Characterization of a novel PepF-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A

An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepFBa oligopeptidase is a secreted protein and may facilitate the process of sporulation.     

Benzylideneacetone and other phenylethylamide bacterial metabolites induce apoptosis to kill insects

Xenorhabdus and Photorhabdus are entomopathogenic bacteria that can induce immunosuppression against target insects by suppressing eicosanoid biosynthesis, leading to fatal septicemia. These bacteria can synthesize and release secondary metabolites such as benzylideneacetone (BZA) and other phenylethylamide compounds that can inhibit phospholipase A₂ (PLA₂) and shut down eicosanoid biosynthesis. However, insecticidal activities of these bacterial metabolites remain unclear. Thus, the objective of this study was to assess cytotoxicities of BZA and seven other bacterial metabolites to insect cells. These eight bacterial metabolites exhibited significant cytotoxicities against an insect cell line Sf9 at micromolar range. Especially, BZA and cPY were highly potent at low micromolar range. When these eight bacterial metabolites were injected to hemocoels of Spodoptera exigua larvae, they significantly decreased total count of hemocytes. In Sf9 cell line and hemocytes, these bacterial metabolites induced cell membrane blebbings, apoptotic vesicles, and genomic DNA fragmentation. Terminal deoxyribonucleotidyl transferase nick end translation assay showed that these bacterial metabolites caused significant DNA breakages in cells in a dose-dependent manner. However, a pan caspase inhibitor treatment significantly rescued the cell death induced by these bacterial metabolites. Cytotoxicities of these bacterial metabolites were highly correlated with their insecticidal activities. These results indicate that the insecticidal activities of the bacterial metabolites may be induced by their apoptotic activities against hemocytes and other insect cells. Taken together, these results suggest that phenylethylamide compounds might have potential as novel insecticides.     

An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.     

A Cytosolic Serine Endopeptidase from Trypanosoma cruzi Is Required for the Generation of Ca2+ Signaling in Mammalian Cells

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca²⁺-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca²⁺ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca²⁺-signaling activity for mammalian cells.     

Crystal Structures of Trypanosoma brucei Oligopeptidase B Broaden the Paradigm of Catalytic Regulation in Prolyl Oligopeptidase Family Enzymes

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.     

Ex-situ enzyme activity and bacterial community diversity through soil depth profiles in penguin and seal colonies on Vestfold Hills, East Antarctica

The soils impacted by sea animal excreta are important sources of nutrients in Antarctic terrestrial ecosystems, and soil microorganisms are the principal drivers of carbon and nitrogen cycling. However, microbial diversity and enzyme activities in these soils have still received little attention. In this paper, we investigated the distribution characteristics of bacterial community in four penguin and seal colony soil profiles collected in East Antarctica, using 16S rDNA-DGGE and real-time quantitative PCR. Soil microbial biomass carbon (C_mic), soil respiration (SR), and enzyme activities involved in carbon, nitrogen, and phosphorus metabolisms were also measured. Overall soil C_mic, SR, enzyme activities, and bacterial abundance decreased with depth. The bacterial abundance had a significant correlation with soil organic carbon and total nitrogen and highly corresponded to the relative content of penguin guano or seal excreta in these soil profiles. The 16S rDNA-DGGE revealed the complicated bacterial community structure in penguin and seal colony soils, and the band richness and dominant bands decreased with soil depth. Cluster analysis of DGGE profiles indicated that bacterial community in those soil profiles were divided into four main categories with the bacterial genetic similarity of 22 %, and the majority of the sequenced bands were Proteobacteria (α, β, γ), Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Chloroflexi, and Firmicutes. Our results indicated that the deposition of penguin guano or seal excreta, which caused the variability in soil soil organic carbon, total nitrogen, pH, and soil moisture, might have an important effect on the vertical distribution pattern of bacterial abundance and diversity in Antarctic soil profiles.     

Structural revision of kynapcin-12 by total synthesis,and inhibitory activities against prolyl oligopeptidase and cancer cells

Kynapcin-12 is a prolyl oligopeptidase (POP) inhibitor isolated from Polyozellus multiplex, and its structure was assigned as 1 having a p-hydroquinone moiety by spectroscopic analyses and chemical means. This Letter describes the total syntheses of the proposed structure 1 for kynapcin-12 and 2′,3′-diacetoxy-1,5′,6′,4″-tetrahydroxy-p-terphenyl 2 isolated from Boletopsis grisea, revising the structure of kynapcin-12 to the latter. These syntheses involved double Suzuki–Miyaura coupling, CAN oxidation, and LTA oxidation as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.     

