Cell Wall Lysis Research Articles

Determination of the efficient enzyme concentration for lytic digestion of vegetative cells but not spores in Schizosaccharomyces pombe

Received: 2016-05-24 | Accepted: 2016-05-30 | Available online: 2017-06-20 http://dx.doi.org/10.15414/afz.2017.20.01.20-22 The fact that lytic enzymes such as b-glucuronidase are capable of cell wall lysis, while the integrity of the spore is not affected, is used in the spore viability tests to investigate meiotic processes. Meiosis as a fundamental feature in all living organisms comprises of a complex tightly linked and mutually dependent processes most of which are scientific targets of many research institutions. The fission yeast Shizosaccharomyces pombe is a powerful tool for studies on eukaryotic meiosis. Mating of yeast strains of opposite mating types on nitrogen free medium results in spores production. Whereas not all cells undergo meiosis, some cells persist in vegetative stage even in the absence of nitrogen, this leads to generation of a mixed suspension of vegetative cells and spores. Thus, in order to separate spores from vegetative cells obtained mixture was exposed to lytic enzyme action. This treatment kills vegetative cells without affecting spores. To obtain the best and reproducible results of spore recovery and viability, different lytic conditions were analysed. Obtained results show, that use of b-glucuronidase as lytic enzyme for random spore analyses in the fission yeast is dose and time dependent. Keywords: b-glucuronidase, cell cycle, spore viability, Schizosaccharomycesn pombe References BAHALUL, M., KANETI, G. and KASHI, Y. (2010) Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascosppores isolation in Saccharomyces cerevisiae yeast. Yeast, vol.27, no.12, pp.999–1003. doi: http://dx.doi.org/10.1002/yea.1808 DAWES, I.W., HARDIE, I.D. (1974) Selective killing of vegetative cells in sporulated cultures by exposure to diethyl ether. Mol Gen Genet, vol.131, no. 4, pp. 281–289. doi: http://dx.doi.org/10.1007/BF00264859 DONOVAN, D.M., KERR, D.E. and WALL, R.J. (2005) Engineering disease resistant cattle. Transgenic Res, vol. 14, pp. 563–567. doi: http://dx.doi.org/10.1007/s11248-005-0670-8 KHARE, A. K., SINGH, B., and SINGH, J. (2011) A fast and inexpensive method for random spore analysis in Schizosaccharomyces pombe . Yeast, vol. 28, pp. 527–533. doi: http://dx.doi.org/10.1002/yea.1855 MORENO, S., KLAR, A. and NURSE, P. (1991) Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe . Methods Enzymol, vol . 194, pp. 795–823. http://dx.doi.org/10.1016/0076-6879(91)94059-L PARENTI-CASTELLI G, et al. (1974) Effect of soluble and membrane proteins upon diethyl ether extraction of aqueous phospholipid dispersions. Lipids, vol . 9, pp.221–228. doi: http://dx.doi.org/10.1007/BF02532197 SALAZAR, O. and ASENJO, J.A. (2007) Enzymatic lysis of microbial cells Review Biotechnology Letters , vol. 29, no. 7, pp. 985-994. doi: http://dx.doi.org/10.1007/s10529-007-9345-2 SMITH, G.R. (2009) Genetic Analysis of Meiotic Recombination in Schizosaccharomyces pombe Methods Mol Biol ., vol. 557, pp. 65–76. doi: http://dx.doi.org/10.1007/978-1-59745-527-5_6 YANG, Y.G, et al. (2000) The application of a novel lytic system to the recovery of recombinant proteins in E. coli. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) Acta biochimica et biophysica Sinica, vol. 32, no. 3, pp. 211–216. ŽUKAITE, V. and BIZIULEVICIUS, G.A. (2000) Acceleration of hyaluronidase production in the course of batch cultivation of Clostridium perfringens can be achieved with bacteriolytic enzymes. Lett Appl Microbiol , vol. 30, pp. 203–206. doi: http://dx.doi.org/10.1046/j.1472-765x.2000.00693.x

Role of Rifampin in Reducing Inflammation and Neuronal Damage in Childhood Bacterial Meningitis: A Pilot Randomized Controlled Trial.

