Expression of spider toxin in entomopathogenic fungus Lecanicillium muscarium and selection of the strain showing efficient secretion of the recombinant protein

2019 ◽  
Vol 366 (14) ◽  
Author(s):  
Sergey Timofeev ◽  
Galina Mitina ◽  
Eugene Rogozhin ◽  
Viacheslav Dolgikh
Keyword(s):  
Signal Peptide ◽  
Entomopathogenic Fungus ◽  
Microscopic Analysis ◽  
Selective Medium ◽  
Gene Encoding ◽  
Lecanicillium Muscarium ◽  
C Terminus ◽  
Recombinant Product ◽  
Toxin Sequence ◽  
Selection Of

ABSTRACT Beta/delta-agatoxin-1 of spider Agelena orientalis was expressed in entomopathogenic fungus Lecanicillium muscarium. To ensure secretion of the recombinant product by the fungus, the signal secretory peptide of the Metarhizium anisopliae Mcl1 protein was inserted into the sequence. For detection of the recombinant product and selection of transformants, the toxin sequence was also fused with eGFP at the C-terminus. The gene encoding the A. orientalis toxin with the Mcl1 protein signal peptide was commercially synthesized, amplified and cloned into the vector pBARGPE1 designed for heterologous expression under the control of the PgpdA promoter and the trpC terminator of Aspergillus nidulans. A double selection on selective medium and microscopic analysis of transformants allowed obtaining a mitotically stable recombinant strain of L. muscarium. The recognition of the Mcl1 derived signal peptide in the cells of transformants and effective secretion of the hybrid product was confirmed by immunoblotting.

scholarly journals Differential Roles of Individual Domains in Selection of Secretion Route of a Streptococcus parasanguinis Serine-Rich Adhesin, Fap1

2007 ◽  
Vol 189 (21) ◽  
pp. 7610-7617 ◽  
Author(s):  
Qiang Chen ◽  
Baiming Sun ◽  
Hui Wu ◽  
Zhixiang Peng ◽  
Paula M. Fives-Taylor
Keyword(s):  
Signal Peptide ◽  
Wild Type ◽  
Secretion Pathway ◽  
C Terminus ◽  
Region I ◽  
Minimal Sequences ◽  
Region Ii ◽  
Dependent Pathway ◽  
Selection Of ◽  
Streptococcus Parasanguinis

ABSTRACT Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

scholarly journals Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Laura Dean Heckman ◽  
Maria H Chahrour ◽  
Huda Y Zoghbi
Keyword(s):  
Transcriptional Repression ◽  
Neurological Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Gene Encoding ◽  
C Terminus ◽  
Mecp2 Duplication Syndrome ◽  
Negative Effect ◽  
Duplication Syndrome

Loss of function of the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) causes the progressive neurological disorder Rett syndrome (RTT). Conversely, duplication or triplication of Xq28 causes an equally wide-ranging progressive neurological disorder, MECP2 duplication syndrome, whose features overlap somewhat with RTT. To understand which MeCP2 functions cause toxicity in the duplication syndrome, we generated mouse models expressing endogenous Mecp2 along with a RTT-causing mutation in either the methyl-CpG binding domain (MBD) or the transcriptional repression domain (TRD). We determined that both the MBD and TRD must function for doubling MeCP2 to be toxic. Mutating the MBD reproduces the null phenotype and expressing the TRD mutant produces milder RTT phenotypes, yet both mutations are harmless when expressed with endogenous Mecp2. Surprisingly, mutating the TRD is more detrimental than deleting the entire C-terminus, indicating a dominant-negative effect on MeCP2 function, likely due to the disruption of a basic cluster.

scholarly journals Chimeric Fusion (F) and Attachment (G) Glycoprotein Antigen Delivery by mRNA as a Candidate Nipah Vaccine

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca J. Loomis ◽  
Anthony T. DiPiazza ◽  
Samantha Falcone ◽  
Tracy J. Ruckwardt ◽  
Kaitlyn M. Morabito ◽  
...  
Keyword(s):  
Nipah Virus ◽  
Neutralizing Antibody ◽  
T Cell Response ◽  
Antigen Delivery ◽  
Design Elements ◽  
C Terminus ◽  
Glycoprotein Antigen ◽  
Pandemic Threat ◽  
Covalently Linked ◽  
Selection Of