Nitrogen uptake of Hypholoma fasciculare and coexisting bacteria

The white-rot fungus Hypholoma fasciculare coexists with a bacterial community that uses low-molecular weight carbon sources provided by fungal, extracellular enzyme activities. Since fungal development on wood is limited by the availability of nitrogen (N), bacteria could contribute to the N supply. To prove or disapprove an interaction in terms of N transfer, N sources of the fungus and the coexisting bacterial isolates were investigated, and the bacterial N₂ fixation was quantified. Fungal, fungal—bacterial and bacterial wood decomposition was analysed by Fourier transform infrared spectroscopy (FTIR), mass loss and surface pH. Microbial N preferences were investigated by elemental analysis isotope ratio mass spectrometry (IRMS). In addition, diazotrophic activity was explored after cultivation under a ¹⁵?N₂/O₂ atmosphere. Decomposition was similar with and without bacteria and both H. fasciculare and coexisting bacteria preferred reduced N species, such as urea, ammonium and organic N. In most of the bacteria, the ¹⁵?N abundance in the biomass increased significantly but to a low extent if they were cultivated under a ¹⁵?N₂/O₂ atmosphere. This effect is considered an artefact and attributed to adsorption rather than to bacterial N₂ fixation activity. Hence, the bacteria coexisting with H. fasciculare rather competed for the same N sources than supported fungal N supply by diazotrophic activity.     

Identification and characterization of a novel prolyl oligopeptidase in filarial parasite Setaria cervi

A 75 kDa serine protease having prolyl oligopeptidase activity has been purified from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 6 peptides showing nearest match S9A (prolyl oligopeptidase) family protein from Plesiocystis pacifica. The ScPOP was found to be unique compared to mammalian POP with respect to its kinetic properties. To elucidate its role, filarial parasites were exposed to specific inhibitor of POP, Z-Pro-prolinal (ZPP) for 8?h. The inhibition of POP induced calcium signaling via phospholipase c stimulation which further triggered mitochondrial mediated apoptosis in filarial parasites.     

Sequential immunosuppressive activities of bacterial secondary metabolites from the entomopahogenic bacterium Xenorhabdus nematophila

The entomopathogenic bacterium Xenorhabdus nematophila secretes at least eight bacterial metabolites that play crucial roles suppressing target insect immune responses by inhibiting eicosanoid biosynthesis. We analyzed sequential changes in bacterial metabolite production during bacterial growth and analyzed their individual immunosuppressive activities against the insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth pattern in both insect host and culture medium, and eight metabolites were secreted at different time points. At the early growth phase (6–12 h), Ac-FGV and PHPP were detected in significant amounts in the culture broth. At this early phase, both Ac-FGV (18 μg/ml) and oxindole (110 μg/ml) levels significantly inhibited phenoloxidase and phospholipase A₂ activities in S. exigua hemolymph. At the late growth phase (12–36 h), all eight metabolites were detected at significant levels (10–140 μg/ml) in the culture broth and were sufficient to induce hemocyte toxicity. These results suggest that X. nematophila sequentially produces immunosuppressive metabolites that might sequentially and cooperatively inhibit different steps of insect immune responses.     

Synthesis of polyozellin,a prolyl oligopeptidase inhibitor,and its structural revision

Polyozellin is a p-terphenyl compound which was isolated from Polyozellus multiplex, and exhibits an inhibitory activity against prolyl oligopeptidase (POP). Its structure was assigned as 1 having a p-terphenyl skeleton including a p-substituted dibenzofuran moiety by spectroscopic analyses and chemical means. This paper describes the total syntheses of the proposed structure 1 for polyozellin and its o-isomer 2, revising the structure of polyozellin to the latter. These syntheses involved a double Suzuki-Miyaura coupling using chlorophenylboronic acid as a common key building block, and Cu mediated Ullmann cyclization as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.     

Isoquinoline Alkaloids from Fumaria officinalis L. and Their Biological Activities Related to Alzheimer's Disease

Two new isoquinoline alkaloids, named fumaranine ( 2 ) and fumarostrejdine ( 10 ), along with 18 known alkaloids were isolated from aerial parts of Fumaria officinalis. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses and by comparison with literature data. The absolute configuration of the new compound 2 was determined by comparing its circular dichroism spectra with those of known analogs. Compounds isolated in sufficient amounts were evaluated for their acetylcholinesterase, butyrylcholinesterase, prolyl oligopeptidase (POP), and glycogen synthase kinase‐3β inhibitory activities. Parfumidine ( 8 ) and sinactine ( 15 ) exhibited potent POP inhibition activities (IC₅₀ 99±5 and 53±2 μM , resp.).     

