Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

Abstract

f Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activities in vitro and/or in vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activities in vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives with Candida albicans cells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in a Galleria mellonella model. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptides in vitro and their therapeutic effects in vivo.

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites

“…Specific mitochondrial labeling and induction of detectable changes in ΔΨm with the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) were confirmed by confocal laser scanning microscopy (CLSM). Then, KP-treated tachyzoites were evaluated for changes in their ΔΨm after TMRM loading, either in end-point or in time-lapse CLSM studies through a LSM 510 Meta scan head integrated with an Axiovert 200M inverted microscope (Carl Zeiss) using a 63 × /1.4 oil objective ( Ciociola et al, 2016 ). TMRM was excited with 543-nm He-Ne laser line.…”

Fungicidal activity of peptides encoded by immunoglobulin genes

“…In vivo toxicity and potential therapeutic efficacy of selected peptides were studied in the G. mellonella model, as previously described 30 . To evaluate peptide toxicity, groups of 16 larvae at their final instar stage (body weight, 400 ± 20 mg) were inoculated (10 μl/larva) directly in the haemocoel, via the last left proleg, with the selected peptides (12.5 mg/kg).…”

“…Peptide-induced apoptosis in C. albicans SC5314 cells was evaluated by the Muse cell analyzer (Merck Millipore, Germany) using the Muse annexin V and dead cell assay kit, as previously described 30 . Briefly, yeast cells were suspended in 100 μl water (5 × 10 5 cells/ml) in the absence (control) or presence of the selected peptides at a concentration equal to 2 × EC 50 and incubated for 30 and 60 min at room temperature.…”

“…For scanning electron microscopy studies, approximately 4 × 10 5 cells, prepared as above, were incubated with the selected peptides (125 μg/ml) for 60 min in a final volume of 20 μl. Slides for microscopic examination were prepared as previously described 30 . Briefly, slides were fixed with a glutaraldehyde-sodium cacodylate buffer, washed in sodium cacodylate, then dehydrated by immersion in alcohol solutions in a 25 to 100% gradient.…”

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases

“…After 120 h the colony forming units (CFUs) were counted. Percent growth inhibition was calculated in comparison to controls as previously described 54 . Three plates for each condition were assayed in two independent experiments.…”