Serine Carboxypeptidase Research Articles

Phylogenomic and synteny analysis of BAHD and SCP/SCPL gene families reveal their evolutionary histories in plant specialized metabolism.

Plant chemical diversity is largely owing to a number of enzymes which catalyse reactions involved in the assembly, and in the subsequent chemical modifications, of the core structures of major classes of plant specialized metabolites. One such reaction is acylation. With this in mind, to study the deep evolutionary history of BAHD and the serine-carboxypeptidase-like (SCPL) acyltransferase genes, we assembled phylogenomic synteny networks based on a large-scale inference analysis of orthologues across whole-genome sequences of 126 species spanning Stramenopiles and Archaeplastida, including Arabidopsis thaliana, tomato (Solanum lycopersicum) and maize (Zea mays). As such, this study combined the study of genomic location with changes in gene sequences. Our analyses revealed that serine-carboxypeptidase (SCP)/serine-carboxypeptidase-like (SCPL) genes had a deeper evolutionary origin than BAHD genes, which expanded massively on the transition to land and with the development of the vascular system. The two gene families additionally display quite distinct patterns of copy number variation across phylogenies as well as differences in cross-phylogenetic syntenic network components. In unlocking the above observations, our analyses demonstrate the possibilities afforded by modern phylogenomic (syntenic) networks, but also highlight their current limitations, as demonstrated by the inability of phylogenetic methods to separate authentic SCPL acyltransferases from standard SCP peptide hydrolases.This article is part of the theme issue 'The evolution of plant metabolism'.

Effector Protein Serine Carboxypeptidase FgSCP Is Essential for Full Virulence in Fusarium graminearum and Is Involved in Modulating Plant Immune Responses.

Fusarium head blight caused by Fusarium graminearum is a significant pathogen affecting wheat crops. During the infection process, effector proteins are secreted to modulate plant immunity and promote infection. The toxin deoxynivalenol is produced in infected wheat grains, posing a threat to human and animal health. Serine carboxypeptidases (SCPs) belong to the α/β hydrolase family of proteases and are widely distributed in plant and fungal vacuoles, as well as animal lysosomes. Research on SCPs mainly focuses on the isolation, purification, and production of a small number of fungi. The role of SCPs in plant secretion, growth and development, and stress resistance has also been extensively studied. However, their functions in F. graminearum, a fungal pathogen, remain relatively unknown. In this study, the biological functions of the FgSCP gene in F. graminearum were investigated. The study revealed that mutations in FgSCP affected the nutritional growth, sexual reproduction, and stress tolerance of F. graminearum. Furthermore, the deletion of FgSCP resulted in reduced pathogenicity and hindered the biosynthesis of deoxynivalenol. The upregulation of FgSCP expression 3 days after infection indicated its involvement in host invasion, possibly acting as a "smokescreen" to deceive the host and suppress the expression of host defensive genes. Subsequently, we confirmed the secretion ability of FgSCP and its ability to inhibit the cell death induced by INF1 in Nicotiana benthamiana cells, indicating its potential role as an effector protein in suppressing plant immune responses and promoting infection. In summary, we have identified FgSCP as an essential effector protein in F. graminearum, playing critical roles in growth, virulence, secondary metabolism, and host invasion.

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-β (TGF-β) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-β superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-β pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Identification of core genes involved in the response of Apocynum venetum to salt stress based on transcriptome sequencing and WGCNA.

