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1.  Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology 
Bioorganic & medicinal chemistry  2010;19(1):490-497.
a-Factor from S. cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS-MS analysis and NMR; the structures of the analogues were confirmed by MS-MS analysis. Using a yeast growth arrest assay, the analogues were found to have activity comparable to a-factor itself.
doi:10.1016/j.bmc.2010.11.006
PMCID: PMC3037452  PMID: 21134758
a-Factor; Benzophenone; C-terminal methyl ester; Farnesyl; Peptide synthesis; Photoaffinity labeling; Prenylation
2.  WdChs2p, a Class I Chitin Synthase, Together with WdChs3p (Class III) Contributes to Virulence in Wangiella (Exophiala) dermatitidis 
Infection and Immunity  2001;69(12):7517-7526.
The chitin synthase structural gene WdCHS2 was isolated by screening a subgenomic DNA library of Wangiella dermatitidis by using a 0.6-kb PCR product of the gene as a probe. The nucleotide sequence revealed a 2,784-bp open reading frame, which encoded 928 amino acids, with a 59-bp intron near its 5′ end. Derived protein sequences showed highest amino acid identities with those derived from the CiCHS1 gene of Coccidioides immitis and the AnCHSC gene of Aspergillus nidulans. The derived sequence also indicated that WdChs2p is an orthologous enzyme of Chs1p of Saccharomyces cerevisiae, which defines the class I chitin synthases. Disruptions of WdCHS2 produced strains that showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms. However, assays showed that membranes of wdchs2Δ mutants were drastically reduced in chitin synthase activity. Other assays of membranes from a wdchs1Δwdchs3Δwdchs4Δ triple mutant showed that their residual chitin synthase activity was extremely sensitive to trypsin activation and was responsible for the majority of zymogenic activity. Although no loss of virulence was detected when wdchs2Δ strains were tested in a mouse model of acute infection, wdchs2Δwdchs3Δ disruptants were considerably less virulent in the same model, even though wdchs3Δ strains also had previously shown no loss of virulence. This virulence attenuation in the wdchs2Δwdchs3Δ mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice. The importance of WdChs2p and WdChs3p to the virulence of W. dermatitidis was then confirmed by reconstituting virulence in the double mutant by the reintroduction of either WdCHS2 or WdCHS3 into the wdchs2Δwdchs3Δ mutant background.
doi:10.1128/IAI.69.12.7517-7526.2001
PMCID: PMC98842  PMID: 11705928
3.  Molecular Cloning and Characterization of WdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella (Exophiala) dermatitidis 
Infection and Immunity  2001;69(3):1781-1794.
1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
doi:10.1128/IAI.69.3.1781-1794.2001
PMCID: PMC98085  PMID: 11179356
4.  WdChs4p, a Homolog of Chitin Synthase 3 in Saccharomyces cerevisiae, Alone Cannot Support Growth of Wangiella (Exophiala) dermatitidis at the Temperature of Infection 
Infection and Immunity  1999;67(12):6619-6630.
By using improved transformation methods for Wangiella dermatitidis, and a cloned fragment of its chitin synthase 4 structural gene (WdCHS4) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus. The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested. Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively. However, no significant loss of virulence was detected when the wdchs4Δ strain was tested in an acute mouse model. Using a wdchs1Δ wdchs2Δ wdchs3Δ triple mutant of W. dermatitidis, which grew poorly but adequately at 25°C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins. Maximal activity required trypsin activation, suggesting a zymogenic nature. The activity also had a pH optimum of 7.5, was most stimulated by Mg2+, and was more inhibited by polyoxin D than by nikkomycin Z. Although the WdChs4p activity had a broad temperature optimum between 30 to 45°C in vitro, this activity alone did not support the growth of the wdchs1Δ wdchs2Δ wdchs3Δ triple mutant at 37°C, a temperature commensurate with infection.
PMCID: PMC97075  PMID: 10569783

Results 1-4 (4)