title: Engineering Candida albicans to secrete a host immunomodulatory factor.
abstract: Gene knockout and transgenic mice are important tools that are widely used to dissect the mammalian hosts' responses to microbial invasion. A novel alternative is to engineer the pathogen itself to secrete host factors that stimulate or suppress specific immune defense mechanisms. Herein, we have described and validated an approach to facilitate the production and export of ectopic host proteins, from the most prevalent human fungal pathogen, Candida albicans. Our strategy utilized a prepropeptide from the C. albicans secreted aspartic proteinase, Sap2p. The prepeptide facilitates entry of Sap2p into the secretory pathway, while the propeptide maintains the protease as an inactive precursor, until proteolytic cleavage in the Golgi apparatus releases the mature protein. The Sap2p prepropeptide coding sequence was linked to that of two mammalian calcium-binding proteins, S100A8 and S100A9, which are associated with symptomatic vaginal candidiasis. The resulting expression constructs were then introduced into C. albicans. While the S100A8 protein is secreted into the growth medium intact, the S100A9 protein is apparently degraded during transit. Nonetheless, culture supernatants from both S100A8 and S100A9 expressing C. albicans strains acted as potent chemoattractants for a macrophage-like cell line and polymorphonuclear leukocytes. Thus, the pathogen-derived mammalian proteins possessed the expected biological activity.
authors.affiliation: Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Johnston
authors.forename: Junko
authors.initials: J
authors.lastname: Yano
authors.forename: Paul L
authors.initials: PL
authors.lastname: Fidel
authors.forename: Karen E
authors.initials: KE
authors.lastname: Eberle
authors.forename: Glen E
authors.initials: GE
authors.lastname: Palmer
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D040501:Calgranulin A / Q000235:genetics / Q000378:metabolism; D040502:Calgranulin B / Q000235:genetics / Q000378:metabolism; D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D002633:Chemotaxis / Q000187:drug effects; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007155:Immunologic Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D040501:Calgranulin A; D040502:Calgranulin B; D005656:Fungal Proteins; D007155:Immunologic Factors; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; C090753:SAP2 protein, Candida
keywords: Candida albicans; S100A8; S100A9; ectopic protein; protein expression; secretion