Development and validation of monoclonal antibodies specific for Candida albicans Als2, Als9-1, and Als9-2

Fungal agglutinin-like sequence (Als) cell-surface glycoproteins, best characterized in Candida albicans, mediate adhesive and aggregative interactions with host cells, other microbes, and abiotic surfaces. Monoclonal antibodies (MAbs) specific for each C. albicans Als protein are valuable reagents for gaining insight into Als protein localization and function. This manuscript describes development and validation of MAbs specific for C. albicans Als2, as well as for C. albicans Als9-1 and Als9-2, two protein variants produced from the ALS9 locus. Native C. albicans ALS9 expression levels were not sufficiently high to produce detectable Als9 protein on the wild-type cell surface so MAb validation required production of overexpression strains, each featuring one of the two ALS9 alleles. An anti-Als2 MAb was raised against an N-glycosylated form of the protein immunogen, as well as an Endoglycosidase H-treated immunogen. The MAb raised against the N-glycosylated immunogen proved superior and immunolabeled C. albicans yeast cells and germ tubes, and the surface of Candida dubliniensis and Candida tropicalis yeasts. Als2 was visible on C. albicans yeast cells recovered from a murine model of oral candidiasis, demonstrating Als2 production both in vivo and in vitro. These new MAbs add to the collection of anti-Als MAbs that are powerful tools to better understand the role of Als proteins in C. albicans biology and pathogenesis.