hLF1-11

Earlier we reported that the peptide corresponding to the first eleven N-terminal amino acids of human lactoferrin (hLF1-11) is active against multi-drug resistant pathogens in mice. The mechanisms underlying this anti-infective activity remain unclear. Since hLF1-11 is ineffective against pathogens at physiological salt concentrations and hLF1-11 directs differentiation of monocytes toward a macrophage subset with enhanced effector functions, we investigated the effects of hLF1-11 on human and murine monocytes. Results revealed that human and murine monocytes exposed for 1 h to hLF1-11 and then stimulated with the Toll-like receptor (TLR)-ligand LPS for 18 h, displayed enhanced cytokine and chemokine production as compared to control (peptide-treated) monocytes. We also found that expression of mRNA, cell-surface receptor expression, and NF-kappaB activation by hLF1-11-exposed human monocytes were enhanced as compared to control (peptide-treated) monocytes. Furthermore, the kinetics of the cytokine production was unchanged as mRNA levels and protein levels paralleled the enhanced response of hLF1-11-exposed monocytes to LPS. The cytokine production by human monocytes in response to TLR4, TLR5, and TLR7 stimulation, but not to TLR2 stimulation, was elevated by hLF1-11. In concordance, translocation of NF-kappaB subunits to the nucleus was enhanced in hLF1-11-exposed monocytes after TLR stimulation, except for TLR2, as compared to control (peptide-exposed) monocytes. In conclusion, monocytes were primed by hLF1-11 for an enhanced inflammatory response upon TLR4, TLR5, and TLR7 stimulation, but not TLR2 stimulation. Such effects of hLF1-11 on monocyte reactivity should be taken into account when considering the clinical development of this peptide for a therapeutic intervention in patients.

Candida species are the main fungal opportunistic pathogens causing systemic infections that are often associated with drug resistance and biofilm production on medical devices. The pressing need for new antifungal agents led to an increased interest in the use of combination therapies. The present study was aimed at investigating potential synergistic activity of the human lactoferrin-derived hLF1-11 peptide with caspofungin against caspofungin-resistant or -susceptible C. albicans, C. parapsilosis, and C. glabrata strains. Synergism was evaluated by the checkerboard assay, measuring cellular metabolic activity against Candida planktonic and sessile cells. A fractional inhibitory concentration (FIC) index of ≤0.5 was interpreted as synergy. Synergism was evaluated by killing assays on planktonic cells. A cell viability assay was performed with biofilm formation inhibition and preformed biofilm. Synergy for killing and viability assays was defined as a ≥2-log-CFU/mL reduction in comparison with the most active constituent. hLF1-11 and caspofungin exerted (i) synergistic effects against planktonic cells of all the tested strains, yielding drastic caspofungin MIC reduction, (ii) synergistic effects on the inhibition of biofilm formation against biofilm producer strains, yielding caspofungin BIC reduction, and (iii) synergistic effects on preformed biofilm assessed by measuring metabolic activity (FIC range, 0.28 to 0.37) against biofilm-producing strains and by cell viability assay in C. albicans SC5314. The synergistic effect observed between caspofungin and hLF1-11 against Candida spp. is of potential clinical relevance, representing a promising novel approach to target caspofungin-resistant Candida species infections. Further studies elucidating the mechanisms of action of such a synergistic effect are needed. IMPORTANCE The present study describes a synergistic effect between a conventional antifungal drug, caspofungin, and a synthetic peptide derived from human lactoferrin, hLF1-11, against Candida species. These yeasts are able to cause severe systemic fungal infections in immunocompromised hosts. In addition, they can form biofilms in medical implanted devices. Recently, caspofungin-resistant Candida strains have emerged, thus highlighting the need to develop different therapeutic strategies. In in vitro studies, this drug combination is able to restore sensitivity to caspofungin in caspofungin-resistant strains of Candida species, both in free-living cells and in cells organized in biofilms. This synergism could represent a promising novel approach to target infections caused by caspofungin-resistant Candida species.

hLF1-11 (GRRRRSVQWCA) is an antimicrobial peptide (AMP) with high activity against methicillin-resistant Staphylococcus aureus (MRSA), the most prevalent species in implant-associated infection. In this work, the effect of the surface immobilization on hLF1-11 antimicrobial activity was studied. Immobilization was performed onto chitosan thin films as a model for an implant coating due to its reported osteogenic and antibacterial properties. Chitosan thin films were produced by spin-coating on gold surfaces. hLF1-11 was immobilized onto these films by its C-terminal cysteine in an orientation that exposes the antimicrobial activity-related arginine-rich portion of the peptide. Two levels of exposure (with and without a polyethylene glycol (PEG) spacer) were analyzed. Covalent immobilization was further compared with the AMP physical adsorption onto chitosan films. Surfaces were characterized using ellipsometry, contact angle measurements, atomic force microscopy, infrared and X-ray photoelectron spectroscopies and using a fluorimetric assay for hLF1-11 quantification. Surface antimicrobial activity was assessed through surface adhesion and viability assays using an MRSA (S. aureus ATCC 33591). The incorporation of hLF1-11 increased significantly bacterial adhesion to chitosan films. However, the presence of hLF1-11, namely when immobilized through a PEG spacer, decreased the viability of adherent bacteria with regard to the control surface. These results demonstrated that hLF1-11 after covalent immobilization by its cysteine can maintain activity, particularly if a spacer is applied. However, further studies, exploring the opposite orientation or the same C-terminal orientation, but non-cysteine related, can help to clarify the potential of the hLF1-11 immobilization strategy.