A Novel RNase 3/ECP Peptide for Pseudomonas aeruginosa Biofilm Eradication That Combines Antimicrobial, Lipopolysaccharide Binding, and Cell-Agglutinating Activities

Abstract

bEradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms.

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana

“…Taken together, our results clearly suggest for the first time that the antimicrobial properties of leech recombinant HvRNASET2 protein are clearly comparable to those expressed by other class A RNases, such as RNase 3 and 7, and correlate to bacterial clumps forming activities (57). Indeed, similarly to RNase 7, HvRNASET2 triggered local blebs in the bacterial wall (58) and promoted a marked inflammatory response, mediated by TLR2 activation and followed by the release of inflammatory stimuli, such as TNF-α (26).…”

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus)

“…The MBC of each peptide was determined as described previously [69,70]. Bacteria were incubated at 37 °C overnight in Luria-Bertani broth (LB) and diluted to approximately 5 × 10 5 CFU/mL.…”

Mechanisms of Action for Antimicrobial Peptides With Antibacterial and Antibiofilm Functions

“…24 h. The remaining colony forming units (CFU) are counted to determine the MBC. In contrast to MIC which provides inhibitory activity information, MBC gives information on the bactericidal activity of the test antibacterial (Schwalbe et al, 2007;Balouiri et al, 2016) or antibiofilm (Pulido et al, 2016) agent at a specific time point.…”

“…The increase in fluorescence indicates the extent of membrane potential depolarization, as well as the integrity of the bacterial cells. The viability of cells upon antimicrobial action can be assessed at different time intervals by plate counting to determine the CFU (Zhang et al, 2000;Pulido et al, 2016).…”

“…Other dyes such as Sytox Green or PI can be used to establish whether HDPs damage cytoplasmic membranes and interact with intracellular nucleic acids (Sträuber and Müller, 2010;Davey and Hexley, 2011;Wenzel et al, 2015;Yang Y. et al, 2015;Pulido et al, 2016;Yasir et al, 2019). These dyes penetrate through membranes in which pores are present and can be used to probe differences in pore size and structure.…”