Comparative antimicrobial activity of granulysin against bacterial biothreat agents

Janice J Endsley, Alfredo G Torres, Christine M Gonzales, Valeri G Kosykh, Vladimir L Motin, Johnny W Peterson, D Mark Estes, Gary R Klimpel, Janice J Endsley, Alfredo G Torres, Christine M Gonzales, Valeri G Kosykh, Vladimir L Motin, Johnny W Peterson, D Mark Estes, Gary R Klimpel

Abstract

Granulysin is a cationic protein produced by human T cells and natural killer cells that can kill bacterial pathogens through disruption of microbial membrane integrity. Herein we demonstrate antimicrobial activity of the granulysin peptide derived from the active site against Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Burkholderia mallei, and show pathogen-specific differences in granulysin peptide effects. The susceptibility of Y. pestis to granulysin is temperature dependent, being less susceptible when grown at the flea arthropod vector temperature (26°C) than when grown at human body temperature. These studies suggest that augmentation of granulysin expression by cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protection against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibility to granulysin may identify important mechanisms that contribute to pathogenesis.

Keywords: B. anthracis; B. mallei; F. tularensis.; Granulysin; Y. pestis; antimicrobial.

Figures

Fig. (1)
Fig. (1)
Antimicrobial activity of granulysin peptide against select agent pathogens. Reduction of S. Typhimurium, B. mallei, F. tularensis (SHU S4), and B. anthracis (Ames) by a peptide derived from the active site of granulysin (1, 10, 100 µM) displayed as a percentage of control (no peptide) growth. Data shown are mean ± SEM of three to four independent experiments performed in triplicate. *p<0.05; **p<0.01, indicate statistically significant differences between peptide treatment and negative control.
Fig. (2)
Fig. (2)
Activity of granulysin peptide against Y. pestis is growth temperature-dependent. CFU reduction of Y. pestis (CO 92) across granulysin peptide concentration when grown at 26°C and 37°C. Reduction of CFU following 3 h incubation with peptide was determined by overnight growth on HIB agar plates. Percentage reduction from control growth at representative peptide concentrations is shown in parenthesis. Data shown are mean ± SEM of five independent experiments. Compared to negative control, growth was significantly (p<0.01) reduced by 5 to 100 µM concentration of granulysin peptide at both 26 and 37°C. Effects of peptide on growth reduction were significantly different (p<0.05) at 26°C and 37°C from 5 to 100 µM concentration.

