Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis.

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.

[1]  R. Cox,et al.  The X‐ray structure of a chitinase from the pathogenic fungus Coccidioides immitis , 2008, Protein science : a publication of the Protein Society.

[2]  S. Sakuda,et al.  Expression and Secretion of a Larval-specific Chitinase (Family 18 Glycosyl Hydrolase) by the Infective Stages of the Parasitic Nematode, Onchocerca volvulus * , 2001, The Journal of Biological Chemistry.

[3]  F. Cozzolino,et al.  A novel secretory pathway for interleukin‐1 beta, a protein lacking a signal sequence. , 1990, The EMBO journal.

[4]  A Bairoch,et al.  Updating the sequence-based classification of glycosyl hydrolases. , 1996, The Biochemical journal.

[5]  J. Rodríguez,et al.  Cell Wall Composition of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis , 1969, Journal of bacteriology.

[6]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[7]  M. Hennig,et al.  The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. , 1996, Journal of Molecular Biology.

[8]  Cathy H. Wu,et al.  InterPro, progress and status in 2005 , 2004, Nucleic Acids Res..

[9]  R. Hussey,et al.  Characterisation and developmental expression of a chitinase gene in Heterodera glycines. , 2002, International journal for parasitology.

[10]  M. D. Berliner,et al.  Vital staining of Histoplasma capsulatum with Janus Green B. , 1966, Sabouraudia.

[11]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[12]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[13]  T. Kirkland,et al.  Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis. , 1995, Gene.

[14]  J. Samuelson,et al.  Chitinase Secretion by Encysting Entamoeba invadensand Transfected Entamoeba histolytica Trophozoites: Localization of Secretory Vesicles, Endoplasmic Reticulum, and Golgi Apparatus , 1999, Infection and Immunity.

[15]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[16]  W. Nickel.  The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. , 2003, European journal of biochemistry.

[17]  S. Matsumoto,et al.  Extracellular Mycobacterial DNA-binding Protein 1 Participates in Mycobacterium-Lung Epithelial Cell Interaction through Hyaluronic Acid* , 2004, Journal of Biological Chemistry.

[18]  P. Jollès,et al.  What's new in chitinase research? , 1992, Experientia.

[19]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Negroni,et al.  [Peripleuritis due to Paracoccidioides brasiliensis in a patient with AIDS]. , 2003, Medicina.

[21]  F. Dromer,et al.  Antifungal drug resistance in pathogenic fungi. , 1998, Medical mycology.

[22]  G. Kunze,et al.  Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells , 1998, Planta.

[23]  J. McEwen,et al.  Experimental murine paracoccidiodomycosis induced by the inhalation of conidia. , 1987, Journal of medical and veterinary mycology : bi-monthly publication of the International Society for Human and Animal Mycology.

[24]  E. Anaissie.  Opportunistic mycoses in the immunocompromised host: experience at a cancer center and review. , 1992, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[25]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[26]  E. Kitamura,et al.  Molecular cloning, sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp. PE2, and its domain structure , 2003, Applied Microbiology and Biotechnology.

[27]  R. Lucius,et al.  Identification of Chitinase as the Immunodominant Filarial Antigen Recognized by Sera of Vaccinated Rodents (*) , 1996, The Journal of Biological Chemistry.

[28]  C. Ulhoa,et al.  Purification and some properties of the extracellular chitinase produced by Trichoderma harzianum , 1992 .

[29]  C. Ouzounis,et al.  Evolution of immunoglobulin-like modules in chitinases: their structural flexibility and functional implications. , 1997, Folding & design.

[30]  A Bairoch,et al.  New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. , 1993, The Biochemical journal.

[31]  G. Del Sal,et al.  The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. , 1989, BioTechniques.

[32]  C Combet,et al.  NPS@: network protein sequence analysis. , 2000, Trends in biochemical sciences.

[33]  C. Bulawa,et al.  Chitin synthase I and chitin synthase II are not required for chitin synthesis in vivo in Saccharomyces cerevisiae. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Mattias Collin,et al.  A Novel Secreted Endoglycosidase from Enterococcus faecalis with Activity on Human Immunoglobulin G and Ribonuclease B* , 2004, Journal of Biological Chemistry.

[35]  C. Locht,et al.  Mycobacterium smegmatis laminin‐binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin‐binding haemagglutinin , 2001, Molecular microbiology.

[36]  Cheng Jin,et al.  A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. , 2001, European journal of biochemistry.

[37]  R. Barton,et al.  Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product. , 2003, Microbiology.

[38]  R. Carter,et al.  Systematic Analysis of the Role of CD19 Cytoplasmic Tyrosines in Enhancement of Activation in Daudi Human B Cells: Clustering of Phospholipase C and Vav and of Grb2 and Sos with Different CD19 Tyrosines1 , 2000, The Journal of Immunology.

[39]  M. Bianchi,et al.  The nuclear protein HMGB1 is secreted by monocytes via a non‐classical, vesicle‐mediated secretory pathway , 2002, EMBO reports.

