Testing a Human Antimicrobial RNase Chimera Against Bacterial Resistance

Abstract

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.

Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa

“…Of initial concern for this study was the potential cytotoxicity of RNases [ 61 ]. However, recent studies demonstrated that the modified RNase 3/1 protein showed low cytotoxicity for concentrations up to 200 μM [ 15 ]. Cytotoxicity assays were conducted for two model cell lines, i.e., THP-1 and Vero.…”

“…As a result, there is an urgent need to develop more effective treatments to avoid the intracellular growth of pathogenic microorganisms. Previously, our research group designed and explored the mechanism of action of a novel hybrid ribonuclease RNase 3/1, towards Gram-negative microorganisms [15]. Additionally, numerous studies have demonstrated the effective activity of the human RNase 3 to inhibit the intracellular growth of several microorganisms, including Mycobacterium [16] and even the proliferation of biofilm structures of P. aeruginosa [12].…”

“…The prepared immobilizates found application in the delivery of RNase A for cancer treatment [49] and RNase H for the detection of pathogenic bacterial DNA [50]. By taking advantage of the potent antimicrobial activity reported for hybrid RNase 3/1 [15], we designed the bionanoconjugate MNP-RNase 3/1.…”

“…Regarding antimicrobial activity, RNase 3 stands out for its high bactericidal action and the efficacy of an N-terminus-derived peptide on Gram-negative bacteria biofilms. This ability to remove biofilms of Gram-negative species can be explained by its specific cell agglutination and lipopolysaccharide-binding properties [ 12 , 13 , 14 , 15 ]. Besides, RNase 3 was proved effective against macrophage intracellular infection.…”

“…Interestingly, we have demonstrated the high performance of RNase 3, not only in bacterial infection removal but also in internalization within macrophage cells and autophagy activation [ 16 ]. In this context, we designed an RNase 3/1 hybrid that combines the unique features of RNase 3 and the high catalytic activity and ribonuclease inhibitor affinity of RNase 1 and tested the construct in an in vitro experimental evolution test with Acinetobacter baumannii , where it demonstrated its ability to delay the acquisition of colistin resistance [ 15 ]. Antimicrobial RNases have been reported to be effective against biofilm-forming pathogens, such as Mycobacterium tuberculosis , A. baumannii , or Pseudomonas aeruginosa [ 12 , 13 ].…”