Treatment of acute bacterial meningitis in children with bactericidal antibiotics causes cell wall lysis and a surge in inflammatory cascade, which in turn contributes to neuronal damage and morbidity. Pretreatment with a nonbacteriolytic antibiotic, such as rifampin, has been shown to attenuate the inflammatory response in experimental models of bacterial meningitis. In a pilot study, in children with bacterial meningitis, we have studied markers of inflammatory response and neuronal damage in 2 groups of children with bacterial meningitis; one group received rifampin pretreatment with ceftriaxone and the other group received ceftriaxone alone. Forty children with bacterial meningitis, who were 3 months to 12 years of age, were randomly assigned to receive either a single dose rifampin (20 mg/kg) 30 minutes before ceftriaxone or ceftriaxone alone was given. The primary outcome variables were cerebrospinal fluid (CSF) concentrations of tumor necrosis factor alpha (TNFα), S100B and neuron-specific enolase on day 1 and day 5, and secondary outcome variables were the values of TNFα and interleukin 6 in serum on day 1 and day 5; hearing and neurologic sequelae at 3 months after recovery from the illness. Children in rifampin pretreatment group had significantly lower CSF TNFα concentrations [median (interquartile range [IQR]): 15.5 (7.2-22.0) vs. 53.0 (9.0-87.5) pg/mL, P = 0.019] and S100B [median (IQR): 145.0 (54.7-450.0) vs. 447.5 (221.0-804.6) pg/mL, P = 0.033] on day 1 and S100B [median (IQR): 109.7 (64.0-287.0) vs. 322 (106.7-578.0) pg/mL, P = 0.048] and neuron-specific enolase [median (IQR): 8.6 (5-14.75) vs. 18.2 (7.0-28.75) ng/mL, P = 0.035] on day 5 when compared with ceftriaxone alone group. The rifampin-treated group also had reduced morbidity and neurologic sequelae; however, these were not statistically significant. Pretreatment with single dose rifampin 30 minutes before ceftriaxone administration reduced the CSF concentrations of markers of inflammation and neuronal damage in children with bacterial meningitis.

Antibacterial Activity and Synergistic Antibacterial Potential of Biosynthesized Silver Nanoparticles against Foodborne Pathogenic Bacteria along with its Anticandidal and Antioxidant Effects.

Silver nanoparticles plays a vital role in the development of new antimicrobial substances against a number of pathogenic microorganisms. These nanoparticles due to their smaller size could be very effective as they can improve the antibacterial activity through lysis of bacterial cell wall. Green synthesis of metal nanoparticles using various plants and plant products has recently been successfully accomplished. However, few studies have investigated the use of industrial waste materials in nanoparticle synthesis. In the present investigation, synthesis of silver nanoparticles (AgNPs) was attempted using the aqueous extract of corn leaf waste of Zea mays, which is a waste material from the corn industry. The synthesized AgNPs were evaluated for their antibacterial activity against foodborne pathogenic bacteria (Bacillus cereus ATCC 13061, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 49444, Escherichia coli ATCC 43890, and Salmonella Typhimurium ATCC 43174) along with the study of its synergistic antibacterial activity. The anticandidal activity of AgNPs were evaluated against Candida species (C. albicans KACC 30003 and KACC 30062, C. glabrata KBNO6P00368, C. geochares KACC 30061, and C. saitoana KACC 41238), together with the antioxidant potential. The biosynthesized AgNPs were characterized by UV-Vis spectrophotometry with surface plasmon resonance at 450 nm followed by the analysis using scanning electron microscope, X-ray diffraction, Fourier-transform infrared spectroscopy and thermogravimetric analysis. The AgNPs displayed moderate antibacterial activity (9.26–11.57 mm inhibition zone) against all five foodborne pathogenic bacteria. When AgNPs were mixed with standard antibacterial or anticandidal agent, they displayed strong synergistic antibacterial (10.62–12.80 mm inhibition zones) and anticandidal activity (11.43–14.33 mm inhibition zones). In addition, the AgNPs exhibited strong antioxidant potential. The overall results highlighted the potential use of maize industrial waste materials in the synthesis of AgNPs and their utilization in various applications particularly as antibacterial substance in food packaging, food preservation to protect against various dreadful foodborne pathogenic bacteria together with its biomedical, pharmaceutical based activities.

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system.

Soft-rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD-dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS-negative mutant grown in a Pels-inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid-dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild-type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD-dependent ROS and callose accumulation, but not cell death.

Biosynthesis of silver nanoparticle and its application in cell wall disruption to release carbohydrate and lipid from C. vulgaris for biofuel production

Microalgae are the fledging feedstocks yielding raw materials for the production of third generation biofuel. Assorted and conventional cell wall disruption techniques were helpful in extracting lipids and carbohydrates, nevertheless the disadvantages have led the biotechnologists to explore new process to lyse cell wall in a faster and an economical manner. Silver nanoparticles have the ability to break the cell wall of microalgae and release biomolecules effectively. Green synthesis of silver nanoparticles was performed using a novel bacterial isolate of Bacillus subtilis. Characterisation of nanosilver and its effect on cell wall lysis of microalgae were extensively analysed. Cell wall damage was confirmed by lactate dehydrogenase assay and visually by SEM analysis. This first piece of research work on direct use of nanoparticles for cell wall lysis would potentially be advantageous over its conventional approaches and a greener, cost effective and non laborious method for the production of biodiesel.

