Antimicrobial Properties of an Immunomodulator - 15 kDa Human Granulysin

<div><p>Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against <i>Pseudomonas aeruginosa</i> within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by <i>P</i>. <i>aeruginosa</i>. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.</p></div

Hydrophobic residues are critical for the helix-forming, hemolytic and bactericidal activities of amphipathic antimicrobial peptide TP4

<div><p>Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of <i>Pseudomonas aeruginosa</i> at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The <i>in vitro</i> binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membrane-like surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an α-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical α-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.</p></div

Effect of cations and pH on the antimicrobial activity, membrane permeability against <i>Pseudomonas aeruginosa</i> PAO1 and secondary structure of 15 kDa granulysin.

<p>The antimicrobial activities of 2 μM 15 kDa granulysin were determined in the presence of NaCl (A) and MgCl₂ (B) at the concentrations indicated. Microbes were incubated with granulysin at different pH values and the remaining colony-forming units were determined (C). SYTOX^™ Green was added to monitor the change in bacterial membrane permeability in the presence of 1 μM granulysin at pH 5.4 and pH 7.4, respectively (D). Fluorescence emission spectra of 8-Anilino-1-naphthalenesulfonic acid (ANS) was excited at 370 nm and measured between 400 and 600 nm in the presence of 1 μM granulysin at pH 7.4 (E) and pH 5.4 (F). Arrow indicates the blue shift (470 nm) of emission spectra of ANS-granulysin hydrophobic complex. Data plotted are normalized by the fluorescence intensity of ANS alone at pH 7.4 or pH 5.4, respectively. NFI, normalized fluorescence intensity. Circular dichroism spectrum of 20 μM 15 kDa granulysin in 20 mM HEPES, 50 mM NaCl, pH 7.4 or pH 5.4 (G). M.E., molar ellipticity.</p

Morphology of <i>Pseudomonas aeruginosa</i> PAO1 after 15 kDa granulysin treatment.

<p>Approximately 1×10⁷ microbes were treated with 2 μM 15 kDa granulysin for 0 min (A-C) and 180 min (D-F) and observed by transmission electron microscopy (TEM).</p

Structural statistics of TP4 in DPC micelles at pH 3.5 and 318 K.

<p>Structural statistics of TP4 in DPC micelles at pH 3.5 and 318 K.</p

MIC and MBC of TP4 mutants against microbes (μg/ml).

<p>MIC and MBC of TP4 mutants against microbes (μg/ml).</p

NMR spectra and NOE connectivity of TP4 bound to DPC micelles.

<p>(A) The fingerprint (the upper panel) and amide-amide (the lower panel) regions of NOESY spectrum recorded at 150 ms mixing time for TP4 in DPC micelles at pH 3.5 and 318 K. Sequential resonance assignments of TP4 are labeled at the positions of NH-C_αH and NH-NH cross-peaks. (B) Summary of NOE connectivity, amide proton exchange, and temperature coefficients for TP4. Opened and filled circles in the exchange row show that the exchangeable amide protons are still visible after 6 hr and 12 hr in D2O. The temperature coefficient values greater than -4.5 ppb/K are shown as filled diamonds. The thickness of the line is relative to the intensity of the NOE as indicated in the bottom.</p

Effect of 15 kDa granulysin on the biofilm formation of <i>Pseudomonas aeruginosa</i> PAO1.

<p>Microbes were grown in M63 minimal media in the presence of SMAP-29 and 15 kDa granulysin at 37°C for 24 hr and quantified by crystal violet staining and measured at the absorbance of 600 nm. Error bars represent the standard errors of the means. The asterisks indicate samples that are significantly different from control. (Student’s t-tests; p≤0.01).</p

OprI-binding ability of TP4-derived peptides.

<p><b>(A)</b> Competition for OprI binding by TP4-derived peptides. Recombinant OprI (12μg, 48μg each) was pre-incubated with free mutated peptide before binding to biotinylated-TP4 which was immobilized on Streptavidin-conjugated gel. The gel pellet was analyzed by non-reducing SDS-PAGE/Coomassie blue staining. rOprI, recombinant OprI; S.t, Streptavidin. (B) Solubility of TP4-derived peptide. TP4-derived peptides (6 μg each) dissolved in 1x sarkosyl buffer (300 μl) were incubated at 37°C for 10 min, then spun at 14,000 x <i>g</i> for 15 min and analyzed by SDS-PAGE/Coomassie blue staining. C, control without treatment; P, precipitation.</p

Hemolytic activity of TP4-derived peptides.

<p>Diluted mouse red blood cells (150 μl) was incubated with TP4-derived peptides at 37°C for 3 hr and spun at 120 x <i>g</i> for 5 minutes. The supernatants (100 μl) were transferred to a 96-well plate for measurement of released hemoglobulin at 414 nm by the SpectraMax 190 microplate reader.</p