A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli.

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

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.

Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa.

Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection.