scholarly journals Site-directed mutagenesis of the 1,3-β glucan synthase catalytic subunit ofPneumocystis jiroveciiand susceptibility assays suggest its sensitivity to caspofungin

2018 ◽  
Author(s):  
A. Luraschi ◽  
S. Richard ◽  
P. M. Hauser
Keyword(s):  
Candida Parapsilosis ◽  
Pneumocystis Carinii ◽  
Culture Method ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Resistant Species ◽  
Rats And Mice

AbstractThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-β glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treatPneumocystisinfections. However, its efficacy againstPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting respectively rats and mice. In absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiae gsc1deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test®and Yeastone®for strains complemented withPneumocystisenzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme ofC. albicansthat is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, asP. cariniiandP. murina.

scholarly journals Site-Directed Mutagenesis of the 1,3-β-Glucan Synthase Catalytic Subunit ofPneumocystis jiroveciiand Susceptibility Assays Suggest Its Sensitivity to Caspofungin

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
A. Luraschi ◽  
S. Richard ◽  
P. M. Hauser
Keyword(s):  
Pneumocystis Carinii ◽  
Culture Method ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Resistant Species ◽  
Rats And Mice

ABSTRACTThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-β-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective againstPneumocystisasci. However, its efficacy against asci ofPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting, respectively, rats and mice. In the absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiaegsc1 deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented withPneumocystisenzymes (respectively, 0.125 and 0.12 μg/ml) were identical to those upon complementation with the enzyme ofC. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25 μg/ml). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, as areP. cariniiandP. murina.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

scholarly journals Prevalent Polymorphisms in Wild-Type HIV-1 Integrase Are Unlikely To Engender Drug Resistance to Dolutegravir (S/GSK1349572)

2013 ◽  
Vol 57 (3) ◽  
pp. 1379-1384 ◽  
Author(s):  
Cindy Vavro ◽  
Samiul Hasan ◽  
Heather Madsen ◽  
Joseph Horton ◽  
Felix DeAnda ◽  
...  
Keyword(s):  
Additional Data ◽  
Integrase Inhibitor ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Functional Roles ◽  
Hiv 1

ABSTRACTThe majority of HIV-1 integrase amino acid sites are highly conserved, suggesting that most are necessary to carry out the critical structural and functional roles of integrase. We analyzed the 34 most variable sites in integrase (>10% variability) and showed that prevalent polymorphic amino acids at these positions did not affect susceptibility to the integrase inhibitor dolutegravir (S/GSK1349572), as demonstrated bothin vitro(in site-directed mutagenesis studies) andin vivo(in a phase IIa study of dolutegravir monotherapy in HIV-infected individuals). Ongoing clinical trials will provide additional data on the virologic activity of dolutegravir across subject viruses with and without prevalent polymorphic substitutions.

scholarly journals Identification of an important cysteine residue in human glutamate–cysteine ligase catalytic subunit by site-directed mutagenesis

1998 ◽  
Vol 336 (3) ◽  
pp. 675-680 ◽  
Author(s):  
Zhongheng TU ◽  
M. W. ANDERS
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Cysteine Residue ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Regulatory Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Glutamate Cysteine Ligase

Glutamate–cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110±12 µmol/h per mg of protein compared with 370±20 µmol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76040-76047 ◽  
Author(s):  
Zhenya Chen ◽  
Ye Li ◽  
Yue Feng ◽  
Liang Chen ◽  
Qipeng Yuan
Keyword(s):  
Active Site ◽  
Simulation Analysis ◽  
Docking Simulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Molecular Docking Simulation ◽  
Activity Enhancement ◽  
First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

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