Novel biomarkers for invasive candidiasis. Selection, characterization and evaluation of Hyr1, a Candida albicans hyphally regulated cell wall protein

Abstract

Invasive candidiasis is a life-threatening disease caused by Candida species. The prognosis of this disease is closely related to early diagnosis; however, blood culture, the current gold standard for the diagnosis of invasive candidiasis, has a low sensitivity and takes days to provide results. Detection of antibodies against Candida albicans germ tubes (CAGTA) by indirect immunofluorescence has emerged as an alternative, but this technique has limitations that can be overcome by identifying the specific targets for these antibodies. With this aim, we analysed a cDNA expression library of C. albicans using immune serum from a rabbit model of infection, and 5 proteins were identified as potential targets: aminopeptidase 2 (Ape2), coatomer subunit gamma (Sec21), cystathionine gamma-lyase (Cys3), enolase 1 (Eno1) and hyphal-regulated cell wall protein (Hyr1). The in silico analysis of the 5 proteins led to the selection of Hyr1 for further study as a potential biomarker for the diagnosis of invasive candidiasis; similarly, a subterminal fragment of the protein, which we named D22b, was also selected. During the characterisation of this protein, we found that the encoding gene HYR1 presents high variability within the C. albicans species, and some of the identified variants showed a statistical association with the anatomical origin of the isolates. Regarding the usefulness of Hyr1 as a biomarker, the detection of antibodies against Hyr1 and the D22b fragment was able to differentiate patients with invasive candidiasis, not only those caused by C. albicans but also by other Candida species, with sensitivity values >70% and specificities >80%, and in some cases, anticipating the diagnosis with respect to blood culture. Finally, the development of monoclonal antibodies against a possible antigenic peptide of the protein resulted in two single-chain antibodies that recognised the recombinant protein and could help characterise, diagnose or combat Candida infections in the future.