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Jin An, Lebin Zhang, Lijuan Li, Dawen Liu, Huiling Cheng, Hengwei Wang, Muhammad Zohaib Nawaz, Hairong Cheng, Zixin Deng
An alternative strategy for synthesizing galactooligosaccharides (GOS) from an erythritol-producing yeast Yarrowia lipolytica using surface display technology was demonstrated. The engineered strain CGMCC11369 was developed by fusion of the β-galactosidase gene from Aspergillus oryzae to the YlPir1 gene, which codes for a cell wall protein. β-Galactosidase was effectively displayed on the cell surface of Yarrowia lipolytica start strain CGMCC7326. This engineered strain with surface-displayed β-galactosidase efficiently synthesized GOS from lactose...
May 18, 2016: Journal of Agricultural and Food Chemistry
Marcelo Victor Holanda Moura, Giulia Pontes da Silva, Antônio Carlos de Oliveira Machado, Fernando Araripe Gonçalves Torres, Denise Maria Guimarães Freire, Rodrigo Volcan Almeida
Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested...
2015: PloS One
Zahra Nasrollahi, Mohammad Hossein Yadegari, Shahla Roudbar Mohammadi, Maryam Roudbary, Maryam Hosseini Poor, Fatemeh Nikoomanesh, Masumeh Rajabi Bazl
BACKGROUND: Some genes may be associated with Candida albicans resistance to azoles. Pir1 gene is described as responsible to induce resistance in C. albicans. OBJECTIVES: The current study aimed to find the relationship between fluconazole resistance and Pir1 protein (Pir1p) overexpression in the females with recurrent C. albicans vaginitis requiring longer fluconazole therapy. PATIENTS AND METHODS: A total of 52l vaginal samples were obtained from the females with C...
September 2015: Jundishapur Journal of Microbiology
Rajeshwer Singh Sankhala, Ravi Kumar Lokareddy, Gino Cingolani
PIR1 is an atypical dual-specificity phosphatase (DSP) that dephosphorylates RNA with a higher specificity than phosphoproteins. Here we report the atomic structure of a catalytically inactive mutant (C152S) of the human PIR1 phosphatase core (PIR1-core, residues 29-205), refined at 1.20 Å resolution. PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5'-phosphatases such as the baculovirus RNA triphosphatase and the human mRNA capping enzyme. The PIR1 active site cleft is wider and deeper than that of VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the γ-phosphate of RNA, and ∼6 Å away, a chloride ion coordinates the general base R158...
February 11, 2014: Biochemistry
Iuliana V Ene, Clemens J Heilmann, Alice G Sorgo, Louise A Walker, Chris G de Koster, Carol A Munro, Frans M Klis, Alistair J P Brown
The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C...
November 2012: Proteomics
Paulo Canessa, Felipe Muñoz-Guzmán, Rafael Vicuña, Luis F Larrondo
Iron, although toxic in excess, is an essential element for biological systems. Therefore, its homeostasis is of critical importance and tight mechanisms participate in its acquisition by microbial organisms. Lately, the relevance of this metal for biomass conversion by wood-degrading fungi has been gaining increasing attention. Iron plays a critical role as cofactor of key enzymes such as lignin and manganese peroxidases in lignin-degrading white-rot fungi, while Fe(II) also serves a pivotal role in Fenton reactions that are central in cellulose depolymerization by brown-rotters...
August 2012: Fungal Genetics and Biology: FG & B
Wael Bahnan, Joseph Koussa, Samer Younes, Marybel Abi Rizk, Bassem Khalil, Sally El Sitt, Samer Hanna, Mirvat El-Sibai, Roy A Khalaf
Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content...
August 2012: Mycopathologia
Joern Linkner, Gregor Witte, Theresia Stradal, Ute Curth, Jan Faix
The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration...
2011: PloS One
Alice G Sorgo, Clemens J Heilmann, Henk L Dekker, Martijn Bekker, Stanley Brul, Chris G de Koster, Leo J de Koning, Frans M Klis
Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth...
August 2011: Eukaryotic Cell
Yogender Pal Khasa, Sara Conrad, Mustafa Sengul, Sarah Plautz, Michael M Meagher, Mehmet Inan
Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins...
