The investigation of nematocidal activity in Stenotrophomonas maltophilia G2 and characterization of a novel virulence serine protease

2009 ◽  
Vol 55 (8) ◽  
pp. 934-942 ◽  
Author(s):  
Xiaowei Huang ◽  
Junwei Liu ◽  
Junmei Ding ◽  
Qiusheng He ◽  
Rui Xiong ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Stenotrophomonas Maltophilia ◽  
Bursaphelenchus Xylophilus ◽  
Panagrellus Redivivus ◽  
Bacterial Culture Supernatant ◽  
Free Living Nematode ◽  
Nematocidal Activity ◽  
Nematode Cuticle

The Gram-negative bacterium Stenotrophomonas maltophilia G2 was isolated from a soil sample and was found to have high nematotoxic activity against a free-living nematode, Panagrellus redivivus, and a plant-parasitic nematode, Bursaphelenchus xylophilus . The analysis of virulence factors revealed that although the small molecular metabolites participated in nematode killing, the crude extracellular protein extract from the bacterial culture supernatant contributed significantly to its nematocidal activity. An extracellular protease was purified by chromatography, and its effects on degrading purified nematode cuticle and killing living nematodes were confirmed experimentally. Characterization of this purified protease revealed that the application of phenylmethylsulphonyl fluoride, an inhibitor of serine proteases, could completely abolish its proteolytic activity. The results from N-terminal amino acid sequencing showed no similarity with any known serine protease in S. maltophilia, suggesting a novel virulence serine protease was obtained. Our study is the first to show the nematocidal activity of S. maltophilia, and we identified a novel serine protease as an important pathogenicity factor.

scholarly journals Molecular cloning and characterization of ornithine decarboxylase cDNA of the nematode Panagrellus redivivus

1995 ◽  
Vol 308 (2) ◽  
pp. 635-640 ◽  
Author(s):  
H von Besser ◽  
G Niemann ◽  
B Domdey ◽  
R D Walter
Keyword(s):  
Amino Acids ◽  
Active Sites ◽  
Cdna Clones ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Panagrellus Redivivus ◽  
Free Living Nematode ◽  
Mixed Stage ◽  
Conserved Amino Acids

In a PCR with degenerate primers encoding highly conserved amino acids within ornithine decarboxylases (ODCs) of several organisms, a fragment of the ODC gene of the free-living nematode Panagrellus redivivus was isolated. Northern blot analysis revealed a single 1.7 kb transcript in a mixed-stage population of animals. From this RNA source, a cDNA library was constructed and screened with the PCR fragment. Several cDNA clones were isolated, one of which encodes the complete 435-amino-acid ODC enzyme with a calculated molecular mass of 47.1 kDa. The P. redivivus ODC possesses 126 of the 136 highly conserved amino acids in the enzymes from fungi, invertebrates and vertebrates. Functional amino acids are conserved, suggesting that the two active sites of the P. redivivus ODC are formed at the interface of a homodimer, as described for mammalian ODCs.

scholarly journals Acanthocytes of Stropharia rugosoannulata Function as a Nematode-Attacking Device

2006 ◽  
Vol 72 (4) ◽  
pp. 2982-2987 ◽  
Author(s):  
Hong Luo ◽  
Xuan Li ◽  
Guohong Li ◽  
Yanbo Pan ◽  
Keqin Zhang
Keyword(s):  
Bursaphelenchus Xylophilus ◽  
Mechanical Force ◽  
Free Living ◽  
Pine Wilt ◽  
Panagrellus Redivivus ◽  
Free Living Nematode

ABSTRACT Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.

Molecular characterization of a partial sequence encoding a novel Schistosoma mansoni serine protease

Parasitology ◽  
1997 ◽  
Vol 115 (4) ◽  
pp. 395-402 ◽  
Author(s):  
C. COCUDE ◽  
C. PIERROT ◽  
C. CETRE ◽  
C. GODIN ◽  
A. CAPRON ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Serine Proteases ◽  
Mrna Level ◽  
Cross Reactivity ◽  
Reading Frame ◽  
Consensus Sequences ◽  
Degenerate Oligonucleotide ◽  
Sequence Encoding

