Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans

Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

[1]  K. To,et al.  Fatal Empyema Thoracis Caused by Schizophyllum commune with Cross-Reactive Cryptococcal Antigenemia , 2013, Journal of Clinical Microbiology.

[2]  S. Brunak,et al.  Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology , 2013, The EMBO journal.

[3]  Teresa R. O’Meara,et al.  The Cryptococcus neoformans Capsule: a Sword and a Shield , 2012, Clinical Microbiology Reviews.

[4]  K. Yuen,et al.  Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models , 2012, European Journal of Clinical Microbiology & Infectious Diseases.

[5]  P. Woo,et al.  Immunoassays Based on Penicillium marneffei Mp1p Derived from Pichia pastoris Expression System for Diagnosis of Penicilliosis , 2011, PloS one.

[6]  V. Chaturvedi,et al.  Cryptococcus gattii: a resurgent fungal pathogen. , 2011, Trends in microbiology.

[7]  S. Brunak,et al.  SignalP 4.0: discriminating signal peptides from transmembrane regions , 2011, Nature Methods.

[8]  T. Kozel,et al.  Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen , 2011, Clinical and Vaccine Immunology.

[9]  I. Xess,et al.  Detection of Cryptococcus by conventional, serological and molecular methods. , 2009, Journal of medical microbiology.

[10]  Hiten D. Madhani,et al.  Systematic Genetic Analysis of Virulence in the Human Fungal Pathogen Cryptococcus neoformans , 2008, Cell.

[11]  P. Woo,et al.  Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity , 2007, Clinical and Vaccine Immunology.

[12]  V. Cheng,et al.  False-negative cerebrospinal fluid cryptococcal antigen test due to small-colony variants of Cryptococcus neoformans meningitis in a patient with cystopleural shunt , 2006, Scandinavian journal of infectious diseases.

[13]  M. Hearn,et al.  Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production , 2005, Journal of molecular recognition : JMR.

[14]  D. Kontoyiannis.  What is the significance of an isolated positive cryptococcal antigen in the cerebrospinal fluid of cancer patients? , 2003, Mycoses.

[15]  F. Dromer,et al.  Antigenic and Biological Characteristics of Mutant Strains of Cryptococcus neoformans Lacking Capsular O Acetylation or Xylosyl Side Chains , 2003, Infection and Immunity.

[16]  K. Yuen,et al.  Cryptococcal infection associated with fludarabine therapy. , 2000, The American journal of medicine.

[17]  K. Nakai,et al.  PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. , 1999, Trends in biochemical sciences.

[18]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[19]  C. Lau,et al.  Cryptococcal meningitis presenting concurrently with systemic lupus erythematosus. , 1998, Clinical and experimental rheumatology.

[20]  K. Carroll,et al.  False-positive cryptococcal antigen latex agglutination caused by disinfectants and soaps , 1995, Journal of clinical microbiology.

[21]  R. Pinner,et al.  Multilocus enzyme typing of Cryptococcus neoformans , 1993, Journal of clinical microbiology.

[22]  Henry D. Isenberg,et al.  Manual of Clinical Microbiology , 1991 .

[23]  J. Jones,et al.  Detection of a Trichosporon beigelii antigen cross-reactive with Cryptococcus neoformans capsular polysaccharide in serum from a patient with disseminated Trichosporon infection , 1985, Journal of clinical microbiology.

[24]  A. Stamm,et al.  False-negative cryptococcal antigen test. , 1980, JAMA.

[25]  Ellen Jo Baron,et al.  Manual of clinical microbiology , 1975 .

[26]  G. Bulmer,et al.  Cryptococcus neoformans I. Nonencapsulated Mutants , 1967, Journal of bacteriology.