Sompong Klaynongsruang

Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria.

We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1-5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 μM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.

Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide.

Peptide lipidation has proven to be an inexpensive and effective strategy for designing next-generation peptide-based drug compounds. In this study, the effect of the acyl chain length of ultrashort LiPs (CX-KYR-NH2; X=10, 12, 14 and 16) on their bacterial killing and membrane disruption kinetics was investigated. The geometric mean of the minimum inhibitory concentration (MIC) values for 4 pathogenic bacterial strains was 25 μM, with a selectivity index of 10.24 for C14-KYR-NH2. LiPs at all concentrations exhibited no cytotoxicity towards human erythrocytes, but towards Vero cells at 80 μM. All the LiPs adopted secondary structure in a membrane mimicking environment. C14-KYR-NH2 aggregated above 256 μM, while C16-KYR-NH2 did above 80 μM. All LiPs showed outer membrane permeabilization within 3 min after treatment, yet the extent and kinetics of inner membrane penetration and depolarization were dependent on the acyl chain length. Cell death subsequently occurred within 10 min, and killing activity appeared to correlate most with depolarization activity but not with outer or inner membrane permeability. AFM imaging of cells treated with C14-KYR-NH2 revealed rupture of the cell surface and cytosolic leakage depending on the length of incubation. This study highlights and follows the progression of events that occur during the membrane disintegration process over time, and determines the optimal amphipathicity of ultrashort LiPs with 12-14 carbon atoms for this membrane disrupting activity. The fast acting bactericidal properties of ultrashort LiPs with optimal chain lengths make them promising candidates for drug lead compounds.

In Vivo Wound Healing Activity of Crocodile (Crocodylus siamensis) Hemoglobin and Evaluation of Antibacterial and Antioxidant Properties of Hemoglobin and Hemoglobin Hydrolysate.

The hydrolysis of proteins constitutes an invaluable tool, granting access to a variety of peptide fragments with potentially interesting biological properties. Therefore, a hemoglobin (Hb) hydrolysate of Crocodylus siamensis was generated by digestion under acidic conditions. The antibacterial and antioxidant activities of the Hb hydrolysate were assessed in comparison with intact Hb. A disc diffusion assay revealed that the Hb hydrolysate exhibited antibacterial activity against eight strains of gram-positive bacteria and showed a higher efficacy than intact Hb. Moreover, the antioxidant activity of intact Hb and its hydrolysate was evaluated using ABTS and DPPH radical scavenging assays. The Hb hydrolysate exhibited free radical scavenging rates of 6-32%, whereas intact Hb showed a slightly higher activity. In addition, non-toxicity to human erythrocytes was observed after treatment with quantities of Hb hydrolysate up to 10 μg. Moreover, active fragmented Hb (P3) was obtained after purifying the Hb hydrolysate by reversed-phase HPLC. Scanning electron microscopy demonstrated the induction of bacterial cell membrane abnormalities after exposure to P3. Antibacterial and antioxidant activities play crucial roles for supporting the wound healing activity. Consequently, an in vivo mice excisional skin wound healing assay was carried out to investigate the effects of intact Hb treatment on wound healing in more detail. The results clearly demonstrate that intact Hb is capable of promoting 75% wound closure within 6 days. These findings imply that intact Hb of C. siamensis and its acid hydrolysate may serve as valuable precursors for food supplementary products benefitting human health.

Biochemical characterization of a new glycosylated protease from Euphorbia cf. lactea latex

A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35 °C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (β-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69 mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (Km) of 3.30 ± 0.26 μM; a maximal velocity (Vmax) of 400.9 ± 0.85 ng min(-1); and a catalytic efficiency (Vmax/Km) of 121.5 ± 9.25 ng μM(-1) min(-1). EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.