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.

scholarly journals Novel Organization of Genes Involved in Prophage Excision Identified in the Temperate Lactococcal Bacteriophage TP901-1

1999 ◽  
Vol 181 (23) ◽  
pp. 7291-7297 ◽  
Author(s):  
Anne Breüner ◽  
Lone Brøndsted ◽  
Karin Hammer
Keyword(s):  
Basic Protein ◽  
Genetic Material ◽  
Gene Encoding ◽  
Low Frequencies ◽  
Uracil Phosphoribosyltransferase ◽  
Phage Integrase ◽  
The Family ◽  
Phage Protein ◽  
Selection Of

ABSTRACT In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated intoattB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10−5 per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.

Phylogeny of the family Pasteurellaceae based on rpoB sequences

2004 ◽  
Vol 54 (4) ◽  
pp. 1393-1399 ◽  
Author(s):  
Bożena Korczak ◽  
Henrik Christensen ◽  
Stefan Emler ◽  
Joachim Frey ◽  
Peter Kuhnert
Keyword(s):  
16S Rdna ◽  
Dna Hybridization ◽  
Gene Encoding ◽  
Phylogenetic Studies ◽  
Study Selection ◽  
Gene Sequence Analysis ◽  
Rdna Sequencing ◽  
The Family ◽  
Type Strains ◽  
Selection Of

Sequences of the gene encoding the β-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA–DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

Efficient transformation of the entomopathogenic fungus Lecanicillium muscarium by electroporation of germinated conidia

Mycoscience ◽  
2019 ◽  
Vol 60 (3) ◽  
pp. 197-200 ◽  
Author(s):  
Sergey Timofeev ◽  
Alexander Tsarev ◽  
Igor Senderskiy ◽  
Eugene Rogozhin ◽  
Galina Mitina ◽  
...  
Keyword(s):  
Entomopathogenic Fungus ◽  
Efficient Transformation ◽  
Lecanicillium Muscarium

scholarly journals Twin-arginine translocation-arresting protein regions contact TatA and TatB

2014 ◽  
Vol 395 (7-8) ◽  
pp. 827-836 ◽  
Author(s):  
Johannes Taubert ◽  
Thomas Brüser
Keyword(s):  
Signal Peptide ◽  
Sequence Motif ◽  
Globular Domain ◽  
Mature Protein ◽  
Linker Region ◽  
Twin Arginine Translocation ◽  
C Terminus ◽  
Translocation Efficiency ◽  
Folded Proteins

Abstract Tat systems translocate folded proteins across biological membranes of prokaryotes and plant plastids. TatBC complexes recognize N-terminal Tat signal peptides that contain a sequence motif with two conserved arginines (RR-motif), and transport takes place after a recruitment of TatA. Unfolded Tat substrate domains lower translocation efficiency and too long linkers lead to translocation arrest. To identify the components that interact with transported proteins during their passage through the translocon, we used a Tat substrate that arrests translocation at a long unfolded linker region, and we chose in vivo site-directed photo cross-linking to specifically detect the interactions of this linker region. For comparison, we included the interactions of the signal peptide and of the folded domain at the C-terminus of this construct. The data show that the linker contacts only two, structurally similar Tat components, namely TatA and TatB. These contacts depend on the recognition of the Tat-specific signal peptide. Only when membrane translocation of the globular domain was allowed – i.e., in the absence of the linker – we observed the same TatAB-contacts also to the globular domain. The data thus suggest that mature protein domains are translocated through a TatAB environment.

scholarly journals Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves

1999 ◽  
Vol 338 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Kazuya YOSHIMURA ◽  
Yukinori YABUTA ◽  
Masahiro TAMOI ◽  
Takahiro ISHIKAWA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Ascorbate Peroxidase ◽  
Differential Splicing ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
C Terminus ◽  
Mrna Variants ◽  
Alternatively Spliced ◽  
Splicing Efficiency ◽  
Spinach Leaves

We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795–800]. To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves. As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified. The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein. Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12. The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX. The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo. The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3´-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes.

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