Tryptophan-substitution antimicrobial peptide temporin-1CEb: in vitro and in vivo antibacterial activity against clinically isolated multidrug-resistant Klebsiella pneumoniae

Tryptophan (Trp)-substitution antimicrobial peptides (AMPs) exhibit enhanced interactions with bacterial cell membranes, potentially improving their antimicrobial efficacy. K. pneumoniae (20.59% of 2054 ICU isolates) is resistant to multiple clinically used antibiotics and presents significant treatment challenges. In the present study, three Trp-modified peptides (I4W, L12W, and I1WL5W) were generated by substituting Ile or Leu residues in temporin-1CEb, a peptide derived from frog skin, with Trp at various sites to assess their antibacterial effects and mechanisms against K. pneumoniae . Compared with L12W, both I4W and I1WL5W displayed superior antimicrobial activity and lower cytotoxicity. Mechanistic studies revealed that the AMPs exerted antibacterial and bactericidal effects through bacterial surface charge neutralization, insertion into bacterial cell membranes, increased permeability of both the inner and outer membranes, and disruption of membrane integrity. Notably, I1WL5W exhibited the most potent membrane-disrupting activity. Assessment of the impact of Trp-containing peptides on bacterial biofilms revealed that these peptides not only inhibited exopolysaccharide production and biofilm formation but also degraded preformed biofilms. A murine lung infection model was established to investigate the therapeutic efficacy of I1WL5W against MDRKP 1203-induced lung infection in mice. Compared with the control treatment, treatment with I1WL5W resulted in reduced bacterial counts and levels of IL-6 and TNF-α in both the blood and lung tissues of MDRKP 1203-infected mice, and treatment with I1WL5W improved lung tissue structure. The present study provides valuable insights for designing Trp-containing peptides with potent antimicrobial properties by facilitating their penetration across bacterial membranes.