Structure-Based Design of an RNase Chimera for Antimicrobial Therapy.

Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs' ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants' capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.

Testing a Human Antimicrobial RNase Chimera Against Bacterial Resistance.

The emergence of bacterial resistance to the most commonly used antibiotics encourages the design of novel antimicrobial drugs. Antimicrobial proteins and peptides (AMPs) are the key players in host innate immunity. They exert a rapid and multifaceted action that reduces the development of bacterial adaptation mechanisms. Human antimicrobial RNases belonging to the vertebrate specific RNase A superfamily participate in the maintenance of tissue and body fluid sterility. Among the eight human canonical RNases, RNase 3 stands out as the most cationic and effective bactericidal protein against Gram-negative species. Its enhanced ability to disrupt the bacterial cell wall has evolved in detriment of its catalytic activity. Based on structure-functional studies we have designed an RNase 3/1 hybrid construct that combines the high catalytic activity of RNase 1 with RNase 3 bactericidal properties. Next, we have explored the ability of this hybrid RNase to target the development of bacterial resistance on an Acinetobacter baumannii cell culture. Synergy assays were performed in combination with colistin, a standard antimicrobial peptide used as an antibiotic to treat severe infections. Positive synergism was observed between colistin and the RNase 3/1 hybrid protein. Subsequently, using an in vitro experimental evolution assay, by exposure of a bacterial culture to colistin at incremental doses, we demonstrated the ability of the RNase 3/1 construct to reduce the emergence of bacterial antimicrobial resistance. The results advance the potential applicability of RNase-based drugs as antibiotic adjuvants.