Exploring the mechanisms of action of human secretory RNase 3 and RNase 7 against Candida albicans

Abstract

Human antimicrobial RNases, which belong to the vertebrate RNase A superfamily and are secreted upon infection, display a wide spectrum of antipathogen activities. In this work, we examined the antifungal activity of the eosinophil RNase 3 and the skin‐derived RNase 7, two proteins expressed by innate cell types that are directly involved in the host defense against fungal infection. Candida albicans has been selected as a suitable working model for testing RNase activities toward a eukaryotic pathogen. We explored the distinct levels of action of both RNases on yeast by combining cell viability and membrane model assays together with protein labeling and confocal microscopy. Site‐directed mutagenesis was applied to ablate either the protein active site or the key anchoring region for cell binding. This is the first integrated study that highlights the RNases’ dual mechanism of action. Along with an overall membrane‐destabilization process, the RNases could internalize and target cellular RNA. The data support the contribution of the enzymatic activity for the antipathogen action of both antimicrobial proteins, which can be envisaged as suitable templates for the development of novel antifungal drugs. We suggest that both human RNases work as multitasking antimicrobial proteins that provide a first line immune barrier.

A Novel RNase 3/ECP Peptide for Pseudomonas aeruginosa Biofilm Eradication That Combines Antimicrobial, Lipopolysaccharide Binding, and Cell-Agglutinating Activities

“…The potential contribution of the protein catalytic activity was blocked by assaying the activity of the RNase 3 H15A variant, in which one of the two catalytic His residues is replaced. Previous work in our laboratory already confirmed that the H15A mutant was an appropriate construct to test the effect of total removal of the RNase enzymatic activity without alteration of the protein's three-dimensional structure (PDB ID 4OWZ) (24). The present results corroborate that the active site-defective mutant achieves an antibiofilm activity equivalent to that of the wild-type protein (Fig.…”

“…Human RNase 3, also known as the eosinophil cationic protein (ECP), is a small, highly cationic protein (pI ϳ11) that is stored in the secondary granules of eosinophils (19,20). RNase 3 is secreted during the infection process, where the protein exerts high antimicrobial activity against a wide range of microorganisms, such as bacteria, yeast, viruses, and parasites (18,(21)(22)(23)(24). Moreover, our laboratory has reported RNase 3's high affinity with negatively charged lipopolysaccharides (LPS) from Gram-negative bacteria outer membranes (25), a feature that mediates the protein's high bacterial agglutination ability (26).…”

Immune Modulation by Human Secreted RNases at the Extracellular Space

“…RNase7 is secreted by a variety of epithelial cells (Table 2 ) and mostly contributes to urinary tract sterility and epidermis protection ( 43 , 80 – 82 ). Together with high antimicrobial activity against a variety of infective microorganisms ( 82 , 87 , 89 , 193 ), some immuno-modulatory properties were reported. RNase7 expression is upregulated during kidney infection ( 84 ).…”

Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa

“…Although the catalytic activity of the protein was reduced, the antibacterial activity was maintained and increased After immobilization on MNP. Recent studies have demonstrated that indeed, the antimicrobial and catalytic activities appear unrelated [16,86]. Finally, it should be noted that the obtained nanobioconjugates were prepared at a single RNases:MNPs ratio, as detailed in the Materials and Methods section.…”