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.

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.

Antimicrobial peptides and proteins (AMPs) constitute ancient host defence mechanisms to preserve tissue sterility and protect the host from infectious diseases. Currently, AMPs are awakening the interest of medical researchers due to their potential to become novel weapons to target multi‐drug resistant (MDR) pathogens and thereby overcome the limitations of traditional antibiotics. Among AMPs, human RNases belonging to the RNase A superfamily stand out as promising agents for therapeutic uses given their high antimicrobial activity, wide spectrum against multiple pathogens and low toxicity. However, a better understanding of how human RNases perform their antimicrobial actions in tissues is necessary to develop novel therapies. Mouse infectious disease models can be extremely useful to study the function of AMPs in vivo and have already provided valuable knowledge about RNase role in tissues such as the intestine and urinary tract. Therefore, it is necessary to understand the genetic and functional divergences that exist between human and mouse RNases to design experiments that are poised for clinical translation. The aim of this review is to present the similarities and differences between human and mouse RNases at genomic, structural and functional levels as a guide for future scientists exploring the roles of RNases in host defence.

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent an-timicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in hu-mans 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 ar-ginine (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 lipopolysac-charide binding and bacterial agglutination abilities, while retaining the same enzymatic effi-ciency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

Biofilms are microbial communities embedded in an extracellular matrix that facilitates their attachment to surfaces. This lifestyle provides advantages to pathogenic bacteria, including increased survival in the presence of antibiotics and an enhanced capacity to develop resistance. Once a biofilm is established, infections get difficult to eradicate and frequently become chronic. There is, therefore, an urgent need to develop novel strategies to counteract biofilm-associated antibiotic resistance. Here, we developed a method to monitor the evolution of antimicrobial resistance, aiming to evaluate novel drugs against bacterial resistance to antibiotics. We validated this methodology using an RNase chimera with antibiofilm activity and a reported ability to hinder colistin resistance in planktonic cultures of Acinetobacter baumannii (A. baumannii). We assessed the emergence of resistance in A. baumannii biofilms by repeated cycles of colistin exposure. This method not only preserves biofilm structure throughout treatment but also enables controlled induction of resistance acquisition while monitoring antimicrobial efficacy. Although the RNase enhanced the antibiotic’s activity against biofilms by reducing by 50% the effective dose, it did not prevent the emergence of colistin resistance, indicating that the protein may use distinct mechanisms against planktonic and biofilm communities. Nonetheless, our findings highlight the potential of this methodology for evaluating antibiotic-adjuvant candidates to combat antibiotic resistance in biofilms.

RNase 2 is the most abundant human RNase A member in macrophages, and its expression is activated upon virus exposure. Here, we explored the protein role by co-transcriptomics of wild-type (WT) and RNase 2-knock-out (KO) macrophages in absence/presence of a virus-derived single-stranded RNA (ssRNA40). Results revealed that RNase 2 is key for maintaining cell homeostasis. RNase 2 deletion induced the expression of stress-response markers and abolished the antiviral response. In contrast, the up-regulated genes in WT macrophages participate in pro-inflammatory response through TLR8-dependent pathways and antiviral immunity, with activation of MAPK and JAK-STAT pathways. We identified five top tRNA-derived small RNAs (tDRs) in response to ssRNA40 in presence of RNase 2, showing preferential cleavage at CA and uridine rich regions of anticodon loops. Another conserved target in all RNase2-generated 3’tDRs was found at the CCA end, releasing truncated 3’-CC. Among tDRs unique for WT exposed to ssRNA40, tRNA-LysTTT 3’-half stands out with the highest significant sequence coverage difference. Last, the immune response profile of RNase 2-KO cells is mostly recovered by addition of tRNA-LysTTT digestion products generated in vitro by recombinant RNase 2, suggesting an activation mechanism mediated by tDRs. Results highlight the essential role of RNase 2 selective tRNA cleavage in antiviral response and inflammation. Graphical Abstract