The role of WSC domain-containing protein encoding gene AOL_s00043g401 in the growth and nematode trapping of Arthrobotrys oligospora.

Arthrobotrys oligospora is a model nematode-trapping fungus that has been extensively investigated to understand the interactions between fungi and nematodes. Nematode capture by A. oligospora is a complex process in which recognition of nematodes is generally believed to be mediated by lectins from the fungi. Lectins are a group of carbohydrate-binding proteins that widely exist in microorganisms and contain specific glycosylation recognition domains. In this work, we report the effect of a putative WSC domain-containing protein encoding gene AOL_s00043g401 (g401) on the growth and nematode-trapping of A. oligospora. The g401 gene was knocked out using the homologous recombination approach, and the △g401 mutant strain was then evaluated for its growth rate, conidial yield and germination rate, adaptation to environmental stresses, and nematocidal activity. Interestingly, the deletion of the putative lectin gene g401 had no significant effect on saprophytic growth, conidial yield and germination rate, responses to high salt, surfactant, and strong oxidative environments, as well as nematode-trapping efficiency of A. oligospora. We speculate that this phenomenon might have been caused by an intrinsic genetic compensation of the g401 in this fungus. For instance, more copies of WSC domain encoding genes or PQQ-DH domain encoding genes were found in the genome of A. oligospora. These findings provide further experimental evidence on the effect of lectin-related functional proteins in A. oligospora on nematode capture and will help further analyze their potential roles in the biological control of nematodes in the future.

A putative F-box-domain-encoding gene AOL_s00076g207 regulates the development and pathogenicity of Arthrobotrys oligospora.

F-box protein is a key component of the Skp1-cullin-F-box-type ubiquitin ligase complex (SCF-ULC) that marks its target proteins with ubiquitin for proteasomal degradation. In this study, we explored the potential role of AOL_s00076g207 (Aog207) in Arthrobotrys oligospora, a model fungus for studying nematodes-fungi interactions. The Aog207 gene encodes a putative F-box protein of the SCF-ULC. Deletion of Aog207 could inhibit mycelial growth in TYGA and PDA media. More importantly, the conidial germination rate of ΔAog207 mutants was remarkably declined compared to that of wild-type (WT) strain, and the mutant strains were more sensitive toward chemical stressors than the WT strain. In addition, ΔAog207 mutants generated fewer traps and captured fewer nematodes than WT strain. In summary, Aog207 disruption significantly affected the pathogenicity, mycelial growth, conidial germination, environmental adaptation and trap formation of A. oligospora. These findings may facilitate a better understanding of the nematode predation mechanism of A. oligospora and provide an experimental basis for developing biological control agents against nematodes.

Purification and antioxidant and anti-Inflammatory activity of extracellular polysaccharopeptide from sanghuang mushroom, Sanghuangporus lonicericola.

BACKGROUND: Sanghuang mushrooms are medicinal fungi widely used in eastern Asia. In this study, the antioxidant and anti-inflammatory activity of a novel extracellular polysaccharopeptide, sanghuang extracellular polysaccharopeptide (SePSP) was investigated. The SePSP was purified from the submerged fermentation broth of a sanghuang mycelium, Sanghuangporus lonicericola strain CBS17, which was isolated from a wild sanghuang fruiting body. RESULTS: The SePSP was extracted using an ethanol precipitation procedure, followed by diethylaminoethanol (DEAE) anion-exchange and size-exclusion chromatography. The mass ratio of the polysaccharide and peptide components in the purified SePSP was approximately 4.87:1. By determining its free radical scavenging abilities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), the hydroxyl free radical, and the superoxide anion free radical, as well as its total reducing power, SePSP was shown to have strong concentration-dependent antioxidant activity in vitro. Further, SePSP effectively alleviated dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice. Administration of 200 mg kg-1 SePSP by gavage for 7 days prevented body weight loss; significantly reduced the mRNA levels of proinflammatory cytokines, including TNF-α and IL-1ß; increased mRNA level of the anti-inflammatory cytokine IL-10 in the colon, and decreased the malondialdehyde concentration from 6.42 to 4.82 µmol L-1 in the blood in UC mice. CONCLUSION: The SePSP had strong concentration-dependent antioxidant activity in vitro and effectively alleviated DSS-induced UC in mice. The in vivo therapeutic efficacy in DSS-induced UC may be mediated by modulating the expression of inflammatory cytokines and inhibiting oxidative stress. The findings provide a scientific rationale for the use of bioactive nutraceuticals from sanghuang mushrooms to develop functional foods for the prevention and treatment of UC. © 2020 Society of Chemical Industry.

Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13.

