Characterization and evaluation of the antifungal activity of antibodies raised against Candida Albicans germ tube in a rabbit model of infection and patients with invasive Candidiasis.
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In the last decades, the incidence of Invasive Candidiasis has dramatically increased. Since Candida is a common component of human microbiota, the distinction between invasion and colonization is complicated. Our group of research developed an immunofluorescence assay to detect antibodies reacting specifically against the surface of the germ tubes of Candida albicans in those patients with Invasive Candidiasis (IC). These antibodies appear when an invasive process occurs and have been referred to as CAGTA. The mortality rate of ICU patients was significantly lower in CAGTA positive patients. Based on this premise, we decided to characterize the antifungal activity of anti-Candida albicans germ tube antibodies (CAGTA). In the first part of this study, we worked with the immune sera obtained from two White New Zealand female rabbits infected intravenously with C. albicans blastospores. We confirmed that both sera antibodies recognize superficial antigens from blastospores and mycelia of C. albicans. In addition, specific IgG titers increased along the infection process, after several C. albicans inoculations. Secondly, we developed several ELISA assays in order to study the kinetics of the antibodies raised in the animal model. We used the following recombinant proteins of C. albicans: 14-3-3, Adh1, Als3-N, Eno-1, Hwp1-N, Met-6, and an eluted fraction of approximately 45 kDa of a C. albicans cell wall extract (CW-Eno). According to the recorded activity, anti Adh1 and anti-Met 6 antibodies could contribute to the immunofluorescence reaction against C. albicans registered for sera of the rabbit model of IC. Contrarily, the early response of anti-Eno 1, anti-CW-Eno and anti-Hwp1 N suggest that their corresponding proteins play an important role as immunogens during the first stages of Candida infection. In addition to the previously identified antigens recognized by CAGTA, we searched for new targets of these antibodies using a cDNA library prepared in the vector Lambda Zap II from mRNA of C. albicans SC5314 growing as mycelia. The screening of the expressed proteins with the CAGTA-enr serum fraction of the IC rabbit model showed 5 clones which correspond to five C. albicans SC5314 proteins: glucose-6-p-isomerase (PGI1), hyphal wall protein (Hwp1), fatty acid synthase (FasI), pH responsive protein 1 (Phr1), and actin (Act1). The CAGTA-enr fraction from patients with IC also recognized the selected clones and therefore, these antigens could be the basis for the future development of immunization protocols that might protect against Candida infections.In the next section of this work, we measured the effect of CAGTA raised in a rabbit model of IC, on C. albicans planktonic cells as well as on the process of biofilm formation and biofilm maturation. We observed that total-IgG and CAGTA-enr IgG fraction from rabbit immune serum reduced the growth of cells in a concentration dependent manner. In addition electron microscopy images of blastospores and hyphal forms of C. albicans treated with CAGTA-enr revealed an altered surface of hyphae. Besides, fluorescent DiBAC and CFDA staining of C. albicans cells treated with CAGTA revealed that these antibodies exerted a fungicidal effect. The same results were obtained using antibodies from patients with IC caused by Candida albicans. However, when patients were infected with C. parapsilosis, C. glabrata or C. tropicalis, their CAGTA-enr serum fractions reduced C. albicans metabolic activity to a different extent, with a mean value of 50-60% inhibition compared to the untreated control. In conclusion the CAGTA-enriched fraction appears as the main responsible for the in vitro antifungal effect on viability and metabolic activity of C. albicans observed for the IC immune sera.We also analyzed the phases of biofilm formation of C. albicans following different time-based protocols of treatment with CAGTA. The presence of CAGTA only during the first 90 minutes of incubation, corresponding to the C. albicans adhesion step, resulted in a significant reduction of the biofilm biomass down to 48% while the metabolic activity remained close to the control group values. In agreement with the observed effect of CAGTA on C. albicans biofilm formation, electron micrographs of 24-hour old biofilms treated with CAGTA exhibited a reduction of the microbial structure density; in addition, fungal filaments appeared rough and with protuberances, when compared to the untreated control. In the last section of this work, the following experiments performed during a stay at Dr Naglik¿s laboratory (King¿s College, London; September-December 2016) aimed to increase our knowledge of C. albicans pathogenicity and the ability of Candidalysin peptide to promote damage and generate an immunological response. First of all, we confirm that Kex1p activity appears as an important requirement for candidalysin maturation being Candidalysin terminating in K residue the mature peptide. In addition, we observed that all Candidalysin variants used in our assays presented a dosage effect on epithelial damage induction (LDH assay). In this regard, based on the results obtained, C terminal K residue seemed not be critical for interacting with the negative charge of epithelial cells surface. These results suggest that despite being different in their amino acid sequences, the function of Candidalysin peptides is conserved