A PCR strategy using degenerate oligonucleotide primers based upon consensus sequences of the active site of serine proteases yielded a 467 bp fragment from genomic DNA from Schistosoma mansoni cercariae. The sequence presented a continuous open reading frame and the deduced amino acid sequence (156 aa) presented homologies with various serine proteases, in particular the highest percentage identity was observed with a mammalian plasma kallikrein. The expression of this serine protease was studied first at the mRNA level and it was only detected by RT-PCR in cercariae and in adult worms. At the protein level we were able to detect it by Western blotting and by using antigen extracts from metabolically radio-isotope labelled worms. The absence of any positive signal in Northern blot and the detection of the protein suggest that the mRNA has a very short half-life, however the protein may be accumulated in the parasite. The significance of identity with mammalian kallikrein was confirmed by cross-immunoreactivity with a native porcine pancreatic kallikrein. However, no cross-reactivity was observed with S. mansoni elastase, another serine protease. Thus, we suggest that the serine protease described in this paper is a kallikrein-like protease.

scholarly journals Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity from Bothrops barnetti Snake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Magaly Alejandra Brousett-Minaya ◽  
Paulo Aparecido Baldasso ◽  
Salomón Huancahuire-Vega ◽  
Sérgio Marangoni
Keyword(s):  
Serine Protease ◽  
Snake Venom ◽  
Serine Proteases ◽  
Kinetic Studies ◽  
Size Exclusion ◽  
Reverse Phase Hplc ◽  
Pharmacological Characterization ◽  
Coagulant Activity ◽  
Exclusion Chromatography

A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of Km and Vmax of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.

Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

2006 ◽  
Vol 52 (2) ◽  
pp. 130-139 ◽  
Author(s):  
Miao Wang ◽  
Jinkui Yang ◽  
Ke-Qin Zhang
Keyword(s):  
Serine Protease ◽  
Catalytic Triad ◽  
Infection Process ◽  
Gene Encoding ◽  
Protein Substrates ◽  
Nematocidal Activity ◽  
Infection Mechanisms ◽  
Biocontrol Potential

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 °C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid – histidine – serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.Key words: extracellular serine protease, Monacrosporium microscaphoides, nematode-trapping fungus, nematocidal activity.

scholarly journals Coprinus comatus Damages Nematode Cuticles Mechanically with Spiny Balls and Produces Potent Toxins To Immobilize Nematodes

2007 ◽  
Vol 73 (12) ◽  
pp. 3916-3923 ◽  
Author(s):  
Hong Luo ◽  
Yajun Liu ◽  
Lin Fang ◽  
Xuan Li ◽  
Ninghua Tang ◽  
...  
Keyword(s):  
Uv Spectrum ◽  
Severe Injuries ◽  
Obvious Effect ◽  
Coprinus Comatus ◽  
Chemical Structures ◽  
Panagrellus Redivivus ◽  
Fungal Structure ◽  
Free Living Nematode ◽  
Nematode Cuticle

ABSTRACT We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. M�ll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus, and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.

Digestion of invertebrate neuropeptides by preparations from the free-living nematodePanagrellus redivivus

2008 ◽  
Vol 82 (3) ◽  
pp. 279-285 ◽  
Author(s):  
E.P. Masler
Keyword(s):  
Serine Proteases ◽  
Free Living ◽  
Peptide Substrates ◽  
Protease Cleavage ◽  
Rp Hplc ◽  
Panagrellus Redivivus ◽  
Free Living Nematode ◽  
Aminopeptidase P ◽  
Hydrolysis Of

AbstractProteases in the soluble fraction of homogenates prepared from the free-living nematodePanagrellus redivivushydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14)in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27°C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss;P < 0.0001), while FLP-14 was digested less (84% loss;P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss;P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss;P < 0.0001) and FLP-12 (59% loss;P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss;P < 0.0005). Further analysis of FMRFa digestion using peptides withd-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss;P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism inP. redivivus.

scholarly journals Substrate-induced changes in dynamics and molecular motions of cuticle-degrading serine protease PL646: a molecular dynamics study

RSC Advances ◽  
2017 ◽  
Vol 7 (67) ◽  
pp. 42094-42104 ◽  
Author(s):  
Li-Quan Yang ◽  
Peng Sang ◽  
Ruo-Peng Zhang ◽  
Shu-Qun Liu
Keyword(s):  
Molecular Dynamics ◽  
Serine Protease ◽  
Serine Proteases ◽  
Nematophagous Fungi ◽  
Molecular Motions ◽  
Nematode Cuticle ◽  
Infection Processes ◽  
Induced Changes

Cuticle-degrading serine proteases secreted by nematophagous fungi can degrade the nematode cuticle during the infection processes.

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