Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of <i>Candida albicans</i> Revealing the Importance of Glycosylation for Antigen Recognition

During infection, the human pathogenic fungus <i>Candida albicans</i> undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host’s immune response. Little is known about the interplay of <i>C. albicans</i> secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC–MS/MS-based approach for the characterization of <i>C. albicans</i> extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive <i>C. albicans</i> secretome map. The serological responses to <i>C. albicans</i> extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of <i>C. albicans</i> was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient’s sera