Abstract
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 Pseudomonas aeruginosa 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 P. aeruginosa. 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.
Highlights
Worldwide emergence of multiple-drug-resistant (MDR) bacteria has led to the urgent need for the development of new antibiotics [1]
The recombinant human 15 kDa granulysin fused with maltose-binding protein (MBP) was expressed in E. coli and purified to homogeneity by several column chromatographies (S1A– S1D Fig)
We found that the 15 kDa granulysin was only detected at the surface of bacteria, but not in the cytosol because a clearly green-fluorescence zone surrounding the DAPI-stained cytosol in blue was found even after 3 hr treatment (Fig 2)
Summary
Worldwide emergence of multiple-drug-resistant (MDR) bacteria has led to the urgent need for the development of new antibiotics [1]. Antimicrobial peptides and proteins (AMPs) are important components of the host innate defensive system that inhibits invading pathogens [2,3,4]. AMPs possess diverse secondary structures, their surfaces are amphipathic with cationic and hydrophobic residues on opposite sides within a hydrophobic environment. These AMPs have various modes of actions that differ from conventional antibiotics [4,5,6]. Disruption of membrane integrity and subsequent condensation of cytoplasmic components usually occur in the AMP-treated bacteria while inhibition of intracellular components can occur without membrane damage [4, 7]. Various targets of AMPs have been extensively proposed, such as the outer surface lipid, outer membrane protein, inner membrane, inner membrane protein, intracellular protein, and nucleic acids [7,8,9,10]
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