Comparative proteomic analysis of leaves, leaf sheaths, and roots of drought-contrasting sugarcane cultivars in response to drought stress

A better understanding of drought response proteins may improve our understanding of the mechanisms underlying drought tolerance in sugarcane. In this research, drought-tolerant (K86-161) and drought-sensitive (B34-164) sugarcane cultivars were grown and exposed to drought stress. The changes in protein expression in leafs, leaf sheaths and roots were analyzed using proteomics techniques. Proteins that responded to drought in both cultivars could be classified into four major categories, including energy and metabolism, photosynthesis, antioxidant, and defense protein. Interestingly, an increased abundance of fructose-bisphosphate aldolase under drought was observed in all three organs of K86-161. Elevated expression of oxygen-evolving enhancer protein was also found in leaves and leaf sheaths of K86-161, when compared with their controls. Additionally, SOD was abundant in the leaves and roots of K86-161. Importantly, the expression level of these proteins decreased in B34-164 under drought stress. These contrasting results suggest that these proteins were inhibited by drought stress in the drought-sensitive cultivar. This proteomic research is the first to combine analyses of leaves, leaf sheaths and roots in sugarcane, which may enhance our understanding of drought responses at the molecular level and lead to selective breeding for enhanced drought tolerance.

Protective Effect of Crocodile Hemoglobin and Whole Blood Against Hydrogen Peroxide-Induced Oxidative Damage in Human Lung Fibroblasts (MRC-5) and Inflammation in Mice

A putative protective effect of cHb and cWb against H2O2-induced oxidative damage was evaluated in detail using MRC-5 cells. In addition, the carrageenan (Carr)-induced mouse paw edema model and the cotton pellet-induced granuloma model were employed to examine the in vivo anti-inflammatory activity of cHb and cWb in mice. It was demonstrated that both cHb and cWb treatments significantly increased cell viability and inhibited morphology alterations in MRC-5 cells exposed to H2O2. Orally administered cHb and cWb significantly reduced Carr-induced paw edema volume and cotton pellet-induced granuloma formation. Moreover, cHb and cWb decreased the expression levels of important pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), while only cWb was found to increase the expression of the anti-inflammatory cytokine IL-10 significantly. Finally, the activity of antioxidant enzymes (SOD, CAT, and GPx) in the liver improved after cHb and cWb treatment under acute and chronic inflammation. Taken collectively, the results of this study suggest that both cHb and cWb protect against hydrogen peroxide-induced damage in fibroblast cells. Moreover, cHb and cWb were found to exhibit anti-inflammatory activity in both the acute and chronic stages of inflammation and appear to enhance antioxidant enzyme activity and decrease lipid peroxidation in the livers of mice. Therefore, this study indicates that cHb and cWb have great potential to be used in the development of dietary supplements for the prevention of oxidative stress related to inflammatory disorders.

Comparison of recombinant α-hemoglobin from Crocodylus siamensis expressed in different cloning vectors and their biological properties

Hemoglobin (Hb) is an important component in red blood cells of the vertebrate. It is a major respiratory protein with oxygen or carbon dioxide transport function. Hb has been reported to contain bioactive peptides which have antibacterial and antioxidant activities. In this study, the alpha-chain hemoglobin(αHb) gene of Crocodylus siamensis was cloned into the three different expression vectors and expressed in Escherichia coli BL21 (DE3). The recombinant αHb proteins from all constructs could be expressed and purified. The result from UV-visible absorption spectra showed a similar pattern of all recombinant proteins to the oxy-hemoglobin form of intact Hb. The different recombinant αHb could exhibit antioxidant activities. All recombinant proteins could inhibit the growth of Bacillus spp. Especially, most of the recombinant proteins could inhibit the growth of Bacillus amyloliquefaciens TISTR 1045 better than intact one. The result obtained from this study can provide us further information about the possibility using of αHb as a supplementary food.

Gallic acid conjugated with gold nanoparticles: antibacterial activity and mechanism of action on foodborne pathogens.

