An integrated transcriptomic and proteomic approach to identify the main Torymus sinensis venom components.

During oviposition, ectoparasitoid wasps not only inject their eggs but also a complex mixture of proteins and peptides (venom) in order to regulate the host physiology to benefit their progeny. Although several endoparasitoid venom proteins have been identified, little is known about the components of ectoparasitoid venom. To characterize the protein composition of Torymus sinensis Kamijo (Hymenoptera: Torymidae) venom, we used an integrated transcriptomic and proteomic approach and identified 143 venom proteins. Moreover, focusing on venom gland transcriptome, we selected additional 52 transcripts encoding putative venom proteins. As in other parasitoid venoms, hydrolases, including proteases, phosphatases, esterases, and nucleases, constitute the most abundant families in T. sinensis venom, followed by protease inhibitors. These proteins are potentially involved in the complex parasitic syndrome, with different effects on the immune system, physiological processes and development of the host, and contribute to provide nutrients to the parasitoid progeny. Although additional in vivo studies are needed, initial findings offer important information about venom factors and their putative host effects, which are essential to ensure the success of parasitism.

Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties.

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Study of Antiviral Activity of Metabolites of a New Serratia species K-57 Strain.

The results of studies of a newly isolated Serratia species K-57 strain are presented. The strain is characterized by antiviral activity towards human influenza A/Aichi/2/68/H3N2, vaccinia, mouse smallpox, and herpes simplex-2 viruses. The detected characteristics of the strain, including the data on activities on nucleolytic enzymes, recommend it for the development of therapeutic and preventive antiviral drugs.

Marine bacterial DNase curtails virulence and disrupts biofilms of Candida albicans and non-albicans Candida species.

Candida is one of the most prevalent fungal pathogens in clinical settings which form antibiotic-resistant biofilms on biomedical devices. Hence, there is a need for non-antimicrobial alternatives to combat these infections. The present study investigates the anti-biofilm effect of marine bacterial DNase by targeting the eDNA present in the biofilms of Candida spp. A strain of Vibrio alginolyticus (AMSII) which showed enhanced DNase activity was isolated from marine sediment. Treatment of young and mature Candida biofilms with purified marine bacterial DNase (MBD) caused a 60-80% reduction in biofilm biomass, similar to treatment with DNase I from Bovine pancreas. Scanning electron microscopy showed that MBD significantly reduced the formation of biofilms on urinary catheters and more importantly prevented the virulent yeast to hyphae dimorphic switch in C. albicans. The present study identified a potential non-antibiotic alternative therapy to eradicate Candida biofilms and can be used to develop enzyme fabricated antifouling indwelling medical devices.

FRET probe-based antibacterial susceptibility testing (F-AST) by detection of bacterial nucleases released by antibiotic-induced lysis.

This study demonstrates a novel and rapid antibacterial susceptibility testing (AST) method, called fluorescence resonance energy transfer (FRET) probe-based AST (F-AST), which relies on a nuclease-activated FRET probe that detects bacterial nucleases released by antibiotic-induced bacterial lysis. Three quality control (QC) strains and two additional clinically important strains were tested, and the minimum inhibitory concentration (MIC) values from both gold standard AST method (broth microdilution (BMD)) and the new F-AST method were compared. The resulting fluorescence signals from the F-AST method were obtained within 3-6 h and were consistent with MIC values obtained from the BMD method, which took more than 16 h. Thus, the F-AST method is a simple and rapid tool to detect antibacterial susceptibility, including MIC values, and provides a basis for rapid clinical treatments.

The Mitochondrial Endonuclease M20 Participates in the Down-Regulation of Mitochondrial DNA in Pollen Cells.

Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20-kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an His-Asn-His/Asn (H-N-H/N) nuclease that degrades linear and circular DNA in the presence of Mg2+ or Mn2+ Arabidopsis (Arabidopsis thaliana) AtM20, which shared high sequence similarity with maize M20, localized to the mitochondria, had a similar H-N-H/N structure, and degraded both linear and circular DNA. AtM20 transcript levels increased during pollen development, in parallel with a rapid reduction in mtDNA. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing techniques were used to generate knockout lines of AtM20 (atm20), which exhibited a significant delay in the reduction in mtDNA levels in pollen vegetative cells but normal mtDNA levels in somatic cells. The delayed reduction in pollen mtDNA levels was rescued by the transgenic expression of AtM20 in atm20 plants. This study thus uncovers an endonucleolytic DNase in plant mitochondria and its crucial role in reducing mtDNA levels, pointing to the complex mechanism regulating mtDNA levels in plants.

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Purification and Characterization of 2S Albumin from Seeds of Wrightia tinctoria Exhibiting Antibacterial and DNase Activity.

2S albumin is a low-molecular-weight seed storage protein belonging to the prolamin superfamily. In the present work a small 2S albumin (WTA) protein of ~16 kDa has been purified from the seeds of Wrightia tinctoria. The WTA is a heterodimer protein with a small subunit of ~5 kDa and a larger subunit of ~11 kDa bridged together through disulphide bonds. The protein exhibits deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The protein also showed antibacterial activity against Morexalla catarrhalis. CD studies indicate a high α-helical content in the protein. The conserved disulphide bonds in the protein suggest that the WTA is highly stable under high pH and temperature like other 2S albumin.

In vitro evaluation of antifungal susceptibility and keratinase, elastase, lipase and DNase activities of different dermatophyte species isolated from clinical specimens in Iran.

The aims of this study were to evaluate the enzymatic activity of various dermatophyte species and their antifungal susceptibility profiles. A total of 60 dermatophyte isolates, including Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum, were examined. Fungal isolates were analysed for the production of keratinase, lipase, elastase and deoxyribonuclease (DNase). A broth microdilution method was performed on the basis of M38-A2 Clinical and Laboratory Standards Institute (CLSI) guidelines. T. mentagrophytes, M. canis and M. gypseum isolates were capable of producing keratinase, lipase, elastase and DNase, while T. rubrum isolates were elastase negative. The highest mean diameter of the clear zone around the colonies (PZ) was associated with keratinase (PZ: 4.56 ± 1.29 mm), followed by lipase (PZ: 1.53 ± 0.90 mm), DNase (PZ: 0.65 ± 0.54 mm) and elastase (PZ: 0.22 ± 0.27 mm) (P < 0.05). The mean minimum inhibitory concentration 90 (MIC90 ) of all strains were as follows: itraconazole (MIC90 : 0.28 ± 0.31 µg ml-1 ), ketoconazole (MIC90 : 0.48 ± 0.51 µg ml-1 ), griseofulvin (MIC90 : 0.86 ± 1.00 µg ml-1 ) and fluconazole (MIC90 : 18.57 ± 20.10 µg ml-1 ). Dermatophyte isolates had higher keratinolytic activity than other enzymes. Itraconazole was the most effective antifungal drug and fluconazole had the poorest activity.

Enzyme promiscuity in earthworm serine protease: substrate versatility and therapeutic potential.

Enzymes are the most versatile molecules in the biological world. These amazing molecules play an integral role in the regulation of various metabolic pathways and physiology subsequently. Promiscuity of an enzyme is the capacity to catalyze additional biochemical reactions besides their native one. Catalytic promiscuity has shown great impact in enzyme engineering for commercial enzyme and therapeutics with natural or engineered catalytic promiscuity. The earthworm serine protease (ESP) is a classic example of enzyme promiscuity and studied for its therapeutic potential over the last few decades. The ESP was reported for several therapeutic properties and fibrinolytic activity has been much explored. ESP, a complex enzyme exists as several isoforms of molecular weight ranging from 14 to 33 kDa. The fibrinolytic capacity of the enzyme has been studied in different species of earthworm and molecular mechanism is quite different from conventional thrombolytics. Cytotoxic and anti-tumor activities of ESP were evaluated using several cancer cell lines. Enzyme had shown tremendous scope in fighting against plant viruses and microbes. ESP is also reported for anti-inflammatory activity and anti-oxidant property. Apart from these, recently, ESP is reported for DNase activity. The daunting challenge for researchers is to understand the molecular mechanism for such diverse properties and possibility of enzyme promiscuity. This review emphasizes molecular mechanism of ESP governing various biochemical reactions. Further, the concept of enzyme promiscuity in ESP towards development of novel enzyme based drugs has been reviewed in this study.

