Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1-25 and 63-78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2-8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition.

Background

Healthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria.

Methods

Liposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low μg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed.

Results

Palmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 μg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 μg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 μg/mL) and a Pseudomonas aeruginosa strain (MIC 8 μg/mL) and lowered the vancomycin MIC for VRE from 32-64 μg/mL to as low as 4 μg/mL. At 90 μg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 μg/mL over 48 h.

Conclusions

Our results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.