Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. However, many AMPs fail to progress into clinics due to unexpected difficulties found in preclinical in vivo phases. Our research has focused on crotalicidin (Ctn), an AMP from snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work, we evaluate the in vivo efficacy of both the native-sequence AMPs and their retroenantio counterparts in a murine infection model with Acinetobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogs either exhibit toxicity or lack efficacy when administered to mice. Our findings underscore the critical importance of in vivo infection model evaluation to fully calibrate the therapeutic potential of AMPs.

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

Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. Our research has focused on crotalicidin (Ctn), an AMP from a snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work we evaluate the in vivo efficacy of both the na-tive-sequence AMPs and their retroenantio counterparts in a murine infection model with Aci-netobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogues either exhibit toxicity or lack efficacy when administered to mice.

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.