Novel amide derivatives containing 1,3,4-thiadiazole moiety: Design,synthesis, nematocidal and antibacterial activities

A series of novel amide derivatives containing 1,3,4-thiadiazole moiety were synthesized and their bioactivities were evaluated. The compound 34 exhibited good nematocidal activities against Meloidogyne incognita in vitro and in vivo, the LC₅₀ value and control effect were 6.5?mg/L and 83.3%, respectively. Meanwhile, it exhibited exciting antibacterial activities against Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. citri, and Ralstonia solanacearum, the EC₅₀ values were 0.4, 6.7 and 5.1?mg/L, respectively, which were better than positive controls. The curative and protection activities under the greenhouse conditions of compound 34 against rice bacterial blight were 47.9 and 55.8%, respectively. The structure-activity relationship were analyzed in detail.     

Design,synthesis, and antibacterial activity against rice bacterial leaf blight and leaf streak of 2,5-substituted-1,3,4-oxadiazole/thiadiazole sulfone derivative

A series of 2,5-substituted-1,3,4-oxadiazole/thiadiazole sulfone derivatives were synthesized and evaluated for their antibacterial activities against rice bacterial leaf blight and leaf streak caused by Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicolaby via the turbidimeter test in vitro. Antibacterial bioassay results indicated that most compounds demonstrated good inhibitory effect antibacterial bioactivities against rice bacterial leaf blight and leaf streak. Among the title compounds, compound 6c demonstrated the best inhibitory effect against rice bacterial leaf blight and leaf streak with half-maximal effective concentration (EC₅₀) values of 1.07 and 7.14 μg/mL, respectively, which were even better than those of commercial agents such as Bismerthiazol and Thiediazole Copper. In vivo antibacterial activities tests at greenhouse conditions demonstrated that the controlling effect of compounds 6c (43.5%) and 6g (42.4%) against rice bacterial leaf blight were better than those of Bismerthiazol (25.5%) and Thiediazole Copper (37.5%).     

A dimeric pyrrocidine from Neonectria ramulariae is an inhibitor of prolyl oligopeptidase

Four novel alkaloids, bispyrrocidine (5), the epoxy derivative of pyrrocidine B (6), 19-O-methyl-pyrrrocidine B (7) and 19-O-ethyl-pyrrrocidine B (8) were isolated from the endophytic fungus Neonectria ramulariae Wollenw KS-246. Their structures were elucidated using 1D- and 2D-NMR spectroscopy. Compound 6 exhibited cytotoxicity against HL60 cells (IC₅₀ 4.6 μM), whereas 5 showed specific inhibitory activity against prolyl oligopeptidase (IC₅₀ 2.6 μM) in a non-competitive manner.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage.

The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257-264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.     

Bioorganic Fertilizer Enhances Soil Suppressive Capacity against Bacterial Wilt of Tomato

Tomato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Many strategies have been taken to improve soil suppressiveness against this destructive disease, but limited success has been achieved. In this study, a novel bioorganic fertilizer revealed a higher suppressive ability against bacterial wilt compared with several soil management methods in the field over four growing seasons from March 2011 to July 2013. The application of the bioorganic fertilizer significantly (P<0.05) reduced disease incidence of tomato and increased fruit yields in four independent trials. The association among the level of disease incidence, soil physicochemical and biological properties was investigated. The soil treated with the bioorganic fertilizer increased soil pH value, electric conductivity, organic carbon, NH₄ ⁺-N, NO₃ ^--N and available K content, microbial activities and microbial biomass carbon content, which were positively related with soil suppressiveness. Bacterial and actinomycete populations assessed using classical plate counts were highest, whereas R. solanacearum and fungal populations were lowest in soil applied with the bioorganic fertilizer. Microbial community diversity and richness were assessed using denaturing gel gradient electrophoresis profile analysis. The soil treated with the bioorganic fertilizer exhibited higher bacterial community diversity but lower fungal community diversity. Redundancy analysis showed that bacterial community diversity and richness negatively related with bacterial wilt suppressiveness, while fungal community richness positively correlated with R. solanacearum population. We concluded that the alteration of soil physicochemical and biological properties in soil treated with the bioorganic fertilizer induced the soil suppressiveness against tomato bacterial wilt.     

A potential processing enzyme in prokaryotes: Oligopeptidase B,a new type of serine peptidase

Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products. Such enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes. Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidases. The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates. The k_cat/K_m is extremely high, 63 μM⁻¹ s⁻¹, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4- methylcoumaryl)amide. The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s). This effect is abolished at 1 M NaCl. In addition, high ionic strength inhibits the reaction considerably by increasing K_m, which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups. In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis. The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms. Proteins 28:375–379, 1997. © 1997 Wiley-Liss, Inc.