Apocynum venetum L. belongs to the Apocynaceae family and is a plant that is highly resistant to stress. It is important in the fields of ecology, feeding, industry and medicine. The molecular mechanism underlying salt tolerance has not been elucidated. In this study, RNA-seq based transcriptome sequencing of A. venetum leaves after 0, 2, 6, 12, 24 and 48 h of treatment with 300 mM NaCl was performed. We conducted a comprehensive analysis of the transcriptome expression profiles of A. venetum under salt stress using the WGCNA method and identified red, black, and brown as the core modules regulating the salt tolerance of A. venetum. A co-expression regulatory network was constructed to identify the core genes in the module according to the correlations between genes. The genes TRINITY_DN102_c0_g1 (serine carboxypeptidase), TRINITY_DN3073_c0_g1 (SOS signaling pathway) and TRINITY_DN6732_c0_g1 (heat shock transcription factor) in the red module were determined to be the core genes. Two core genes in the black module, TRINITY_DN9926_c0_g1 and TRINITY_DN7962_c0_g1, are pioneer candidate salt tolerance-associated genes in A. venetum. The genes in the brown module were mainly enriched in two pathways, namely photosynthesis and osmotic balance. Among them, the TRINITY_DN6321_c0_g2 and TRINITY_DN244_c0_g1 genes encode aquaporin, which is helpful for maintaining the cell water balance and plays a protective role in defending A. venetum under abiotic stress. Our findings contribute to the identification of core genes involved in the response of A. venetum to salt stress.

Sprayable RNAi for silencing of important genes to manage red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

The red palm weevil, Rhynchophorus ferrugineus (Oliver, 1970) (Coleoptera: Dryophthoridae) is the most devastating insect-pest of palm trees worldwide. Synthetic insecticides are the most preferred tool for the management of RPW. Alternatively, RNA interference (RNAi) mediated silencing of crucial genes provides reasonable control of insect pests. Recently, we have targeted four important genes; ecdysone receptor (EcR), serine carboxypeptidase (SCP), actin and chitin-binding peritrophin (CBP) in the 3rd and 5th instar larvae RPW. The results from 20 days trial showed that the survival rate of 3rd instar larvae fed on SCP and actin dsRNAs exhibited the lowest survival (12-68%). While, in the 5th instar larvae, the lowest survival rate (24%) was recorded for SCP after 20 days of incubation. Similarly, the weight of the 3rd and 5th instar larvae treated with SCP and actin was significantly reduced to 2.30-2.36 g and 4.64-4.78 g after 6 days of dsRNA exposure. The larval duration was also decreased significantly in the larvae treated with all the dsRNA treatments. The qRT-PCR results confirmed a significant suppression of the targeted genes as 90-97% and 85-93% in the 3rd and 5th instar larvae, respectively. The results suggest that the SCP and the actin genes can be promising targets to mediate RNAi-based control of RPW.

Comparative transcriptome and proteome reveal the unique genes and proteins of female parasitic wasps, Lysiphlebia japonica Ashmead.

Lysiphlebia japonica Ashmead (Hymenoptera, Braconidae) is an endophagous parasitoid wasp and its host, Aphis gossypii Glover (Hemiptera, Aphididae) is a major cotton pest. L. japonica affects the growth and fatty acid metabolism of cotton aphids after parasitization and has been widely used as a biocontrol agent. However, there are currently few reports about the molecular characteristics of L. japonica, especially the differences between male and female. In this study, using transcriptome and proteome analysis of the abdomen of female and male parasitic wasps, respectively, we obtained a total of 27,169 DEGs and 1,194 DEPs, then a total of 909 positively correlated high-expression proteins and genes were obtained by combined omics analysis. Subsequently, 20 differentially expressed abdomen specific proteins were selected for validation by RT-qPCR and Multiple Reaction Monitoring (MRM) protein verification. The result of RT-qPCR demonstrated that all 20 genes were highly expressed in the abdomen of females, and five target proteins with unique peptide fragments and identification profiles were identified by MRM, which were venom protease, tropomyosin, lipase member I, venom serine carboxypeptidase and calreticulin, respectively. Overall, these results provided molecular resources for the differences between males and females in L. japonica and the screened 20 abdomen specific proteins were verified to demonstrate the validity of the data, which offered important molecular data resources for further studies on the related functional genes of parasitic wasps and the mechanism of parasitoids regulating the growth of aphids. © 2023 Society of Chemical Industry.

Identification of Loci for Four Important Agronomic Traits in Loose-Curd Cauliflower Based on Genome-Wide Association Studies

Cauliflower is a nutritious vegetable with inflorescences that are specialized to form the edible organs called curds. Uncovering key genes underlying important traits is crucial for the genetic improvement of this important crop. However, the genetic basis of many important agronomic traits, including curd performance and plant architecture in cauliflower, remains unclear. GWASs have proved to be powerful tools to study agronomic traits in many crops. To reveal the genetic basis of four important agronomic traits, namely, the main stem height (MSH), purplish curd (PC), external leaf wing (ELW) and weight of a single curd (WSC), we selected 220 core accessions of loose-curd cauliflower for resequencing, phenotypic investigation and GWAS. The approach revealed significant novel loci. We detected several significant associations: on C02 for MSH and PC, on C06 for ELW and on C01 for WSC. More interestingly, we identified a significant single-peak signal for the weight of a single curd (WSC), an important yield trait, and within this signal interval, we identified the BOB01G136670 gene with five SNPs encoding nonsynonymous mutations in the CDS region; these mutations resulted in two haplotypes with significant differences in curd weight. The weight of a single curd was significantly increased in the varieties with the BOB01G136670 Hap1 allele compared to those with BOB01G136670 Hap2. BOB01G136670 was highly conserved with the homologous genes that encode serine carboxypeptidase and belong to the S10 family in other species, including GS5, which functions as a positive regulator of grain size in rice, wheat and maize. Additionally, BOB01G136670 was highly expressed specifically at the curd enlargement stage, with low or even no expression at all in other tissues and stages, indicating that BOB01G136670 is a plausible candidate gene for WSC. Overall, this study identified genomic loci for four important agronomic traits that are relevant for accelerating biological breeding and the improvement of cauliflower varieties.

Unraveling the transcriptional network regulated by miRNAs in blast-resistant and blast-susceptible rice genotypes during Magnaporthe oryzae interaction

The plant pathogen Magnaporthe oryzae poses a significant threat to global food security, and its management through the cultivation of resistant varieties and crop husbandry practices, including fungicidal sprays, has proven to be inadequate. To address this issue, we conducted small-RNA sequencing to identify the roles of miRNAs and their target genes in both resistant (PB1637) and susceptible (PB1) rice genotypes. We confirmed the expression of differentially expressed miRNAs using stem-loop qRT-PCR analysis and correlated them with rice patho-phenotypic and physio-biochemical responses. Our findings revealed several noteworthy differences between the resistant and susceptible genotypes. The resistant genotype exhibited reduced levels of total chlorophyll and carotenoids compared to the susceptible genotype. However, it showed increased levels of total protein, callose, H2O2, antioxidants, flavonoids, and total polyphenols. Additionally, among the defense-associated enzymes, guaiacol peroxidase and polyphenol oxidase responses were higher in the susceptible genotypes. In our comparative analysis, we identified 27 up-regulated and 43 down-regulated miRNAs in the resistant genotype, while the susceptible genotype exhibited 44 up-regulated and 62 down-regulated miRNAs. Furthermore, we discovered eight up-regulated and five down-regulated miRNAs shared between the resistant and susceptible genotypes. Notably, we also identified six novel miRNAs in the resistant genotype and eight novel miRNAs in the susceptible genotype. These novel miRNAs, namely Chr8_26996, Chr12_40110, and Chr12_41899, were found to negatively correlate with the expression of predicted target genes, including Cyt-P450 monooxygenase, serine carboxypeptidase, and zinc finger A20 domain-containing stress-associated protein, respectively. The results of our study on miRNA and transcriptional responses provide valuable insights for the development of future rice lines that are resistant to blast disease. By understanding the roles of specific miRNAs and their target genes in conferring resistance, we can enhance breeding strategies and improve crop management practices to ensure global food security.

Hallmarks of the relationship between host and Trypanosoma cruzi sulfated glycoconjugates along the course of Chagas disease.

