Title : Production and biological characterization of egg yolk antibodies (IgY) against Ftr1 iron permease of Candida albicans
Abstract:
Iron is essential for the functionality of biological processes, however, excess iron is toxic. In humans, due to its toxicity, this metal is found coupled to proteins, such as transferrin, which transports iron to tissues, and ferritin, an intracellular storage molecule of this metal. During an infection, microorganisms obtain iron from the host to survive. Candida albicans, one of the major pathogens responsible for severe fungal diseases, contains a transmembrane protein, iron Ftr1 high-affinity permease, which uses to get iron from ferritin, transferrin, and iron chelators synthesized by other organisms. In the search for new mechanisms to contain fungal multiplication, antibodies are applicable since it is possible to produce specific antibodies against a microbial structure. In this context, the work objective was to produce and perform the biological characterization of egg yolk antibodies (IgY) against the Ftr1 iron permease of C. albicans. A peptide derived from Ftr1 was synthesized and used to immunize laying hens seven times. Eggs from pre-immunized chickens and post-3rd, 5th, and 7th immunizations were collected. IgY antibodies were extracted from egg yolks by the ammonium sulfate precipitation technique. Subsequently, they were purified by molecular exclusion chromatography using Sephadex G-100 resin. The samples containing proteins or peptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10%. After this step, the samples were concentrated by filtration using a 50 kDa cut-off ultra centrifugal filter. Then, an avidity enzyme-linked immunosorbent assay (ELISA) was performed to establish the binding strength of the antigen with the antibodies, using urea as a chaotropic agent. To determine the antifungal effect of immunoglobulins extracted after the 7th immunization, in vivo test was performed on larvae of the moth Galleria mellonella, an alternative model of systemic infection. Regarding the results, the production, extraction, and purification occurred successfully, being possible to observe the presence of bands corresponding to the heavy (65 kDa) and light (25 kDa) IgY chains in the electrophoresis gel. Besides, after purification, contaminants (38 kDa) were removed. The immunoglobulin was reactive to the antigen and the avidity was considered low for pre-immunization antibodies, moderate for antibodies extracted post-3rd and 5th immunizations, and high after the 7th immunization. Concerning the challenge in the larvae, at 96 h after the beginning of the experiment, the survival of larvae treated with 80 mg/kg of anti-Fr1 IgY was 90%. In contrast, all larvae that did not receive treatment died (p < 0.0001). Meanwhile, only 16% of the larvae that received 80 mg/kg of pre-immunized IgY survived, and there was no difference between this group and the untreated group (p = 0.2359). This work showed that an antibody produced against C. albicans Ftr1 was able to increase the survival of G. mellonella larvae infected with the yeast.Iron is essential for the functionality of biological processes, however, excess iron is toxic. In humans, due to its toxicity, this metal is found coupled to proteins, such as transferrin, which transports iron to tissues, and ferritin, an intracellular storage molecule of this metal. During an infection, microorganisms obtain iron from the host to survive. Candida albicans, one of the major pathogens responsible for severe fungal diseases, contains a transmembrane protein, iron Ftr1 high-affinity permease, which uses to get iron from ferritin, transferrin, and iron chelators synthesized by other organisms. In the search for new mechanisms to contain fungal multiplication, antibodies are applicable since it is possible to produce specific antibodies against a microbial structure. In this context, the work objective was to produce and perform the biological characterization of egg yolk antibodies (IgY) against the Ftr1 iron permease of C. albicans. A peptide derived from Ftr1 was synthesized and used to immunize laying hens seven times. Eggs from pre-immunized chickens and post-3rd, 5th, and 7th immunizations were collected. IgY antibodies were extracted from egg yolks by the ammonium sulfate precipitation technique. Subsequently, they were purified by molecular exclusion chromatography using Sephadex G-100 resin. The samples containing proteins or peptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10%. After this step, the samples were concentrated by filtration using a 50 kDa cut-off ultra centrifugal filter. Then, an avidity enzyme-linked immunosorbent assay (ELISA) was performed to establish the binding strength of the antigen with the antibodies, using urea as a chaotropic agent. To determine the antifungal effect of immunoglobulins extracted after the 7th immunization, in vivo test was performed on larvae of the moth Galleria mellonella, an alternative model of systemic infection. Regarding the results, the production, extraction, and purification occurred successfully, being possible to observe the presence of bands corresponding to the heavy (65 kDa) and light (25 kDa) IgY chains in the electrophoresis gel. Besides, after purification, contaminants (38 kDa) were removed. The immunoglobulin was reactive to the antigen and the avidity was considered low for pre-immunization antibodies, moderate for antibodies extracted post-3rd and 5th immunizations, and high after the 7th immunization. Concerning the challenge in the larvae, at 96 h after the beginning of the experiment, the survival of larvae treated with 80 mg/kg of anti-Fr1 IgY was 90%. In contrast, all larvae that did not receive treatment died (p < 0.0001). Meanwhile, only 16% of the larvae that received 80 mg/kg of pre-immunized IgY survived, and there was no difference between this group and the untreated group (p = 0.2359). This work showed that an antibody produced against C. albicans Ftr1 was able to increase the survival of G. mellonella larvae infected with the yeast.