[40]  B. Henrissat,et al.  A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037 , 1996, Journal of bacteriology.

[41]  C. Locht,et al.  Enhanced bacterial virulence through exploitation of host glycosaminoglycans , 2002, Molecular microbiology.

[42]  Maristela Pereira,et al.  The glyceraldehyde-3-phosphate dehydrogenase homologue is differentially regulated in phases of Paracoccidioides brasiliensis: molecular and phylogenetic analysis. , 2004, Fungal genetics and biology : FG & B.

[43]  T. Fujii,et al.  Purification and characterization of chitinase and chitobiase produced by Aeromonas hydrophila subsp. anaerogenes A52. , 1986 .

[44]  N. Seidah,et al.  Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[45]  D. Barrett.  From natural products to clinically useful antifungals. , 2002, Biochimica et biophysica acta.

[46]  J. McCarthy,et al.  Human immune responses to infective stage larval-specific chitinase of filarial parasite, Onchocerca volvulus, Ov-CHI-1. , 2003, Filaria journal.

[47]  U. Reichard,et al.  Disruption of the Gene Which Encodes a Serodiagnostic Antigen and Chitinase of the Human Fungal PathogenCoccidioides immitis , 2000, Infection and Immunity.

[48]  K. Zen,et al.  Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta , 1992 .

[49]  A. Kurosky,et al.  Monoclonal Antibody against the Plasmodium falciparum Chitinase, PfCHT1, Recognizes a Malaria Transmission-Blocking Epitope in Plasmodium gallinaceum Ookinetes Unrelated to the Chitinase PgCHT1 , 2002, Infection and Immunity.

[50]  Frédéric Massicotte,et al.  Mammalian chitinase-like proteins. , 1999, EXS.

[51]  Leszek Rychlewski,et al.  ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins , 2003, Nucleic Acids Res..

[52]  Eugene W. Myers,et al.  Basic local alignment search tool. Journal of Molecular Biology , 1990 .

[53]  T. Watanabe,et al.  High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2). , 1999, Bioscience, biotechnology, and biochemistry.

[54]  T. Okamoto,et al.  Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system. , 1999, FEMS microbiology letters.

[55]  K S Wilson,et al.  Crystal structure of a bacterial chitinase at 2.3 A resolution. , 1994, Structure.

[56]  J. Hamdan,et al.  Disseminated paracoccidioidomycosis: correlation between clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole , 2003, Mycoses.

[57]  B. Mayer,et al.  SH3 domains: complexity in moderation. , 2001, Journal of cell science.

[58]  C. Beck-Sague,et al.  Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. , 1993, The Journal of infectious diseases.

[59]  P. Robbins,et al.  Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. , 1991, The Journal of biological chemistry.

[60]  Keqin Zhang,et al.  Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host. , 2004, Research in microbiology.

[61]  B. Henrissat,et al.  Structures and mechanisms of glycosyl hydrolases. , 1995, Structure.

[62]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[63]  Amos Bairoch,et al.  ScanProsite: a reference implementation of a PROSITE scanning tool. , 2002, Applied bioinformatics.

[64]  David J Adams,et al.  Fungal cell wall chitinases and glucanases. , 2004, Microbiology.

[65]  A Elofsson,et al.  Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. , 1997, Protein engineering.

[66]  Paul Horton,et al.  Better Prediction of Protein Cellular Localization Sites with the it k Nearest Neighbors Classifier , 1997, ISMB.

[67]  N. Gow,et al.  Regulation of chitin synthesis during dimorphic growth of Candida albicans. , 1998, Microbiology.

[68]  P. Tongaonkar,et al.  A semi‐empirical method for prediction of antigenic determinants on protein antigens , 1990, FEBS letters.

[69]  Nalvo F. Almeida,et al.  Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells* , 2005, Journal of Biological Chemistry.

[70]  M. Ashburner.  A Laboratory manual , 1989 .

[71]  Maristela Pereira,et al.  The cell wall of Paracoccidioides brasiliensis: insights from its transcriptome. , 2005, Genetics and molecular research : GMR.

[72]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[73]  A. Flores-Carreón,et al.  β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis , 2004, Antonie van Leeuwenhoek.

[74]  Maristela Pereira,et al.  Therapeutic targets in Paracoccidioides brasiliensis: post-transcriptome perspectives. , 2005, Genetics and molecular research : GMR.

[75]  Peer Bork,et al.  SMART 4.0: towards genomic data integration , 2004, Nucleic Acids Res..

[76]  D. Kontoyiannis,et al.  Antifungal drug resistance of pathogenic fungi , 2002, The Lancet.

[77]  T. Watanabe,et al.  Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. , 1993, The Journal of biological chemistry.

[78]  Bin Tian,et al.  A large-scale analysis of mRNA polyadenylation of human and mouse genes , 2005, Nucleic acids research.

[79]  A. Ohta,et al.  Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. , 1998, Bioscience, biotechnology, and biochemistry.

[80]  L. Goldani,et al.  Paracoccidioidomycosis and AIDS: an overview. , 1995, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.