Antimycobacterial activity of methanolic plant extract of Artemisia capillaris containing ursolic acid and hydroquinone against Mycobacterium tuberculosis.

In order to protect against Mycobacterium tuberculosis (MTB) infection, novel drugs and new targets should be screened from the vast source of plants. We investigated the potentiality of the herbal plant of Artemisia capillaris extract (AC) against Mycobacterium tuberculosis. In this study, we isolated ursolic acid and hydroquinone by bio-activity guided fractionation from the methanol extracts of AC, and tested the inhibitory effects against several strains of MTB. Anti-mycobacterial evaluation of these compounds was carried out using the MGIT™ 960 and resazurin assay. Mycobacterial morphological changes due to the treatment of these compounds were further evaluated by Transmission electron microscopy (TEM). Ursolic acid (UA) and hydroquinone (HQ) inhibited the growth of both susceptible and resistant strains of M.tuberculosis. The MIC (minimum inhibitory concentration) values of both UA and HQ were 12.5μg/ml against the susceptible strains of M.tuberculosis. Also both UA and HQ showed 12.5-25μg/ml of MIC values against MDR/XDR MTB strains. However, against clinical strains of MTB, UA was found sensitive against those strains that are sensitive against both INH and RFP but resistant against those strains that are resistant to INH. On the other hand HQ was sensitive against all clinical strains. TEM image-analysis of the strain H37Ra after treatment with UA revealed cell wall lysis, whereas HQ-treated cells showed deformed cytoplasmic morphology. All these results indicate that AC extracts containing UA and HQ possess promising chemotherapeutic potency against MTB for future use.

Efficacy of a combination of nisin and p-Anisaldehyde against Listeria monocytogenes

Nisin is used as a food-safe antimicrobial agent and has been widely applied to daily food products to prevent bacterial growth. However, the practical application of nisin is limited. Although nisin inhibits the growth of Listeria monocytogenes, exposure to this agent is also a risk factor for the development of nisin resistance. p-Anisaldehyde (AS) is used in the pharmaceutical industry to manufacture antimicrobial drugs, but few studies have been reported describing the antibacterial activity of AS in the food field. Combining nisin with AS may enhance antimicrobial activity. The synergy observed in vitro was confirmed by checkerboard microdilution method, with the fractional inhibitory concentration index (FICI) values ranging from 0.125 to 0.281. When AS was combined with nisin, a strong synergistic effect was observed using the time-kill and agar-diffusion assays. SEM and LIVE/DEAD BacLight experiment results also suggest that the bactericidal mechanism of the drug combination involves cell wall lysis and membrane damage. We can use this synergy to achieve a stable antimicrobial effect to overcome antimicrobial drug resistance.

Enhanced antimicrobial activity of silver nanoparticles-Lonicera Japonica Thunb combo.

Silver metals have long been known to possess antimicrobial properties. Recently, even the nanoparticle version of silver (AgNPs) has also been established as antimicrobials. In this study AgNPs were combined with extracts of the medicinal plant Chinese honeysuckle, Lonicera japonica Thunb. The antimicrobial activity of the AgNPs-herb was tested against pathogenic Escherichia coli CMCC44113. Using different AgNPs or herb (honeysuckle water extract or HWE) ratios in the presence of a fixed concentration of E. coli CMCC44113, potencies were found to be proportional with concentrations. The antimicrobial activities of AgNPs-HWE combo were significant enhanced, when compared with solely AgNPs or HWE. Thus, atomic force microscopic and propidium monoazide-PCR were used to probe the damages caused by AgNPs-HWE combo on the cell morphology and cell membrane integrity of E. coli. The mechanism of AgNPs-HWE combo against E. coli may attribute to AgNPs leads to cell wall lysis and damages cell membrane integrity, and thus increases the penetration of HWE into the bacterium, which results in more serious damage to bacterial cells. These findings indicated that AgNPs-herb was more potent than the AgNPs alone and holds promise for the development of nanoparticle enhanced herbal pharmaceuticals.