Very stable high molecular mass multiprotein complex with DNase and amylase activities in human milk.

For breastfed infants, human milk is more than a source of nutrients; it furnishes a wide array of proteins, peptides, antibodies, and other components promoting neonatal growth and protecting infants from viral and bacterial infection. It has been proposed that most biological processes are performed by protein complexes. Therefore, identification and characterization of human milk components including protein complexes is important for understanding the function of milk. Using gel filtration, we have purified a stable high molecular mass (~1000 kDa) multiprotein complex (SPC) from 15 preparations of human milk. Light scattering and gel filtration showed that the SPC was stable in the presence of high concentrations of NaCl and MgCl2 but dissociated efficiently under the conditions that destroy immunocomplexes (2 M MgCl2 , 0.5 M NaCl, and 10 mM DTT). Such a stable complex is unlikely to be a casual associate of different proteins. The relative content of the individual SPCs varied from 6% to 25% of the total milk protein. According to electrophoretic and mass spectrometry analysis, all 15 SPCs contained lactoferrin (LF) and α-lactalbumin as major proteins, whereas human milk albumin and ß-casein were present in moderate or minor amounts; a different content of IgGs and sIgAs was observed. All SPCs efficiently hydrolyzed Plasmid supercoiled DNA and maltoheptaose. Some freshly prepared SPC preparations contained not only intact LF but also small amounts of its fragments, which appeared in all SPCs during their prolonged storage; the fragments, similar to intact LF, possessed DNase and amylase activities. LF is found in human epithelial secretions, barrier body fluids, and in the secondary granules of leukocytes. LF is a protein of the acute phase response and nonspecific defense against different types of microbial and viral infections. Therefore, LF complexes with other proteins may be important for its functions not only in human milk.

Enzymatic and hemolytic activity in different Candida species.

BACKGROUND: Candida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. AIMS: This study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. METHODS: Fifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. RESULTS: Forty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p<0.0001) and proteinase (p<0.05) production. CONCLUSIONS: It is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis.

Purification and characterization of a novel earthworm DNase.

A new deoxyribonuclease (DNase), referred to as EWDNase, was isolated from earthworm tissues. The purification protocol included acetone precipitation, chromatography on CM-Sepharose, and gel electrophoresis. The overall purification was 73-fold with a recovery rate of 2.3% and a final specific activity of 2039 U/mg. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested a molecular mass of 30 kD for EWDNase, with an isoelectric point of approximately 7.0. Maximum activity was detected at a pH of 5.6 and a temperature of 40°C. Addition of Mg(2+) and Ca(2+) ions promoted enzyme activity strongly, while Zn(2+) and ethylenediamine tetraacetic acid (EDTA) acted as inhibitors. Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis indicated that there was no known matching sequence. The properties of EWDNase were sufficiently different from previously reported enzymes to suggest that it is a new enzyme requiring further confirmation and characterization.

Characterization of a recently purified thermophilic DNase from a novel thermophilic fungus.

A newly isolated thermophilic fungus was found to produce a partially inducible extracellular DNase. This manuscript focuses on the characterization of this novel thermophilic DNase in terms of optimal enzyme conditions, molecular weight, and certain kinetic properties. The DNase was found to be inactivated by the presence of EDTA demonstrating its dependence on metal cofactors for activity. Maximum activity occurred at pH 6.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65 °C. The thermophilic DNase was found to be an exonuclease with an estimated molecular weight of 56 kDa.

Purification of an inducible DNase from a thermophilic fungus.

