Turning a Collagenesis-Inducing Peptide Into a Potent Antibacterial and Antibiofilm Agent Against Multidrug-Resistant Gram-Negative Bacteria

Abstract

Antimicrobial resistance is becoming one the most serious health threats worldwide, as it not only hampers effective treatment of infectious diseases using current antibiotics, but also increases the risks of medical procedures like surgery, transplantation, bone and dental implantation, chemotherapy, or chronic wound management. To date, there are no effective measures to tackle life-threatening nosocomial infections caused by multidrug resistant bacterial species, of which Gram-negative species within the so-called “ESKAPE” pathogens are the most worrisome. Many such bacteria are frequently isolated from severely infected skin lesions such as diabetic foot ulcers (DFU). In this connection, we are pursuing new peptide constructs encompassing antimicrobial and collagenesis-inducing motifs, to tackle skin and soft tissue infections by exerting a dual effect: antimicrobial protection and faster healing of the wound. This produced peptide 3.1-PP4 showed MIC values as low as 1.0 and 2.1 μM against Escherichia coli and Pseudomonas aeruginosa , respectively, and low toxicity to HFF-1 human fibroblasts. Remarkably, the peptide was also potent against multidrug-resistant isolates of Klebsiella pneumoniae, E. coli, and P. aeruginosa (MIC values between 0.5 and 4.1 μM), and hampered the formation of/disaggregated K. pneumoniae biofilms of resistant clinical isolates. Moreover, this notable hybrid peptide retained the collagenesis-inducing behavior of the reference cosmeceutical peptide C 16 -PP4 (“Matrixyl”). In conclusion, 3.1-PP4 is a highly promising lead toward development of a topical treatment for severely infected skin injuries.

“Clicking” an Ionic Liquid to a Potent Antimicrobial Peptide: On the Route towards Improved Stability

“…To produce the target IL-peptide conjugate, hereafter termed MeIm-3.1-PP4, the synthetic route shown in Scheme 1 was employed, starting with the synthesis of the alkyne-modified IL, by reacting 1-methylimidazole (MeIm) with propargyl bromide (step i), according to Hu et al [ 15 ]; the structure of the target propargyl-MeIm (Pr-MeIm) was confirmed by proton ( 1 H) and carbon ( 13 C) nuclear magnetic resonance (NMR) and electrospray ionization-ion trap mass spectrometry (ESI-IT MS), as described in detail in the Supplementary Materials (Figures S1–S3) . In parallel, the amino acid sequence of 3.1-PP4 was assembled by solid-phase peptide synthesis (SPPS, steps ii and iii) as previously reported by us [ 12 ]. Cleavage of half of the peptidyl-resin (step iv) afforded the parent peptide 3.1-PP4.…”

“…50 in most cases. In face of the excellent antibacterial properties exhibited by the MeIm-3.1-PP4 conjugate, especially against Gram-negative bacteria, we next evaluated its antibiofilm activity using the K. pneumoniae MDR isolate, KP010, since the parent peptide, 3.1-PP4, had previously displayed a more notable effect against this isolate [ 12 ]. This activity was assessed in two different ways: (i) inhibition of biofilm formation, and (ii) effect on a preformed biofilm.…”

“…This activity was assessed in two different ways: (i) inhibition of biofilm formation, and (ii) effect on a preformed biofilm. For evaluation of the inhibition of biofilm formation, both conjugate and parent peptide were tested at MIC and sub-inhibitory concentrations (½×MIC and at ¼×MIC) following previously established procedures [ 12 , 18 , 19 , 20 ], and the mass of biofilm formed at these concentrations was assessed through the crystal violet assay. Figure 1 shows results obtained in absence (control) and in presence of the test compounds at the three different concentrations.…”

“…MIC values were also determined against MDR clinical isolates (antibiotic resistance patterns given in Supplementary Materials, Table S1 ) of P. aeruginosa (PA004, Pa3, Pa4), E. coli (Ec2, EC001, EC002, EC003), K. pneumoniae (KP004, KP010), S. aureus (SA007), and Enterococcus faecalis (Ef1). Along with the MIC values, MBC values were also determined as previously reported [ 12 ].…”

“…In view of the above, and based on our recent disclosure of a collagenesis-inducing peptide with potent antibacterial and antibiofilm properties [ 12 ], we now investigated the effect of coupling an antimicrobial methylimidazolium IL to the N -terminus of that peptide on antibacterial and antibiofilm activities, as well as on stability. To this end, we opted to use the well-known copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction, given its chemoselectivity and mildness, and the fact that it produces a stable triazole ring [ 13 ].…”