Purification and characterization of a heteromultimeric glycoprotein from Artocarpus heterophyllus latex with an inhibitory effect on human blood coagulation

Plant latex has many health benefits and has been used in folk medicine. In this study, the biological effect of Artocarpus heterophyllus (jackfruit) latex on human blood coagulation was investigated. By a combination of heat precipitation and ion-exchange chromatography, a heat stable heteromultimeric glycoprotein (HSGPL1) was purified from jackfruit milky latex. The apparent molecular masses of the monomeric proteins on SDS/PAGE were 33, 31 and 29 kDa. The isoelectric points (pIs) of the monomers were 6.63, 6.63 and 6.93, respectively. Glycosylation and deglycosylation tests confirmed that each subunit of HSGPL1 formed the native multimer by sugar-based interaction. Moreover, the multimer of HSGPL1 also resisted 2-mercaptoethanol action. Peptide mass fingerprint analysis indicated that HSGPL1 was a complex protein related to Hsps/chaperones. HSGPL1 has an effect on intrinsic pathways of the human blood coagulation system by significantly prolonging the activated partial thrombin time (APTT). In contrast, it has no effect on the human extrinsic blood coagulation system using the prothrombin time (PT) test. The prolonged APTT resulted from the serine protease inhibitor property of HSGPL1, since it reduced activity of human blood coagulation factors XIa and α-XIIa

Antibacterial activity of plasma from crocodile (<it>Crocodylus siamensis</it>) against pathogenic bacteria

Abstract Background The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains. Methods Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity. Results Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates. Conclusions The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.</p

Antimicrobial Action of the Cyclic Peptide Bactenecin on <i>Burkholderia pseudomallei</i> Correlates with Efficient Membrane Permeabilization

<div><p><i>Burkholderia pseudomallei</i> is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against <i>B. pseudomallei</i> and examine the membrane disrupting abilities of the potent antimicrobial peptides: bactenecin, RTA3, BMAP-18 and CA-MA. All peptides exhibited >97% bactericidal activity at 20 µM, with bactenecin having slightly higher activity. Long term time-kill assays revealed a complete inhibition of cell growth at 50 µM bactenecin and CA-MA. All peptides inhibited biofilm formation comparable to CAZ, but exhibited faster kinetics (within 1 h). Bactenecin exhibited stronger binding to LPS and induced perturbation of the inner membrane of live cells. Interaction of bactenecin with model membranes resulted in changes in membrane fluidity and permeability, leading to leakage of dye across the membrane at levels two-fold greater than that of other peptides. Modeling of peptide binding on the membrane showed stable and deep insertion of bactenecin into the membrane (up to 9 Å). We propose that bactenecin is able to form dimers or large β-sheet structures in a concentration dependent manner and subsequently rapidly permeabilize the membrane, leading to cytosolic leakage and cell death in a shorter period of time compared to CAZ. Bactenecin might be considered as a potent antimicrobial agent for use against <i>B. pseudomallei</i>.</p></div

Long-term killing kinetics of four antimicrobial peptides against <i>B. pseudomallei</i>.

<p>Bacterial suspensions were incubated with bactenecin (2A), BMAP-18 (2B), CA-MA (2C), RTA3 (2D), or CAZ (2E) at concentrations of 0 (black square), 20 (black circle), 50 (black triangle), 100 (black diamond) µM and samples were taken at 1, 2, 3, 4, 5, 6, and 24 h. Colonies were counted and the bactericidal effects (dashed line) were defined as a ≥3-log reduction in colony-forming units (CFU)/ml compared to the initial inoculum. Data are the mean of two independent experiments performed in duplicate.</p

LPS binding and membrane permeabilization of four peptides on living bacterial cells.

<p>(A) LPS binding of peptides measured with a polymyxin B-BY displacement assay. <i>B. pseudomallei</i> suspensions at 1×10⁶ CFU/ml were incubated with PMB-BY for 1 hour and treated with the peptides at indicated concentrations. Fluorescence of PMB-BY was monitored 30 min after adding peptides. (B) Permeabilization of the outer membrane by the peptides was observed by NPN uptake. <i>B. pseudomallei</i> cell suspension was incubated with NPN in the presence of 50 µM peptide, and NPN fluorescence was monitored at ex/em 350/429 nm at 0–60 min. The fluorescence intensity upon peptide treatment is reported as a percentage of the maximum fluorescence intensity upon TTX-100 treatment. (C) Permeabilization of the inner membrane by the peptides was assayed by ONPG hydrolysis. The <i>E. coli</i> MG 1655 cell suspension was incubated with 50 µM peptides. Activity of leaked β-galactosidase was measured using ONPG as substrate. ONPG hydrolysis was monitored for 0–2 h. Data are the mean of two independent experiments performed in triplicate. Data are the mean of two independent experiments performed in triplicate.</p

Parameters of spatial orientations in the lipid bilayer of antimicrobial peptides.

<p>The parameters were calculated by the PPM server.</p>a<p>Depth of the deepest non-hydrogen atom relative to position of membrane boundary located at the level of lipid carbonyl group.</p>b<p>Angle between membrane normal and main inertia axis of the molecule.</p>c<p>Binding energy (ΔG_binding) was calculated as the sum of the energy of peptide transfer from water to the lipid bilayer (ΔG_transfer) estimated by PPM and the folding energy (ΔG_folding) of an α-helix estimated by Framework; the folding energy of a β-hairpin stabilized by the disulfide was assumed to be zero.</p

Electron micrographs of <i>B. pseudomallei</i> deformation and cytoplasmic leakage caused by four antimicrobial peptides.

<p>Bacterial suspensions at 1×10⁶ CFU/ml were incubated with or without 50 µM of the peptides for 2 h in a 37°C incubator. Untreated bacterial cells had smooth membranes and uniform shape (A) but shrunk when they were treated with CAZ (B). Cell deformation and leakage of cytosol was observed upon peptide treatment (C: bactenecin, D: BMAP-18, E: CA-MA, and F: RTA3). Scale bar represents 500 nm.</p

β-hairpin model of bactenecin in the oxidized state.

<p>Cys³-Cys¹¹ disulfide bond in gauche-gauche-gauche conformation is formed in the non hydrogen-bonding pair; the 3-residue β-turn is formed by Val⁶-Val⁷-Ile⁸ residues in α_Rγ_Rα_L conformation <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002267#pntd.0002267-Sibanda2" target="_blank">[56]</a>.</p

Dose and time dependent anti-biofilm activity of four antimicrobial peptides.

<p>Bacterial suspensions at 1×10⁶ CFU/ml were incubated with 20, 50 and 100 µM peptides for 1 (black), 2 (dark grey), 3 (light grey) and 4 (white) hrs at 37°C. The cells were collected and cultured for 2 days and biofilm mass was measured. The anti-biofilm activities were calculated by [(1-A_T/A_C)×100%], where A_T is the absorbance of the biofilm mass from <i>B. pseudomallei</i> treated with the peptides or CAZ, and A_C is the absorbance (550 nm) of biofilm mass from bacteria only. Data are the mean of two independent experiments performed in triplicate.</p