The ability to induce an extracellular DNase from a novel thermophilic fungus was studied and the DNAse purified using both traditional and innovative purification techniques. The isolate produced sterile hyphae under all attempted growing conditions, with an average diameter of 2 µm and was found to have an optimal temperature of 45 °C and a maximum of 65 °C. Sequencing of the internal transcribed region resulted in a 91% match with Chaetomium sp., suggesting a new species, but further clarification on this point is needed. The optimal temperature for DNase production was found to be 55 °C and was induced by the presence of DNA and/or deoxyribose. Static growth of the organism resulted in significantly higher DNase production than agitated growth. The DNase was purified 145-fold using a novel affinity membrane purification system with 25% of the initial enzyme activity remaining. Electrophoresis of the purified enzyme resulted in a single protein band, indicating DNase homogeneity.

Development of a novel affinity membrane purification system for deoxyribonuclease.

A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes.

Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties.

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases.

Nuclease released by Verticillium dahliae is a signal for non-host resistance.

A DNase released from the fungal pathogen of bean, Fusarium solani f. sp. phaseoli (Fsph), was previously shown to signal the activation of total disease resistance and activate pathogenesis-related (PR) genes in pea. Data in the current study which used the pea-endocarp model to research non-host resistance, indicated that DNase released by Verticillium dahliae (Vd), pathogenic on potato also has non-host resistance-inducing capabilities in peas. Other strains of Vd that release DNase are pathogenic on other plant species. DNase catalytic activity was also released from representative genera of other pathogenic fungi. Purified VdDNase induced pisatin and pea gene DRR49 (PR-10 gene) in pea endocarp tissue. VdDNase reduced the in vitro growth of Vd but completely inhibited that of F. solani f. sp. pisi (Fspi) and a Colletotrichum pathogen of potato. VdDNase (2 units) applied to pea endocarp tissue both broke resistance to Fsph and increased resistance to Fspi. Pea DNA damage generated both by the VdDNase enzyme and the intact Vd spores indicated that the host DNA alteration is a component of the non-host resistance response (innate immunity). These data support the previously reported inductive potential of fungal DNase and further implicate fungal DNases as signals in activating non-host resistance responses.

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label free nuclease enzyme detection.

The double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) with high fluorescence, whereas the single-strand DNA (ssDNA) cannot support the formation of Cu NPs. This difference in fluorescent signal generation can be used for the detection of nuclease cleavage activity. Thus, a label-free strategy for sensitive detection of nuclease has been developed. The sensor contains a complete complementary dsDNA which acts as a template for the formation of Cu NPs and generation of fluorescence signal. The enzyme S1 nuclease was taken as the model analyte. Upon addition of S1 nuclease into the sensing system, the DNA was cleaved into fragments, preventing the formation of the Cu NPs and resulting in low fluorescence. In order to achieve the system's best sensing performance, a series of experimental conditions were optimized. Under the optimized experimental conditions, the sensor exhibits excellent performance (e.g., a detection limit of 0.3 U mL⁻¹ with high selectivity). This possibly makes it an attractive platform for the detection of S1 nuclease and other biomolecules.

Fungal mitochondrial DNases: effectors with the potential to activate plant defenses in nonhost resistance.

Previous reports on the model nonhost resistance interaction between Fusarium solani f. sp. phaseoli and pea endocarp tissue have described the disease resistance-signaling role of a fungal DNase1-like protein. The response resulted in no further growth beyond spore germination. This F. solani f. sp. phaseoli DNase gene, constructed with a pathogenesis-related (PR) gene promoter, when transferred to tobacco, generated resistance against Pseudomonas syringe pv. tabaci. The current analytical/theoretical article proposes similar roles for the additional nuclear and mitochondrial nucleases, the coding regions for which are identified in newly available fungal genome sequences. The amino acid sequence homologies within functional domains are conserved within a wide array of fungi. The potato pathogen Verticillium dahliae nuclease was divergent from that of the saprophyte, yeast; however, the purified DNase from yeast also elicited nonhost defense responses in pea, including pisatin accumulation, PR gene induction, and resistance against a true pea pathogen. The yeast mitochondrial DNase gene (open reading frame) predictably codes for a signal peptide providing the mechanism for secretion. Mitochondrial DNase genes appear to provide an unlimited source of components for developing transgenic resistance in all transformable plants.