrane, with an orientation essentially parallel to the bilayer plane. These findings, together with experimental results on peptide-induced leakage from large and giant vesicles, lipid flip-flop and peptide exchange between vesicles, support a mechanism of action consistent with the “carpet” model. Furthermore, the atomic detail provided by the simulations suggested the occurrence of an additional, more specific and novel mechanism of bilayer destabilization by PMAP-23, involving the unusual insertion of charged side chains into the hydrophobic core of the membrane.     

Responses of Candida albicans to the human antimicrobial peptide LL-37

Candida albicans is amajor fungal pathogen in humans. Antimicrobial peptides (AMPs) are critical components of the innate immune response in vertebrates and represent the first line of defense against microbial infection. LL-37 is the only member of the human family of cathelicidin AMPs and is commonly expressed by various tissues and cells, including surfaces of epithelia. The candidacidal effects of LL-37 have been well documented, but the mechanisms by which LL-37 kills C. albicans are not completely understood. In this study, we examined the effects of LL-37 on cell wall and cellular responses in C. albicans. Using transmission electron microscopy, carbohydrate analyses, and staining for β-1,3-glucan, changing of C. albicans cell wall integrity was detected upon LL-37 treatment. In addition, LL-37 also affected cell wall architecture of the pathogen. Finally, DNA microarray analysis and quantitative PCR demonstrated that sub-lethal concentrations of LL-37 modulated the expression of genes with a variety of functions, including transporters, regulators for biological processes, response to stress or chemical stimulus, and pathogenesis. Together, LL-37 induces complex responses in C. albicans, making LL-37 a promising candidate for use as a therapeutic agent against fungal infections.     

Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans

Pleurocidin (GWGSFFKKAAHVGKHVGKAALTHYL-NH₂), found in skin mucous secretions of the winter flounder Pleuronectes americanus, is known to possess a high potency and broad-spectrum antimicrobial peptide without cytotoxicity. In this study, to investigate the impact of pleurocidin on apoptotic progress, we observed morphological and physiological changes in Candida albicans. In cells exposed to pleurocidin, intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased, and hydroxyl radicals were especially a large part of ROS. The increase of ROS induced oxidative stress and mitochondrial membrane depolarization which causes release of pro-apoptotic factors. Using FITC-VAD-FMK staining, we confirmed activation of yeast metacaspases which lead to apoptosis and phosphatidylserine externalization at early stage apoptosis was observed using annexin V FITC. In addition, pleurocidin induced-apoptotic cells underwent apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC) in flow cytometry dot plots. Under the influence of oxidative stress, DNA and nuclei were fragmented and condensed in cells, and they were visualized by 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These apoptotic phenomena represent that oxidative stress by inducing pleurocidin must be an important factor of the apoptotic process in C. albicans.     

Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans

Lactobacillus pentosus TV35b, isolated from the posterior fornix secretions of the vagina of a prenatal patient, produced a bacteriocin-like peptide (pentocin TV35b), which is inhibitory to Clostridium sporogenes, Cl. tyrobutyricum, Lact. curvatus, Lact. fermentum, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp. and Candida albicans. The mechanism of activity of pentocin TV35b is bactericidal, as shown by a decrease in the viable cell numbers of Lact. sake from approximately 4 x 108 to less than 10 cfu ml - 1 over a period of 4 h. Pentocin TV35b added to the growth medium of C. albicans stimulated the formation of pseudohyphae during the first 36 h, followed by a slight repression in cell growth. Production of pentocin TV35b was at its maximum towards the end of the logarithmic growth phase of strain TV35b. The peptide was purified by ammonium sulphate precipitation, followed by SP-Sepharose cation exchange chromatography. The molecular size of pentocin TV35b was estimated to be between 2.35 and 3.4 kDa, according to tricine-SDS PAGE. However, results obtained by electrospray ionization mass spectroscopy indicated that the peptide is 3930.2 Da in size. Amino acid analysis performed by using the Pico-Tag(R) method and a Nova-Pak C18 HPLC column indicated that pentocin TV35b consists of 33 amino acids with a total mass of 3929.63 Da. Pentocin TV35b is inactivated when treated with papain and Proteinase K, but remains active after incubation at pH 1-10 for 2 h at 25 degrees C, and when heat-treated for 30 min at 100 degrees C.     

Serotyping of Candida albicans isolated from clinical specimens

A specific antiserum to Candida albicans serotype A was prepared absorbing a total antiserum with Candida albicans serotype B cells. This specific antiserum was used for serotyping C. albicans strains obtained from patients in different hospitals of Havana City, Cuba. Two hundred strains (95.2%) were serotype A, the remaining 10 (4.8%) were serotype B. Results were also correlated with strains isolated from the specimen origin, sex and race of the patient. The usefulness of this specific antiserum to determine C. albicans serotypes and its therapeutic value are pointed out.     

Diorcinol D Exerts Fungicidal Action against Candida albicans through Cytoplasm Membrane Destruction and ROS Accumulation

Candida albicans, which is the most common human fungal pathogen, causes high mortality among immunocompromised patients. Antifungal drug resistance becomes a major challenge for the management of Candida infection. Diorcinol D (DD), a diphenyl ether derivative isolated from an endolichenic fungus, exerted fungicidal action against Candida species. In this study, we investigated the possible mechanism of its antifungal activity. The change of membrane dynamics and permeability suggested that the cell membrane was disrupted by the treatment of DD. This was further supported by the evidences of intracellular glycerol accumulation, alteration of cell ultrastructure, and down-regulation of genes involved in cell membrane synthesis. In addition, the treatment of C. albicans with DD resulted in the elevation of reactive oxygen species (ROS), which caused the dysfunction of mitochondria. These altogether suggested that DD exerted its antifungal activity through cytoplasmic membrane destruction and ROS accumulation. This finding is helpful to uncover the underlying mechanisms for the diphenyl ether derivatives and provides a potential application in fighting clinical fungal infections.     

The antimicrobial peptide, psacotheasin induces reactive oxygen species and triggers apoptosis in Candida albicans

Previously, the antimicrobial effects and membrane-active action of psacotheasin in Candida albicans were investigated. In this study, we have further found that a series of characteristic cellular changes of apoptosis in C. albicans can be induced by the accumulation of intracellular reactive oxygen species, specifically hydroxyl radicals, the well-known important regulators of apoptosis. Cells treated with psacotheasin showed diagnostic markers in yeast apoptosis at early stages: phosphatidylserine externalization from the inner to the outer membrane surface, visualized by Annexin V-staining; mitochondrial membrane depolarization, observed by DiOC₆(3) staining; and increase of metacaspase activity, measured using the CaspACE FITC-VAD-FMK. Moreover, DNA fragmentation and condensation also revealed apoptotic phenomena at late stages through the TUNEL assay staining and DAPI staining, respectively. Taken together, our findings suggest that psacotheasin possess an antifungal property in C. albicans via apoptosis as another mode of action.