American Trypanosomiasis or Chagas disease (ChD), a major problem that is still endemic in large areas of Latin America, is caused by Trypanosoma cruzi. This agent holds a major antigen, cruzipain (Cz). Its C-terminal domain (C-T) is retained in the glycoprotein mature form and bears several post-translational modifications. Glycoproteins containing sulfated N-linked oligosaccharides have been mostly implicated in numerous specific procedures of molecular recognition. The presence of sulfated oligosaccharides was demonstrated in Cz, also in a minor abundant antigen with serine-carboxypeptidase (SCP) activity, as well as in parasite sulfatides. Sulfate-bearing glycoproteins in Trypanosomatids are targets of specific immune responses. T. cruzi chronically infected subjects mount specific humoral immune responses to sulfated Cz. Unexpectedly, in the absence of infection, mice immunized with C-T, but not with sulfate-depleted C-T, showed ultrastructural heart anomalous pathological effects. Moreover, the synthetic anionic sugar conjugate GlcNAc6SO3-BSA showed to mimic the N-glycan-linked sulfated epitope (sulfotope) humoral responses that natural Cz elicits. Furthermore, it has been reported that sulfotopes participate via the binding of sialic acid Ig-like-specific lectins (Siglecs) to sulfosialylated glycoproteins in the immunomodulation by host-parasite interaction as well as in the parasite infection process. Strikingly, recent evidence involved Cz-sulfotope-specific antibodies in the immunopathogenesis and infection processes during the experimental ChD. Remarkably, sera from chronically T. cruzi-infected individuals with mild disease displayed higher levels of IgG2 antibodies specific for sulfated glycoproteins and sulfatides than those with more severe forms of the disease, evidencing that T. cruzi sulfotopes are antigenic independently of the sulfated glycoconjugate type. Ongoing assays indicate that antibodies specific for sulfotopes might be considered biomarkers of human cardiac ChD progression, playing a role as predictors of stability from the early mild stages of chronic ChD.

Genome-wide analysis of the serine carboxypeptidase-like protein family reveals Ga09G1039 is involved in fiber elongation in cotton

The Gossypium is a model genus for understanding polyploidy and the evolutionary pattern of inheritance. This study aimed to investigate the characteristics of SCPLs in different cotton species and their role in fiber development. A total of 891 genes from one typical monocot and ten dicot species were naturally divided into three classes based on phylogenetic analysis. The SCPL gene family in cotton has undergone intense purifying selection with some functional variation. Segmental duplication and whole genome duplication were shown to be the two main reasons for the increase in the number of genes during cotton evolution. The identification of Gh_SCPL genes exhibiting differential expression in particular tissues or response to environmental stimuli provides a new measure for the in-depth characterization of selected genes of importance. Ga09G1039 was involved in the developmental process of fibers and ovules, and it is significantly different from proteins from other cotton species in terms of phylogenetic, gene structure, conserved protein motifs and tertiary structure. Overexpression of Ga09G1039 significantly increased the length of stem trichomes. Ga09G1039 may be a serine carboxypeptidase protein with hydrolase activity, according to functional region, prokaryotic expression, and western blotting analysis. The results provide a comprehensive overview of the genetic basis of SCPLs in Gossypium and further our knowledge in understanding the key aspects of SCPLs in cotton with their potential role in fiber development and stress resistance.

Identification and analysis of the secretome of plant pathogenic fungi reveals lifestyle adaptation.

The secretory proteome plays an important role in the pathogenesis of phytopathogenic fungi. However, the relationship between the large-scale secretome of phytopathogenic fungi and their lifestyle is not fully understood. In the present study, the secretomes of 150 plant pathogenic fungi were predicted and the characteristics associated with different lifestyles were investigated. In total, 94,974 secreted proteins (SPs) were predicted from these fungi. The number of the SPs ranged from 64 to 1,662. Among these fungi, hemibiotrophic fungi had the highest number (average of 970) and proportion (7.1%) of SPs. Functional annotation showed that hemibiotrophic and necrotroph fungi, differ from biotrophic and symbiotic fungi, contained much more carbohydrate enzymes, especially polysaccharide lyases and carbohydrate esterases. Furthermore, the core and lifestyle-specific SPs orthogroups were identified. Twenty-seven core orthogroups contained 16% of the total SPs and their motif function annotation was represented by serine carboxypeptidase, carboxylesterase and asparaginase. In contrast, 97 lifestyle-specific orthogroups contained only 1% of the total SPs, with diverse functions such as PAN_AP in hemibiotroph-specific and flavin monooxygenases in necrotroph-specific. Moreover, obligate biotrophic fungi had the largest number of effectors (average of 150), followed by hemibiotrophic fungi (average of 120). Among these effectors, 4,155 had known functional annotation and pectin lyase had the highest proportion in the functionally annotated effectors. In addition, 32 sets of RNA-Seq data on pathogen-host interactions were collected and the expression levels of SPs were higher than that of non-SPs, and the expression level of effector genes was higher in biotrophic and hemibiotrophic fungi than in necrotrophic fungi, while secretase genes were highly expressed in necrotrophic fungi. Finally, the secretory activity of five predicted SPs from Setosphearia turcica was experimentally verified. In conclusion, our results provide a foundation for the study of pathogen-host interaction and help us to understand the fungal lifestyle adaptation.

