Structure-function analyses involving palindromic analogs of tritrypticin suggest autonomy of anti-endotoxin and antibacterial activities

Abstract

Neutralization of invading pathogens by gene-encoded peptide antibiotics has been suggested to manifest in a variety of different modes. Some of these modes require internalization of the peptide through a pathway that involves LPS-mediated uptake of the peptide antibiotics. Many proline/tryptophan-rich cationic peptides for which this mode has been invoked do, indeed, show LPS (endotoxin) binding. If the mechanism of antibiotic action involves the LPS-mediated pathway, a positive correlation ought to manifest between the binding to LPS, its neutralization, and the bacterial killing. No such correlation was evident based on our studies involving minimal active analogs of tritrypticin. The anti-endotoxin activities of these analogs appear not to relate directly to their antibiotic potential. The two palindromic analogs of tritrypticin, NT7 (RRFPWWW) and CT7 (WWWPFRR), showed comparable antibacterial activities. However, while NT7 exhibited anti-endotoxin activity, CT7 did not. The LPS binding of two tritrypticin analogs correlated with their corresponding structures, but the antibacterial activities did not. Further structure-function analysis indicated specific structural implications of the antibacterial activity at the molecular level. Studies involving designed analogs of NT7 incorporating either rigid or flexible linkers between the specifically distanced hydrophobic and cationic clusters modulate the LPS binding. On the other hand, not knowing the target receptor for antibacterial activity is a drawback since the precise epitope for antibacterial activity is not definable. It is apparent that the anti-endotoxin and antibacterial activities represent two independent functions of tritrypticin, consistent with the emerging multifunctionality in the nature of cathelicidins.

Figure 1.

Comparison of dose-dependent antibacterial activity of NT7 and CT7 against (A) S. typhimurium, (B) E. coli, (C) A. tumefaciens, and (D) P. aeruginosa expressed in terms of inhibition zone area in the radial diffusion assay.

Figure 2.

Surface plasmon resonance analysis of different peptides for binding to LPS and kinetic analysis of the binding of NT7 as a function of temperature. The sensograms representing the binding at 25°C to the immobilized endotoxin by (A) tritrypticin, (B) NT7, and (C) CT7 are shown as a function of peptide concentration. (Inset) Corresponding linear fit plots of the k _on (s⁻¹) as a function of peptide concentration. Analysis of plots of variation of (D) k _ass and (E) k _diss as a function of temperature for NT7.

Figure 3.

Comparison of circular dichroism profiles of (A) NT7 and (B) CT7, in free and LPS-bound forms.

Figure 4.

Comparison of NT7 and CT7 for LPS binding by competitive displacement of dansyl polymyxin B in a dose-dependent manner.

Figure 5.

Functional comparison of endotoxin binding of NT7 and CT7. Dose-dependent inhibition of (A) B-cell proliferation and (B) NO release induced by endotoxin.

Figure 6.

Functional comparison of various analogs containing different linker residues. Antibacterial activity against S. typhimurium of NT7 analogs containing different lengths of linker of (A) flexible Gly residues and (B) constrained Pro residues, in between cationic and aromatic clusters. Dose-dependent inhibition of B-cell proliferation of NT7 analogs containing different lengths of linker of (C) flexible Gly residues and (D) constrained Pro residues in between cationic and aromatic clusters.