Application of 2'-OMethylRNA' Antisense Oligomer to Control Candida albicans EFG1 Virulence Determinant

Abstract

Antisense oligomers and their analogs have been successfully utilized to silence gene expression for the treatment of many human diseases; however, the control of yeast's virulence determinants has never been exploited before. In this sense, this work is based on the key hypothesis that if a pathogen's genetic sequence is a determinant of virulence, it will be possible to synthesize a nucleic acid mimic based on antisense therapy (AST) that will bind to the mRNA produced, blocking its translation into protein and, consequently, reducing the pathogen virulence phenotype. EFG1 is an important determinant of virulence that is involved in the regulation of the Candida albicans switch from yeast to filamentous form. Thus, our main goal was to design and synthesize an antisense oligonucleotide (ASO) targeting the EFG1 mRNA and to validate its in vitro applicability. The results show that the anti-EFG1 2'-OMethylRNA (2'OMe) oligomer was able to significantly reduce the levels of EFG1 gene expression and of Efg1p protein translation (both approximately 60%), as well as effectively prevent filamentation of C. albicans cells (by 80%). Moreover, it was verified that anti-EFG1 2'OMe keeps the efficacy in different simulated human body fluids. Undeniably, this work provides potentially valuable information for future research into the management of Candida infections, regarding the development of a credible and alternative method to control C. albicans infections, based on AST methodology.

Keywords: 2′-OMethylRNA modification; Candidiasis; filamentation; nucleic acid mimics.

Figure 1

Anti-EFG1 2′OMe Sensitivity and Specificity Obtained by FISH (A) List of strains and species used and their origin, as well as the respective results obtained by FISH at 37°C, during 3 h. (B) Illustrative images obtained by epifluorescence microscopy. The exposure time was the same for each strain: Candida albicans SC5314 was obtained with 218.7 ms; Candida albicans HLC52 (ΔΔefg1 mutant strain) was obtained with 713.2 ms, and Candida tropicalis ATCC750 was obtained with 293.9 ms of exposure. Negative controls were prepared only with 20 μL hybridization solution without probe.

Figure 2

Anti-EFG1 2′OMe Effect on Candida albicans Filamentation (A) Relative cell viability (%) determined by the absorbance values (Abs; 490 nm cm⁻²) of formazan product obtained from 3T3 cells treated with different concentrations of ASO (10, 20, 40, and 60 nM). The control is related to the cells without ASO treatment. (B) Percentage of inhibition (%) of filamentous forms, after treatment with different concentrations of ASO (10, 20, and 40 nM). (C) Levels of EFG1 gene expression obtained by the Pfaffl method, after application of 40 nM ASO, at different time points (4, 6, and 8 h) in RPMI. Error bars represent SD. *Significant differences among 10 nM and the other concentrations of ASO tested (p < 0.05). ⁺Significant difference between untreated and treated cells (p < 0.05).

Figure 3

Anti-EFG1 2′OMe Effect on Efg1p Translation (A) Percent inhibition (%) of filamentous forms at different time points (6, 8, 10, and 24 h). (B) Levels of EFG1 gene expression obtained by the Pfaffl method at 24 h. (C) Levels of Efg1p translation normalized with the translation of Act1p at 24 h. (D) Epifluorescence microscopy images of Candida cells stained with Calcofluor after treatment with 40 nM ASO (control was prepared only with cells in RPMI; without ASO). The assays were performed for C. albicans SC5314. Error bars represent SD. *Significant difference between 6 h and the other times tested (p < 0.05). ⁺Significant difference between untreated and treated cells (p < 0.05).

Figure 4

Anti-EFG1 2′OMe Effect on Simulated Human Body Fluids (AS, AU, and Horse Blood) (A) Percent inhibition (%) of filamentous forms at different time points (6, 8, 10, and 24 h for AS and AU; 48 h for horse blood). (B) Levels of EFG1 gene expression for C. albicans SC5314 obtained by the Pfaffl method, after treatment with 40 nM ASO in the presence of different simulated human body fluids (AS and AU at 24 h and horse blood at 48 h). Error bars represent SD. *Significant difference between 6 h and the other times tested (p < 0.05); ⁺Significant difference between untreated and treated cells (p < 0.05).