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

Abstract

Tryptophan (Trp)-substituted antimicrobial peptides (AMPs) exhibit enhanced interactions with bacterial cell membranes, potentially improving their antimicrobial efficacy. Klebsiella 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) are 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 display superior antimicrobial activity and lower cytotoxicity. Mechanistic studies reveal that AMPs exert antibacterial and bactericidal effects through bacterial surface charge neutralization, insertion into bacterial cell membranes, increase permeability of both the inner and outer membranes, and disruption of membrane integrity. Notably, I1WL5W exhibit the most potent membrane-disrupting activity. Assessment of the impact of Trp-containing peptides on bacterial biofilms reveals that these peptides not only inhibit exopolysaccharide production and biofilm formation but also degrade preformed biofilms. A murine lung infection model is established to investigate the therapeutic efficacy of I1WL5W against MDRKP 1203-induced lung infection in mice. Compared with the control treatment, treatment with I1WL5W results 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 improves 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.

1. Introduction

The emergence of antimicrobial resistance, driven by widespread antimicrobial utilization since the late 20th century, has precipitated a global public health crisis through accelerated evolution of multidrug-resistant (MDR) bacterial pathogens [1]. Klebsiella pneumoniae, a ubiquitous gram-negative opportunistic pathogen, colonizes environmental reservoirs, cutaneous surfaces, mucosal interfaces, and gastrointestinal ecosystems. This organism represents a critical nosocomial pathogen associated with severe clinical manifestations, including urinary tract infections, bacteremia, intra-abdominal infections, and ventilator-associated pneumonia [2,3]. The increasing prevalence of multidrug-resistant strains, coupled with a limited arsenal of effective antibiotics, has contributed to increased mortality associated with gram-negative bacterial infections, particularly those caused by K. pneumoniae. There is an increasing trend in the clinical identification of carbapenem-resistant K. pneumoniae, prompting the creation of new treatment options and the investigation of strategies involving multiple drugs [4,5]. Antimicrobial peptides (AMPs), which are typically composed of 8–15 amino acid residues, represent an early component of innate immunity across diverse organisms and serve as regulators of the adaptive immune system in higher eukaryotes [6,7]. AMPs possess a range of biological functions, including antibacterial, antiviral, and cancer-fighting attributes, along with the capacity to prevent biofilm development and regulate immune reactions. Additionally, compared with traditional antibiotics, AMPs generally exhibit distinct advantages, including minimal molecular mass, excellent solubility, low cytotoxic effects, and heat resistance [8]. However, the clinical translation of AMPs is hindered by challenges related to stability, production cost, toxicity, bioavailability, distribution, and metabolic stability [9–11]. Recent research endeavors have focused on enhancing AMP efficacy through structural modifications and synergistic combinations with other compounds. Elucidating the specific mechanism of action for each peptide is crucial for optimizing its therapeutic potential [12]. Consequently, the development of novel AMPs with simplified sequences, emphasizing key core residues, has been pursued [13].

Gram-negative and gram-positive bacteria exhibit anionic surface constituents—lipopolysaccharides (LPS) and teichoic acids, respectively—combined with negatively charged phospholipid bilayers. The cationic nature of AMPs facilitates electrostatic interactions with these bacterial surfaces [14]. This interaction contributes to the multifunctional and broad-spectrum antimicrobial activity of AMPs, positioning them as promising candidates for novel antimicrobial drug development [15,16]. While classical models attribute AMP bactericidal effects to membrane permeabilization and cytosol leakage [17], contemporary studies have identified nonlytic mechanisms, including intracellular translocation without membrane compromise [18]. Tryptophan (Trp) residues, which are prevalent in natural AMPs, mediate membrane interfacial interactions via indole side chain hydrogen bonding (dipole moment ~21 D) [19]. Building on the temporin-1CEb scaffold [20–22], we engineered Trp-substituted analogs through systematic replacement of Ile/Leu residues at positions 1, 4, 5, 11, and 12, generating mono-substituted (I1W, I4W, L5W, L11W, and L12W) and di-substituted (I1WL5W and I4WL5W) variants. Structure-activity analysis revealed enhanced Gram-negative/-positive potency in I1W/I4W analogs versus attenuated activity in L11W/L12W derivatives. Di-substituted peptides exhibit expanded antimicrobial spectra, with positional Trp placement rather than stoichiometry emerging as the critical determinant of bactericidal efficacy [21,22].