title: Listeriolysin O-dependent bacterial entry into the cytoplasm is required for calpain activation and interleukin-1 alpha secretion in macrophages infected with Listeria monocytogenes.
abstract: Listeriolysin O (LLO), an hly-encoded cytolysin of Listeria monocytogenes, plays an essential role in the entry of L. monocytogenes into the host cell cytoplasm. L. monocytogenes-infected macrophages produce various proinflammatory cytokines, including interleukin-1 alpha (IL-1 alpha), that contribute to the host immune response. In this study, we have examined IL-1 alpha production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Delta hly). Expression of IL-1 alpha mRNA and accumulation of pro-IL-1 alpha in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1 alpha from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Delta hly mutant. A recovery of the ability to induce IL-1 alpha secretion was shown in a mutant strain complemented with the hly gene. The Toll-like receptor (TLR)/MyD88 signaling pathway was exclusively required for the expression of pro-IL-1 alpha, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1 alpha was abolished by addition of calcium chelators, and only LLO-producing L. monocytogenes strains were able to induce elevation of the intracellular calcium level in infected macrophages. A calcium-dependent protease, calpain, was implicated in the maturation and secretion of IL-1 alpha induced by LLO-producing L. monocytogenes strains based on the effect of calpain inhibitor. Functional activation of calpain was detected in macrophages infected with LLO-producing L. monocytogenes strains but not with a mutant strain lacking LLO. These results clearly indicated that LLO-mediated cytoplasmic entry of bacteria could induce the activation of intracellular calcium signaling, which is essential for maturation and secretion of IL-1 alpha in macrophages during L. monocytogenes infection through activation of a calcium-dependent calpain protease. In addition, recombinant LLO, when added to macrophages infected with the Delta hly strain, could induce calcium influx and IL-1 alpha secretion at doses exhibiting cytolytic activity, suggesting that LLO produced by intracellular L. monocytogenes may be implicated in induction of calcium influx through pore formation.
authors.affiliation: Department of Microbiology, Kyoto University Graduate School of Medicine, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Sita R
authors.initials: SR
authors.lastname: Dewamitta
authors.forename: Takamasa
authors.initials: T
authors.lastname: Nomura
authors.forename: Ikuo
authors.initials: I
authors.lastname: Kawamura
authors.forename: Hideki
authors.initials: H
authors.lastname: Hara
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Tsuchiya
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kurenuma
authors.forename: Yanna
authors.initials: Y
authors.lastname: Shen
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daim
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Huixin
authors.initials: H
authors.lastname: Qu
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Sakai
authors.forename: Yanting
authors.initials: Y
authors.lastname: Xu
authors.forename: Masao
authors.initials: M
authors.lastname: Mitsuyama
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002154:Calpain / Q000096:biosynthesis*; D003593:Cytoplasm / Q000382:microbiology*; D005260:Female; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000172:deficiency / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000172:deficiency / Q000378:metabolism*; D053582:Interleukin-1alpha / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000472:pathogenicity*; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D053582:Interleukin-1alpha; D037521:Virulence Factors; D002154:Calpain; C038705:hlyA protein, Listeria monocytogenes