G13 is a 19-residue cationic antimicrobial peptide derived from granulysin. In order to achieve high-level expression of G13 in Escherichia coli cells, and to reduce downstream processing costs, we introduced an Asp-Pro acid labile bond between the His-Patch thioredoxin and G13 and constructed the recombinant plasmid pThiohisA-DP-G13. The plasmid was transformed into E. coli BL21 (DE3). After induction with isopropyl-ß-d-thiogalactopyranoside for 5 h, the fusion protein accumulated up to 200 mg/L in soluble form. The fusion protein was released by a high pressure homogenizer, cleaved using 13% acetic acid at 50 °C hydrolysis for 72 h. The recombinant G13 (r-G13) was then successively purified by fractional precipitation with ammonium sulfate and trichloroacetic acid, followed by one-step cation exchange chromatography. The purified r-G13 displayed a single band (about 2.2 kDa) as analyzed by Tris-Tricine buffered SDS-PAGE, and its precise molecular weight was confirmed using tandem mass spectrometry. Analysis of r-G13 by circular dichroism (CD) indicated that r-G13 contained predominantly ß-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli.

High-level secretory expression of metchnikowin in Escherichia coli.

Metchnikowin is a proline-rich peptide from Drosophila with antibacterial and antifungal activities. Its commercial application is limited due to the low immune-inducible expression in vivo. Here we present a method to produce high-yield metchnikowin in recombinant Escherichia coli. The genes coding for metchnikowin and the fusion partner Cherry tag were subcloned into the pET22b vector to construct a recombinant expression plasmid and transformed into E. coli BL21 (DE3). The fusion protein was successfully expressed and secreted with a yield of 300µg/ml after 18-h induction. Active metchnikowin was released by cleavage of the Asp-Pro bond between fused proteins by 72-h formic acid hydrolysis at 50°C. After 24-h dialysis, metchnikowin was purified to electrophoretic homogeneity and showed significant antibacterial activities against both Bacillus subtilis and E. coli DH5α. It is the first report on efficient production of metchnikowin in recombinant E. coli.

The fucose-specific lectin gene AOL_s00054g276 affects trap formation and nematocidal activity of the nematophagous fungus Arthrobotrys oligospora.

Nematode-trapping fungi are natural enemies of nematodes in nature. Arthrobotrys oligospora, a typical nematode-trapping fungus with a clear genetic background, can capture and infect nematodes by forming adhesive three-dimensional networks. Lectins, a class of glycoproteins containing glycosyl-specific recognition domains, play an important role in biological recognition. However, the fucose-specific lectins have rarely been studied regarding the process of preying on nematodes. In this study, we characterized the biological role of the fucose-specific lectin-encoding gene AOL_s00054g276 (g276) in A. oligospora. The gene g276 was first deleted based on homologous recombination, then the phenotype and nematocidal activity of the Δg276 mutant was evaluated. The results showed that the deletion of gene g276 delayed trap formation and weakened its nematocidal activity; however, mycelial growth, conidia production, conidial germination rates and adaption to environmental stresses were not affected. Our results suggest that the fucose-specific lectin-encoding gene g276 might be associated with the morphogenesis of this fungus, and its deletion resulted in a significantly low density of three-dimensional traps (P < 0.05) and a significantly low nematode-trapping efficiency (P < 0.001). These findings provide a basis for further elucidating the mechanism of A. oligospora preying on nematodes and lay a foundation for the development and utilization of fungal-derived lectins for nematode control in the future.

Probiotic Bacillus cereus Alleviates Dextran Sulfate Sodium-Induced Colitis in Mice through Improvement of the Intestinal Barrier Function, Anti-Inflammation, and Gut Microbiota Modulation.

Dysbiosis leads to continuous progress of inflammatory bowel disease (IBD). However, current therapeutic approaches for IBD have limited efficacy and are associated with various side effects. This study focused on exploring the positive effect of a new Bacillus cereus (B. cereus) strain (HMPM18123) in a colitis mouse model and elucidate the underlying molecular mechanisms. The colitis symptoms were alleviated by the B. cereus administration as evidenced by decreased body weight loss, colon length shortening, disease activity index score, and histopathological score. The B. cereus mitigated intestinal epithelial barrier damage by upregulating tight junction protein expression. Moreover, B. cereus exerted anti-inflammatory effects by regulating macrophage polarization and suppressing the TLR4-NF-κB-NLRP3 inflammasome signaling pathways. B. cereus also rebalanced the damaged gut microbiota. Thus, the molecular mechanism of alleviating colitis by B. cereus treatment involved the regulation of the TLR4-NF-κB-NLRP3 inflammasome signaling pathways in intestinal mucosal barriers by modulating gut microbiota composition.