Lost genome segments associate with trait diversity during rice domestication

BackgroundDNA mutations of diverse types provide the raw material required for phenotypic variation and evolution. In the case of crop species, previous research aimed to elucidate the changing patterns of repetitive sequences, single-nucleotide polymorphisms (SNPs), and small InDels during domestication to explain morphological evolution and adaptation to different environments. Additionally, structural variations (SVs) encompassing larger stretches of DNA are more likely to alter gene expression levels leading to phenotypic variation affecting plant phenotypes and stress resistance. Previous studies on SVs in rice were hampered by reliance on short-read sequencing limiting the quantity and quality of SV identification, while SV data are currently only available for cultivated rice, with wild rice largely uncharacterized. Here, we generated two genome assemblies for O. rufipogon using long-read sequencing and provide insights on the evolutionary pattern and effect of SVs on morphological traits during rice domestication.ResultsIn this study, we identified 318,589 SVs in cultivated and wild rice populations through a comprehensive analysis of 13 high-quality rice genomes and found that wild rice genomes contain 49% of unique SVs and an average of 1.76% of genes were lost during rice domestication. These SVs were further genotyped for 649 rice accessions, their evolutionary pattern during rice domestication and potential association with the diversity of important agronomic traits were examined. Genome-wide association studies between these SVs and nine agronomic traits identified 413 candidate causal variants, which together affect 361 genes. An 824-bp deletion in japonica rice, which encodes a serine carboxypeptidase family protein, is shown to be associated with grain length.ConclusionsWe provide relatively accurate and complete SV datasets for cultivated and wild rice accessions, especially in TE-rich regions, by comparing long-read sequencing data for 13 representative varieties. The integrated rice SV map and the identified candidate genes and variants represent valuable resources for future genomic research and breeding in rice.

Biobran/MGN-3, an Arabinoxylan Rice Bran, Protects against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An In Vitro and In Silico Study.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), poses a serious global public health threat for which there is currently no satisfactory treatment. This study examines the efficacy of Biobran/MGN-3 against SARS-CoV-2. Biobran is an arabinoxylan rice bran that has been shown to significantly inhibit the related influenza virus in geriatric subjects. Here, Biobran's anti-SARS-CoV-2 activity was assessed using MTT and plaque reduction assays, RT-PCR, ELISA techniques, and measurements of SARS-CoV-2-related gene expression and protein levels. For Vero E6 cells infected with SARS-CoV-2, Biobran reduced the viral load by 91.9% at a dose of 100 μg/mL, it reduced viral counts (PFU/mL) by 90.6% at 50 μg/mL, and it exhibited a significant selectivity index (EC50/IC50) of 22.5. In addition, Biobran at 10 μg/mL inhibited papain-like proteinase (PLpro) by 87% and ACE2 SARS-CoV-2 S-protein RBD by 90.5%, and it significantly suppressed SARS-CoV-2 gene expression, down-regulating E-gene and RdRp gene expression by 93% each at a dose of 50 μg/mL and inhibiting the E-protein by 91.3%. An in silico docking study was also performed to examine the protein-protein interaction (PPI) between SARS-CoV-2 RBD and DC-SIGN as well as between serine carboxypeptidase and papain-like protease PLpro. Serine carboxypeptidase, an active ingredient in Biobran, was found to interfere with the binding of SARS-CoV-2 to its receptor DC-SIGN on Vero cells, thus preventing the cell entry of SARS-CoV-2. In addition, it impairs the viral replication cycle by binding to PLpro. We conclude that Biobran possesses potent antiviral activity against SARS-CoV-2 in vitro and suggest that Biobran may be able to prevent SARS-CoV-2 infection. This warrants further investigation in clinical trials.