Suppressive effects of Bacillus spp. on mycelia, apothecia and sclerotia formation of Sclerotinia sclerotiorum and potential as biological control of white mold on mustard

Biological control, especially with Bacillus-based biocontrol agents, offers an attractive alternative to synthetic pesticides for sustainable management of white mold disease caused by Sclerotinia sclerotiorum. In this study, eight effective Bacillus isolates were isolated from rhizospheric soil samples as potential bacterial biocontrol agents. Cultural, biochemical, and molecular analyses of 16S rDNA and gyrase subunit A (gyrA) confirmed that all isolates were identified as Bacillus amyloliquefaciens subsp. plantarum. The production of hydrolytic enzymes and the plant growth-promotional attributes of these isolates confirmed their multifaceted potential. Molecular analysis of the eight biosynthetic genes, which are related to antibiotic properties of bacilli, revealed that all of the isolates possess five genes: bacA for bacilysin, dfnM for difficidin, fenA for fengycin, ituA for iturin, and sfp for surfactin. The Bacillus isolates inhibited mycelial growth and suppressed formation of sclerotia during an in vitro test against S. sclerotiorum. Deformities and cell-wall lysis of mycelia, abnormalities of apothecia, and germination failure of ascospores through interaction with the Bacillus isolates were observed with light and scanning electron microscopes, suggesting that they have high antagonistic effects against S. sclerotiorum. Seed bacterization with the Bacillus isolates protected mustard seedlings in vitro up to 98 % against S. sclerotiorum. In a pot experiment, damages of mustard plants against the pathogen decreased up to 90 % after foliar spray of the Bacillus isolates. In addition, the isolates increased seed germination and accelerated seedling vigor of mustard, suggesting that they have plant growth-promoting functions.

The direct anti-MRSA effect of emodin via damaging cell membrane.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important bacterium for nosocomial infection. Only a few antibiotics can be effective against MRSA. Therefore, searching for new drugs against MRSA is important. Herein, anti-MRSA activities of emodin and its mechanisms were investigated. Firstly, in vitro antimicrobial activity was investigated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-growth curve, and multipassage resistance testing was performed. Secondly, protection of emodin on mice survival and blood bacterial load in mice challenged with lethal or sublethal dose of MRSA were investigated. Subsequently, the influences of emodin on the bacterial morphology, messenger RNA (mRNA) expressions related to cell wall synthesis and lysis, β-lactamase activity, drug accumulation, membrane fluidity, and integrity were performed to investigate its mechanisms. Lastly, in vitro cytotoxicity assay were performed using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. The results showed MICs and MBCs of emodin against MRSA252 and 36 clinical MRSA strains were among 2-8 and 4-32μg/mL, respectively. There was no MIC increase for emodin during 20 passages. In vivo, emodin dose-dependently protected mice challenged with lethal dose of MRSA and decreased bacterial load in mice challenged with sublethal dose of MRSA. Morphology observation showed emodin might disrupt cell wall and membrane of MRSA. Although emodin had no influence on genes related to cell wall synthesis and lysis as well as β-lactamase activity and drug accumulation, emodin reduced membrane fluidity and disrupted membrane integrity. Based on the fact that emodin had no significant cytotoxicity against mammalian cells, it could be further investigated as a membrane-damage bactericide against MRSA in the future.

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells.

Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane

Gram-negative Escherichia coli (ATCC 8735) and Gram-positive Staphylococcus aureus (ATCC 3101) were selected as model bacteria to determine the antimicrobial mechanism of cinnamaldehyde. Several techniques were utilized to investigate the effects of cinnamaldehyde on food-borne bacterial membranes. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. β-Galactosidase activity was determined to detect inner membrane permeability. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to observe bacterial morphology. Samples from both strains exposed to cinnamaldehyde showed higher UV absorptions, conductivity values, and β-Galactosidase activities compared with the control group and displayed a rapid rise trend. Thereafter, the values stabilized at a relatively steady state. SEM shows that treated E. coli and S. aureus cell samples exhibited rough cell membranes with particulate matter, and some of the S. aureus cells split due to deep wrinkle formation and distortion, unlike the control group. TEM shows that the bacteria treated with cinnamaldehyde exhibited numerous abnormalities, including cytoplasmic membrane separation from the cell wall, cell wall and cell membrane lysis, cytoplasmic content leakage, cytoplasmic content polarization, cell distortion, and cytoplasmic content condensation. These results indicate that bacterial cell morphology, membrane integrity, and permeability are damaged when the E. coli and S. aureus cells are exposed to the minimum inhibitory concentrations of cinnamaldehyde (0.31 mg/mL). In addition, the higher the cinnamaldehyde concentration, the more serious the bacterial membrane damage is.