References

    1. Houchins JP, Kricek F, Chujor CS, et al. Genomic structure of NKG5, a human NK and T cell-specific activation gene. Immunogenetics. 1993;37:102–7.
    1. Jongstra J, Schall TJ, Dyer BJ, et al. The isolation and sequence of a novel gene from a human functional T cell line. J Exp Med. 1987;165:601–14.
    1. Yabe T, McSherry C, Bach FH, Houchins JP. A cDNA clone expressed in natural killer and T cells that likely encodes a secreted protein. J Exp Med. 1990;172:1159–63.
    1. Stegelmann F, Bastian M, Swoboda K, et al. Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis. J Immunol. 2005;175:7474–83.
    1. Stenger S, Hanson DA, Teitelbaum R, et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science. 1998;282:121–5.
    1. Andreu D, Carreno C, Linde C, Boman HG, Andersson M. Identification of an anti-mycobacterial domain in NK-lysin and granulysin. Biochem J. 1999;344(Pt 3):845–9.
    1. Ernst WA, Thoma-Uszynski S, Teitelbaum R, et al. Granulysin, a T cell product, kills bacteria by altering membrane permeability. J Immunol. 2000;165:7102–8.
    1. Farouk SE, Mincheva-Nilsson L, Krensky AM, Dieli F, Troye-Blomberg M. Gamma delta T cells inhibit in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that requires granulysin. Eur J Immunol. 2004;34:2248–56.
    1. Ma LL, Spurrell JC, Wang JF, et al. CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15. J Immunol. 2002;169:5787–95.
    1. Wang Z, Choice E, Kaspar A, et al. Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J Immunol. 2000;165:1486–90.
    1. Chen X, Howe J, Andra J, et al. Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins. Biochim Biophys Acta. 2007;1768:2421–31.
    1. Endsley JJ, Furrer JL, Endsley MA, et al. Characterization of bovine homologues of granulysin and NK-lysin. J Immunol. 2004;173:2607–14.
    1. Bengoechea JA, Lindner B, Seydel U, Diaz R, Moriyon I. Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica. Microbiology. 1998;144:1509–15.
    1. Anisimov AP, Dentovskaya SV, Titareva GM, et al. Intraspecies and temperature-dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B. Infect Immun. 2005;73:7324–31.
    1. Bengoechea JA, Diaz R, Moriyon I. Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides. Infect Immun. 1996;64:4891–9.
    1. Galvan EM, Lasaro MA, Schifferli DM. Capsular antigen fraction 1 and Pla modulate the susceptibility of Yersinia pestis to pulmonary antimicrobial peptides such as cathelicidin. Infect Immun. 2008;76:1456–64.
    1. Andersson M, Gunne H, Agerberth B, et al. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity. EMBO J. 1995;14:1615–25.
    1. Davis EG, Sang Y, Rush B, Zhang G, Blecha F. Molecular cloning and characterization of equine NK-lysin. Vet Immunol Immunopathol. 2005;105:163–9.
    1. Hong YH, Lillehoj HS, Dalloul RA, et al. Molecular cloning and characterization of chicken NK-lysin. Vet Immunol Immunopathol. 2006;110:339–47.
    1. Wang Q, Wang Y, Xu P, Liu Z. NK-lysin of channel catfish: gene triplication, sequence variation, and expression analysis. Mol Immunol. 2006;43:1676–86.
    1. Praveen K, Evans DL, Jaso-Friedmann L. Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells. J Mol Evol. 2004;58:449–59.
    1. Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM. Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol. 2005;174:5243–8.
    1. Chung WH, Hung SI, Yang JY, et al. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med. 2008;14:1343–50.
    1. Gamen S, Hanson DA, Kaspar A, Naval J, Krensky AM, Anel A. Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways. J Immunol. 1998;161:1758–64.
    1. Okada S, Li Q, Whitin JC, Clayberger C, Krensky AM. Intracellular mediators of granulysin-induced cell death. J Immunol. 2003;171:2556–62.
    1. Savoia D, Scutera S, Raimondo S, Conti S, Magliani W, Polonelli L. Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes. Exp Parasitol. 2006;113:186–92.
    1. Anderson DH, Sawaya MR, Cascio D, et al. Granulysin crystal structure and a structure-derived lytic mechanism. J Mol Biol. 2003;325:355–65.
    1. Guo L, Lim KB, Poduje CM, et al. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell. 1998;95:189–98.
    1. Bengoechea JA, Brandenburg K, Arraiza MD, Seydel U, Skurnik M, Moriyón I. Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure. Infect Immun. 2003;71:2014–21.
    1. Rebeil R, Ernst RK, Jarrett CO, Adams KN, Miller SI, Hinnebusch BJ. Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation. J Bacteriol. 2006;188:1381–8.
    1. Llobet E, Tomas JM, Bengoechea JA. Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiology. 2008;154:3877–86.
    1. Lieberman J. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol. 2003;3:361–70.
    1. Walch M, Latinovic-Golic S, Velic A, et al. Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells. BMC Immunol. 2007;8:14–0.
    1. Miteva M, Andersson M, Karshikoff A, Otting G. Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin. FEBS Lett. 1999;462:155–8.
    1. Forestal CA, Malik M, Catlett SV, et al. Francisella tularensis has a significant extracellular phase in infected mice. J Infect Dis. 2007;196:134–7.

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