Engineering a carboxypeptidase from Aspergillus oryzae M30011 to improve the terminal-specific enzymatic hydrolysis of aromatic amino acids

Due to unique amino acid composition and anti-fatigue properties, high Fischer ratio oligopeptides have shown pharmacological effects on multiple diseases. Currently, preparing oligopeptides with high Fisher ratios by enzymic hydrolysis has many limitations such as complexity of optimization conditions of enzymes, time costing, and process inconvenience. Therefore, it is necessary to investigate the specificity mechanism of enzymatic digestion to obtain high Fischer ratio oligopeptides. In this study, serine carboxypeptidase (CPY) from the fungus Aspergillus Oryzae M30011 was cloned and expressed in Escherichia coli (E. coli). Based on prediction from docking by AutoDock Vina which clarified the binding mode of the CPY with synthetic peptides to improve its specificity to recognize aromatic amino acids, we deduced that the key substrate binding sites 271Tyr, 464Ile and 517Met in the hydrophobic cavity of CPY are associated with substrate specificity of the enzyme. Through a series of mutations, we found that the triple mutant Y271R/I464R/M517R had an activity of 1065 U/mg for the synthetic peptide Phe-Gly-Leu-Gly-Phe. In the combination of a specific chymotrypsin mutant P308R and CPY mutant M517R, the Fisher ratio of the oligopeptides from rice bran protein was 43.16, which was 54.09% higher than that of the non-specific enzyme combination.

Transcriptomic Analysis Insight into the Immune Modulation during the Interaction of Ophiocordyceps sinensis and Hepialus xiaojinensis.

Ophiocordyceps sinensis (Berk.) is an entomopathogenic fungus that can infect the larva of the ghost moth, Hepialus xiaojinensis, causing mummification after more than one year. This prolonged infection provides a valuable model for studying the immunological interplay between an insect host and a pathogenic fungus. A comparative transcriptome analysis of pre-infection (L) and one-year post-infection (IL) larvae was performed to investigate the immune response in the host. Here, a total of 59,668 unigenes were obtained using Illumina Sequencing in IL and L. Among the 345 identified immune-related genes, 83 out of 86 immune-related differentially expressed genes (DEGs) had a much higher expression in IL than in L. Furthermore, the immune-related DEGs were classified as pathogen recognition receptors (PRRs), signal modulators or transductors, and immune effector molecules. Serpins and protease inhibitors were found to be upregulated in the late phase of infection, suppressing the host’s immune response. Based on the above analysis, the expression levels of most immune-related genes would return to the baseline with the immune response being repressed in the late phase of infection, leading to the fungal immunological tolerance after prolonged infection. Meanwhile, the transcriptomes of IL and the mummified larva (ML) were compared to explore O. sinensis invasion. A total of 1408 novel genes were identified, with 162 of them annotated with putative functions. The gene families likely implicated in O. sinensis pathogenicity have been identified, primarily including serine carboxypeptidase, peroxidase, metalloprotease peptidase, aminopeptidases, cytochrome P450, and oxidoreductase. Furthermore, quantitative real-time PCR (qPCR) was used to assess the expression levels of some critical genes that were involved in immune response and fungal pathogenicity. The results showed that their expression levels were consistent with the transcriptomes. Taken together, our findings offered a comprehensive and precise transcriptome study to understand the immune defense in H. xiaojinensis and O. sinensis invasion, which would accelerate the large-scale artificial cultivation of this medicinal fungus.

Multilocus Sequence Typing of Eimeria maxima in Commercial Broiler Flocks.