March 2011: Yeast
Lydia Schild, Antje Heyken, Piet W J de Groot, Ekkehard Hiller, Marlen Mock, Chris de Koster, Uwe Horn, Steffen Rupp, Bernhard Hube
The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues...
January 2011: Eukaryotic Cell
Mark Held, Md Shakhawat Hossain, Keisuke Yokota, Paola Bonfante, Jens Stougaard, Krzysztof Szczyglowski
Great advances have been made in our understanding of the host plant's common symbiosis functions, which in legumes mediate intracellular accommodation of both nitrogen-fixing bacteria and arbuscular mycorrhiza (AM) fungi. However, it has become apparent that additional plant genes are required specifically for bacterial entry inside the host root. In this opinion article, we consider Lotus japonicus nap1 and pir1 symbiotic mutants within the context of other deleterious mutations that impair an intracellular accommodation of bacteria but have no impact on the colonization of roots by AM fungi...
October 2010: Trends in Plant Science
Alice G Sorgo, Clemens J Heilmann, Henk L Dekker, Stanley Brul, Chris G de Koster, Frans M Klis
The pathogenic fungus Candida albicans secretes a considerable number of hydrolases and other proteins. In-depth studies of the C. albicans secretome could thus provide new candidates for diagnostic markers and vaccine development. We compared various growth conditions differing in pH, temperature and the presence of the hyphal inducer N-acetylglucosamine. The polypeptide content of the growth media was ca. 0.1-0.2% of the total biomass. Using LC-tandem mass spectrometry, we identified 44 secretory proteins, the transmembrane protein Msb2, six secretory pathway-associated proteins and 28 predicted cytosolic proteins...
August 2010: Yeast
Keisuke Yokota, Eigo Fukai, Lene H Madsen, Anna Jurkiewicz, Paloma Rueda, Simona Radutoiu, Mark Held, Md Shakhawat Hossain, Krzysztof Szczyglowski, Giulia Morieri, Giles E D Oldroyd, J Allan Downie, Mette W Nielsen, Anna Maria Rusek, Shusei Sato, Satoshi Tabata, Euan K James, Hiroshi Oyaizu, Niels Sandal, Jens Stougaard
Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se...
January 2009: Plant Cell
Grazyna J Sosinska, Piet W J de Groot, M Joost Teixeira de Mattos, Henk L Dekker, Chris G de Koster, Klaas J Hellingwerf, Frans M Klis
Proteins that are covalently linked to the skeletal polysaccharides of the cell wall of Candida albicans play a major role in the colonization of the vaginal mucosal surface, which may result in vaginitis. Here we report on the variability of the cell-wall proteome of C. albicans as a function of the ambient O(2) concentration and iron availability. For these studies, cells were cultured at 37 degrees C in vagina-simulative medium and aerated with a gas mixture consisting of 6 % (v/v) CO(2), 0.01-7 % (v/v) O(2) and N(2), reflecting the gas composition in the vaginal environment...
February 2008: Microbiology
Qingjie Wang, Lei Li, Min Chen, Qingsheng Qi, Peng George Wang
A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study...
April 2008: Current Microbiology
Yoh-Ichi Shimma, Fumie Saito, Fumi Oosawa, Yoshifumi Jigami
Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library...
November 2006: Applied and Environmental Microbiology
Saija Kiljunen, Kristo Hakala, Elise Pinta, Suvi Huttunen, Patrycja Pluta, Aneta Gador, Harri Lönnberg, Mikael Skurnik
Bacteriophage piR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulence-plasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9...
December 2005: Microbiology
Toru Sumita, Takehiko Yoko-o, Yoh-ichi Shimma, Yoshifumi Jigami
We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1Delta cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars. Stepwise deletion of the eight units of the repetitive sequence of Pir1p revealed that one unit is enough for the protein to bind bud scars and that the extent of binding of Pir1p to the cell wall depends on the number of these repetitive units...
November 2005: Eukaryotic Cell
Ana I Martínez, Luis Castillo, Ana Garcerá, María V Elorza, Eulogio Valentín, Rafael Sentandreu
Searches in a Candida albicans database ( identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts...
October 2004: Microbiology
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