Foodborne pathogens, including Plesiomonas shigelloides and Shigella flexneri B, are the major cause of diarrheal endemics worldwide. Antibiotic drug resistance is increasing. Therefore, bioactive compounds with antibacterial activity, such as gallic acid (GA), are needed. Gold nanoparticles (AuNPs) are used as drug delivery agents. This study aimed to conjugate and characterize AuNP–GA and to evaluate the antibacterial activity. AuNP was conjugated with GA, and the core–shell structures were characterized by small-angle X-ray scattering and transmission electron microscopy. Antibacterial activity of AuNP–GA against P. shigelloides and S. flexneri B was evaluated by well diffusion method. AuNP–GA bactericidal mechanism was elucidated by Fourier transform infrared microspectroscopic analysis. The results of small-angle X-ray scattering showed that AuNP–GA conjugation was successful. Antibacterial activity of GA against both bacteria was improved by conjugation with AuNP because the minimum inhibitory concentration value of AuNP–GA was significantly decreased (P<0.0001) compared to that of GA. Fourier transform infrared analysis revealed that AuNP–GA resulted in alterations of lipids, proteins, and nucleic acids at the bacterial cell membrane. Our findings show that AuNP–GA has potential for further application in biomedical sciences.

Inhibitory Effects of Gallic Acid Isolated from Caesalpinia mimosoides Lamk on Cholangiocarcinoma Cell Lines and Foodborne Pathogenic Bacteria

Gallic acid was isolated from Caesalpinia mimosoides Lamk and the structure s identified based on spectroscopic analysis and comparison with authentic compound. In this study we compared the ability of natural gallic acid (nGA) and commercial gallic acid (cGA) to inhibit the proliferation of cholangiocarcinoma cell lines (M213, M214) and foodborne pathogenic bacteria (Salmonella spp. and Plesiomonas shigelloides). Both nGA and cGA had the same inhibitory effects on cell proliferation by inducing apoptosis of cholangiocarcinoma cell lines. In addition, nGA inhibited growth of foodborne pathogenic bacteria in the same manner as cGA. Our results suggest that nGA from Caesalpinia mimosoides Lamk is a potential anticancer and antibacterial compound. However, in vivo studies are needed to elucidate the specific mechanisms involved.

Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei.

Silver nanoparticles (AgNPs) have a strong antimicrobial activity against a variety of pathogenic bacteria. The killing mechanism of AgNPs involves direct physical membrane destruction and subsequent molecular damage from both AgNPs and released Ag+. Burkholderia pseudomallei is the causative agent of melioidosis, an endemic infectious disease primarily found in northern Australia and Southeast Asia. B. pseudomallei is intrinsically resistant to most common antibiotics. In this study, the antimicrobial activity and mechanism of AgNPs (10–20 nm) against B. pseudomallei were investigated. The MIC and MBC for nine B. pseudomallei strains ranged from 32–48 μg/mL and 96–128 μg/mL, respectively. Concentrations of AgNPs less than 256 μg/mL were not toxic to human red blood cells. AgNPs exhibited a two-phase mechanism: cell death induction and ROS induction. The first phase was a rapid killing step within 5 min, causing the direct damage of the cytoplasmic membrane of the bacterial cells, as observed by a time-kill assay and fluorescence microscopy. During the period of 5–30 min, the cell surface charge was rapidly neutralized from -8.73 and -7.74 to 2.85 and 2.94 mV in two isolates of B. pseudomallei, as revealed by zeta potential measurement. Energy-dispersive X-ray (EDX) spectroscopy showed the silver element deposited on the bacterial membrane, and TEM micrographs of the AgNP-treated B. pseudomallei cells showed severe membrane damage and cytosolic leakage at 1/5 MIC and cell bursting at MBC. During the killing effect the released Ag+ from AgNPs was only 3.9% from the starting AgNPs concentration as observed with ICP-OES experiment. In the second phase, the ROS induction occurred 1–4 hr after the AgNP treatment. Altogether, we provide direct kinetic evidence of the AgNPs killing mechanism, by which cell death is separable from the ROS induction and AgNPs mainly contributes in the killing action. AgNPs may be considered a potential candidate to develop a novel alternative agent for melioidosis treatment with fast action.