Effect of Chitosan on Lysostaphin Lysis of Staphylococcal Cells

The studied stimulation of lysostaphin-mediated lysis of staphylococcal cell walls under the effect of chitosan. The results suggested a mechanism of antibacterial activity of chitosan consisting in stimulation of bacterial cell wall degradation by its own autolysins in the presence of chitosan polymer.

Assessing the essentiality of the decaprenyl‐phospho‐d‐arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants

In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

Prolotherapy in Temperomandibular Disorders: an Overview

Prolotherapy reinitiates the inflammatory process thereby augmenting the natural healing process of the body by stimulating fibroblastic activity. It is a promising approach in the management of TMDs, especially in refractory cases where conservative management has failed and in patients where surgical management is not possible. The osmotic agent used is dextrose. It produces a hypertonic extracellular environment causing lysis of adjacent cell walls resulting in migration of the macrophages and granulocytes to the area, thus initiating the process of localized inflammation and fibrous healing.

Adjunctive Corticosteroid Therapy Improves Lung Immunopathology and Survival During Severe Secondary Pneumococcal Pneumonia in Mice

Secondary bacterial pneumonia is a significant cause of morbidity and mortality during influenza, despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a well-established murine model of secondary bacterial pneumonia (SBP) following influenza, we stratified disease severity based on pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. Adjunctive dexamethasone therapy improved ampicillin-induced immunopathology and improved outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired lung adaptive immunity as manifest by increased viral titers, with an associated loss of its protective functions in SBP. These data support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia.

Isolation and characterization of a c-type lysozyme from the nurse shark

Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein.

In silico optimization of a fragment-based hit yields biologically active, high-efficiency inhibitors for glutamate racemase.

A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase (GR), was optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid molecular dynamics-docking and scoring scheme, FERM-SMD, was used to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high- and low-affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency was increased approximately fourfold against GR from the model organism, B. subtilis. Lead derivatives show two- to fourfold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis over E. coli and S. aureus depends on the substituent added to the parent scaffold. Finally, insight was gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The outcome of this study is a novel small-molecule inhibitor of GR with the following characteristics: Ki=2.5 μM, LE=0.45 kcal mol(-1) atom(-1), LiPE=6.0, MIC50=260 μg mL(-1) against B. subtilis, EC50, lysis=520 μg mL(-1) against B. subtilis.

Antibacterial and antifungal activity of photoactivated ZnO nanoparticles in suspension

Antibacterial activity of photoactivated zinc oxide nanoparticles (ZnO NPs) against human pathogens Escherichia coli O157:H7, Listeria monocytogenes ATCL3C 7644 and plant pathogen Botrytis cinerea was investigated. Data indicate that photoactivated (λ=400nm) ZnO NPs at concentration 1×10−3M and incubation time 60min reduced population of both bacteria by 7 log (CFU/ml). Clear dependence of antimicrobial properties of ZnO NPs on used concentration and incubation time was found. Scanning electron microscopy (SEM) images of treated bacteria indicate that treatment induced cell wall disintegration and lysis. Results obtained on examination of antifungal activity of ZnO NPs reveal that significant photoinactivation (58%) of B. cinerea was observed at NPs concentration 5×10−3M and incubation time of 24h. SEM analysis confirmed that substantial morphological changes occur in the microfungus after treatment.The data suggest that ZnO NPs in the presence of visible light exhibit strong antibacterial and antifungal activity. Such ZnO NPs properties obviously could be used for the development of effective fungicides in agriculture or innovative physical antibacterial agents, so important in medicine and food microbial control.

Investigating the bioactivity of cells and cell-free extracts of Streptomyces griseus towards Fusarium oxysporum f. sp. cubense race 4

The antifungal activity of viable cells of Streptomyces griseus (St 4) and its cell-free extracts were investigated against the pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), causal agent of wilt disease in bananas. Results from in vitro and soil assays showed cells and cell-free extracts of S. griseus were able to inhibit FOC race 4 with varying degree of success. Antifungal activity was attributed to chitinase and β-1,3-glucanase, detected in both cells and cell-free extracts, which caused lysis of fungal cell wall and inhibited sporulation. Interestingly, β-1,3-glucanase and chitinase activities were significantly higher in cell-free extracts compared to cells, with 8.30 and 5.43 against 7.96 and 4.95UmL−1, respectively. Application to soil however, showed inoculation using S. griseus cells were more effective in suppressing growth of FOC race 4 than crude extracts, with 6 log10 CFU of FOC race 4g−1 soil enumerated compared to 7 log10 CFU of FOC race 4g−1 soil after 20days. To summarize, this study has shown that cell-free extracts of S. griseus have antifungal properties but may not be suitable for soil application in its current form (liquid suspension). Further investigations on bioformulation may address this limitation.