Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

The ability of human CD1b molecules to present nonpeptide antigens is suggested by the T cell recognition of microbial lipids and lipoglycans in the presence of CD1b-expressing antigen-presenting cells. We demonstrate the high-affinity interaction of CD1b molecules with the acyl side chains of known T cell antigens, lipoarabinomannan, phosphatidylinositol mannoside, and glucose monomycolate. Furthermore, CD1b-antigen binding was optimal at acidic pH, consistent with the known requirement for endosomal acidification in CD1b-restricted antigen presentation. The mechanism for CD1b-ligand interaction involves the partial unfolding of the alpha helices of CD1b at acidic pH, revealing a hydrophobic binding site that could accommodate lipid. These data provide direct evidence that the CD1b molecule has evolved unique biochemical properties that enable the binding of lipid-containing antigens from intracellular pathogens.

We have purified soluble mouse and human CD1d molecules to assess the structural requirements for lipid antigen presentation by CD1. Plate-bound CD1d molecules from either species can present the glycolipid α-galactosyl ceramide (α-GalCer) to mouse natural killer T cells, formally demonstrating both the in vitro formation of antigenic complexes, and the presentation of α-GalCer by these two CD1d molecules. Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized α-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding. The CD1d molecules can also bind both to the nonantigenic β-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d. These studies provide the first quantitative analysis of monomeric lipid antigen–CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.