The role of Jacalin-related lectin gene AOL_s00083g511 in the development and pathogenicity of the nematophagous fungus Arthrobotrys oligospora.

Arthrobotrys oligospora is a model species of nematophagous fungi and has great potential for the biological control of nematode diseases. Lectin is a protein that binds to carbohydrates and their complexes with high specificity, which mediates recognition events in various physiological and pathological processes. This study aimed to investigate the role of the Jacalin-related lectin (JRL) gene, AOL_s00083g511, in A. oligospora development. Through a homology recombination approach, we obtained the AOL_s00083g511 knockout mutant strain (Ag511). Next, the biological characteristics of the Ag511 mutant strain, including growth rate, conidia germination rate, adaptation to environmental stresses, and nematocidal activity, were compared with those of the wild-type (WT) strain. The results showed that the JRL gene AOL_s00083g511 did not affect fungal growth, conidia germination, 3D-trap formation, and the ability of A. oligospora to prey on nematodes significantly. We speculate that this phenomenon may be caused by a loss of the key ß1-ß2 loops in the AOL_ s00083g511-encoded JRL domain and an intrinsic genetic compensation of AOL_s00083g511 in this fungus. The growth rates of both strains on high salt or surfactant media were similar; however, in the strong oxidation medium, the growth rate of the Ag511 mutant was significantly lower than that of the WT strain, indicating that AOL_s00083g511 might play a role in oxidative stress resistance. These findings provide a basis for further analysis of the related functions of the JRL gene in A. oligospora and their potential roles in the biological control of nematodes in the future.

Grape seed proanthocyanidin extract ameliorates dextran sulfate sodium-induced colitis through intestinal barrier improvement, oxidative stress reduction, and inflammatory cytokines and gut microbiota modulation.

It is widely believed that grape seed proanthocyanidin extract (GSPE) exerts antioxidant and anti-inflammatory effects. Dietary supplementation with GSPE has been reported to alleviate colitis signs in mice, but the mechanisms involved require further exploration. The present study investigated how the oral administration of GSPE ameliorates colitis signs and reduces colitis-associated inflammation. C57BL/6 mice were treated with GSPE for 21 days. During the final 7 days of treatment, the mice were administered dextran sulfate sodium (DSS) dissolved in drinking water to induce experimental colitis. We found that GSPE treatment improved DSS-induced colitis, which was evidenced by decreases in disease activity index (DAI) scores, pathological scores, and oxidative stress and increases in zonula occludens-1 (ZO-1), occludin, and claudin-1 mRNA levels of colon tissue. Notably, the proinflammatory cytokines TNF-α and IL-1ß were significantly downregulated as a result of GSPE treatment in colon tissues. GSPE treatment also reduced NLR family pyrin domain-containing 3 (NLRP3) inflammasome mRNA levels of colon tissue. Furthermore, an analysis of 16S rRNA sequences showed that GSPE rebalanced the DSS-damaged gut microbiota, including reducing Bacteroidetes, Dubosiella, and Veillonella, increasing Verrucomicrobia and Akkermansia, and elevating the Firmicutes to Bacteroidetes ratio. In conclusion, GSPE supplementation alleviates DSS-induced colitis by modulating inflammatory cytokines and oxidation stress, maintaining the intestinal barrier, and improving the microbial community. These results indicate that GSPE might be a new dietary strategy for the treatment of ulcerative colitis.

Associations of the ABCA1 and LPL Gene Polymorphisms With Lipid Levels in a Hyperlipidemic Population.

We conducted a cross-sectional study to investigate the effects of the adenosine triphosphate-binding cassette transporter 1 (ABCA1) I883M and lipoprotein lipase (LPL) HindIII polymorphisms on lipid levels in patients with hyperlipidemia. A total of 533 patients were enrolled. Serum lipid parameters were determined by an automatic biochemistry analyzer. Genotyping of the ABCA1 I883M and LPL HindIII was carried out using the polymerase chain reaction-restriction fragment length polymorphism technique. Multiple linear regression analysis was used to estimate the associations between serum lipid levels and the genetic polymorphisms. The frequency distribution of the ABCA1 I883M and LPL HindIII polymorphisms did not deviate from Hardy-Weinberg equilibrium. The major finding of our regression analysis showed that neither the ABCA1 I883M nor the LPL HindIII polymorphism was associated with baseline serum lipid levels in the total population. However, among patients with elevated alanine aminotransferase (ALT) levels (ALT ≥ 40 U/L), carriers of the M allele of the ABCA1 gene had lower levels of high-density lipoprotein cholesterol (HDL-C) and higher levels of low-density lipoprotein cholesterol (LDL-C) after adjusting for age, sex, smoking status, alcohol consumption, education level, occupation, and work intensity ( P < .05 for both). A test on interaction terms between the ABCA1 I833M polymorphism and ALT on HDL-C and LDL-C levels also remained significant ( P = .001 and P = .014, respectively). Our data suggest that there are significant interactive effects between ABCA1 I883M and ALT levels on HDL-C and LDL-C levels. However, the LPL HindIII polymorphism did not influence lipid levels.

