title: Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.
abstract: Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P. boydii provides important new information for understanding immune modulation and provides important new perspectives on the biology of this intriguing fungus.
authors.affiliation: Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazil.
authors.forename: Bianca Alcântara
authors.initials: BA
authors.lastname: da Silva
authors.forename: Cátia Lacerda
authors.initials: CL
authors.lastname: Sodré
authors.forename: Ana Luiza
authors.initials: AL
authors.lastname: Souza-Gonçalves
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Aor
authors.forename: Lucimar Ferreira
authors.initials: LF
authors.lastname: Kneipp
authors.forename: Beatriz Bastos
authors.initials: BB
authors.lastname: Fonseca
authors.forename: Sonia
authors.initials: S
authors.lastname: Rozental
authors.forename: Maria Teresa Villela
authors.initials: MT
authors.lastname: Romanos
authors.forename: Mauro
authors.initials: M
authors.lastname: Sola-Penna
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
authors.forename: Dário Eluan
authors.initials: DE
authors.lastname: Kalume
authors.forename: André Luis Souza
authors.initials: AL
authors.lastname: dos Santos
mesh_terms: D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000097:blood; D000946:Antigens, Fungal / Q000276:immunology; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D016681:Genome, Fungal; D006801:Humans; D020128:Inhibitory Concentration 50; D050296:Microbial Viability; D008969:Molecular Sequence Data; D025282:Mycelium / Q000254:growth & development / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D009181:Mycoses / Q000097:blood / Q000382:microbiology*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D020543:Proteome / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D040901:Proteomics; D011541:Pseudallescheria / Q000254:growth & development / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D005656:Fungal Proteins; D010446:Peptide Fragments; D020543:Proteome