About 35% of all broiler flocks in the United States receive an anticoccidial vaccine, but it is not possible to easily differentiate Eimeria vaccine strains from Eimeria field isolates. Being able to do that would allow using vaccines in a more targeted way. The objective of this study was to collect Eimeria maxima isolates from broiler flocks that received anticoccidial feed additives and flocks that had been vaccinated against coccidia and then test them with a multilocus sequencing typing (MLST) scheme developed for this study. Fecal samples were obtained from commercial broiler flocks in Alabama and Tennessee. Oocyst counts in samples tended to be lower in flocks receiving anticoccidial feed additives and higher in vaccinated flocks. Selected samples were screened for presence of E. maxima by quantitative PCR, and Eimeria spp. composition was investigated by next-generation amplicon sequencing (NGAS) in 37 E. maxima positive samples. Other detected Eimeria spp. besides E. maxima were Eimeria acervulina in 35 samples, Eimeria praecox in 23 samples, Eimeria mitis or Eimeria mivati in 17 samples, and Eimeria necatrix or Eimeria tenella in 10 samples. Six partial E. maxima genes (dnaJ domain containing protein, 70-kDa heat shock protein, prolyl endopeptidase, regulator of chromosome condensation domain containing protein, serine carboxypeptidase, and vacuolar proton-translocating ATPase subunit) of 46 samples were sequenced. The MLST scheme was able to differentiate two vaccines from each other. Three of 17 samples from vaccinated flocks differed from the vaccine used in the flock, while 16 of 29 samples from unvaccinated flocks differed from the vaccine. However, there was also a large number of low-quality, ambiguous chromatograms and negative PCRs for the selected genes. If and when more advanced, possibly next-generation sequencing-based methods will be developed, the genes should be considered as targets.

In Silico Structural Analysis of Serine Carboxypeptidase Nf314, a Potential Drug Target in Naegleria fowleri Infections.

Naegleria fowleri, also known as the “brain-eating” amoeba, is a free-living protozoan that resides in freshwater bodies. This pathogenic amoeba infects humans as a casual event when swimming in contaminated water. Upon inhalation, N. fowleri invades the central nervous system and causes primary amoebic meningoencephalitis (PAM), a rapidly progressive and often fatal disease. Although PAM is considered rare, reducing its case fatality rate compels the search for pathogen-specific proteins with a structure–function relationship that favors their application as targets for discovering new or improved drugs against N. fowleri infections. Herein, we report a computational approach to study the structural features of Nf314 (a serine carboxypeptidase that is a virulence-related protein in N. fowleri infections) and assess its potential as a drug target, using bioinformatics tools and in silico molecular docking experiments. Our findings suggest that Nf314 has a ligand binding site suitable for the structure-based design of specific inhibitors. This study represents a further step toward postulating a reliable therapeutic target to treat PAM with drugs specifically aimed at blocking the pathogen proliferation by inhibiting protein function.

Anabaenopeptins from Nostoc edaphicum CCNP1411.

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.

Profiling the secretomes of Penicillium expansum reveals that a serine carboxypeptidase (PeSCP) is required for the fungal virulence on apple fruit

Phytopathogenic fungi often secrete effector proteins that suppress host immunity and facilitate infection. Penicillium expansum is an important postharvest fruit rot pathogen that caused blue mold. However, little is known about the function of effectors secreted by P. expansum. In this study, a LC-MS/MS based proteomic approach was used to analyze the secretomes of P. expansum grown in apple juice, a medium used to mimic natural in planta condition. A total of 103 and 148 effector candidates were identified after incubation for 24 and 72 h, respectively. As expected, the majority of the proteins identified with known function were hydrolytic enzymes, such as cell-wall degrading enzymes and peptidases/proteases. One of the identified effector candidates is a serine carboxypeptidase (PeSCP) which was detected at 24 h after incubation and significantly up-regulated at the early stage of infection on apple fruit. Deletion of PeSCP gene led to reduced conidiation, germination as well as affected fungal growth and morphology, and also resulted in altered tolerance to environmental stresses, greatly reduced extracellular carboxypeptidase activity and virulence of P. expansum on apple fruit. Scanning electron microscopy revealed that the knockout of PeSCP in P. expansum impaired its ability to penetrate host plant tissues. Together, our findings demonstrate an important role of PeSCP in the virulence of P. expansum on apple fruit.