A gender-specific association of the polymorphism Ile197Met in the kininogen 1 gene with plasma irbesartan concentrations in Chinese patients with essential hypertension.

This study was conducted to explore interactions in the association of the kininogen (KNG1) Ile197Met polymorphism and gender with plasma concentrations of irbesartan in Chinese patients with essential hypertension. A total of 1100 subjects with essential hypertension received a daily oral dose of 150 mg irbesartan for twenty-eight consecutive days. High-performance liquid chromatography-fluorescence (HPLC) was used to detect plasma irbesartan concentrations on day 28. The KNG1 Ile197Met gene polymorphism was determined using high-throughput TaqMan technology. The frequency distribution of KNG1 Ile197Met genotype conformed to the Hardy-Weinberg equilibrium. After 28 days of treatment, patients with the GG genotype had significantly lower irbesartan concentrations (P = 0.033) compared to homozygous TT genotype carriers. After stratifying by gender, male G allele carriers had significantly lower irbesartan concentrations (GG, P = 0.015; TG, P = 0.015, respectively) relative to TT genotype after adjusting for age, region, body mass index (BMI), smoking, and alcohol consumption. However, there was no significant difference in female subjects. A further test for a multiplicative interaction between the KNG1 Ile197Met polymorphism and gender in association with ln-plasma irbesartan concentrations in a multiple linear regression model was also significant (P for interaction = 0.033). This is the first study to suggest that gender may influence the association of the Ile197Met variant of KNG1 with ln-plasma irbesartan concentration. This finding may indicate that the interaction of gender and the KNG1 Ile197Met gene polymorphism can influence plasma trough irbesartan concentrations, which may contribute to a better development of personalized hypertensive treatment in Chinese patients.

Associations of the SLCO1B1 Polymorphisms With Hepatic Function, Baseline Lipid Levels, and Lipid-lowering Response to Simvastatin in Patients With Hyperlipidemia.

Our goal was to examine the associations of the 388A>G and 521T>C polymorphisms in the solute carrier organic anion transporter 1B1 (SLCO1B1) gene with hepatic function, baseline lipid levels, and the lipid-lowering efficiency of simvastatin. We recruited 542 patients with hyperlipidemia. The 388A>G and 521T>C polymorphisms were genotyped. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST), Serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured before and after an oral 20-mg dose of simvastatin. Individuals with the 388AA genotype had higher ALT and AST levels than those with the 388AG or 388GG genotypes (P = .037 and P = .002, respectively). Individuals with both the 388AA and the 521TT genotypes had the highest levels of ALT and AST (P = .001 and P = .001, respectively). Moreover, we divided all patients into normal and abnormal subgroups based on elevated ALT and AST values (≥ 40 U/L), participants in the abnormal subgroup had a higher frequency of the 388A/521T haplotype and a lower frequency of the 388G/521T haplotype compared to those in the normal subgroup. In addition, compared to 388G allele and 521C allele carriers, individuals with the 388G allele and 521TT genotype carriers had greater TC and LDL-C reduction in response to simvastatin after 4 weeks of treatment. Our conclusion suggests that the interaction between the SLCO1B1 388A>G and 521T>C polymorphisms could be an important genetic determinant of hepatic function and the therapeutic efficiency of simvastatin in Chinese patients with hyperlipidemia.

[Expression of Pleurocidin from winter flounder in Escherichia coli and optimization of culture conditions].

To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.

[Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli].

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.

Thrombin-like enzymes from venom gland of Deinagkistrodon acutus: cDNA cloning, mechanism of diversity and phylogenetic tree construction.

AIM: To clone cDNAs of thrombin-like enzymes (TLEs) from venom gland of Deinagkistrodon acutus and analyze the mechanisms by which their structural diversity arose. METHODS: Reverse transcription-polymerase chain reaction and gene cloning techniques were used, and the cloned sequences were analyzed by using bioinformatics tools. RESULTS: Novel cDNAs of snake venom TLEs were cloned. The possibilities of post-transcriptional recombination and horizontal gene transfer are discussed. A phylogenetic tree was constructed. CONCLUSION: The cDNAs of snake venom TLEs exhibit great diversification. There are several types of structural variations. These variations may be attributable to certain mechanisms including recombination.

cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus.

AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template. The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100). Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.