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Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1α Secretion in Macrophages Infected with Listeria monocytogenes

Dewamitta, Sita R., Nomura, Takamasa, Kawamura, Ikuo, Hara, Hideki, Tsuchiya, Kohsuke, Kurenuma, Takeshi, Shen, Yanna, Daim, Sylvia, Yamamoto, Takeshi, Qu, Huixin, Sakai, Shunsuke, Xu, Yanting, Mitsuyama, Masao
Infection and immunity 2010 v.78 no.5 pp. 1884-1894
Listeria monocytogenes, bacteria, calcium, calcium signaling, calpain, chelating agents, cytokines, cytoplasm, genes, immune response, macrophages, messenger RNA, mutants, secretion
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-1α (IL-1α), that contribute to the host immune response. In this study, we have examined IL-1α production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Δhly). Expression of IL-1α mRNA and accumulation of pro-IL-1α in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1α from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Δhly mutant. A recovery of the ability to induce IL-1α 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-1α, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1α 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-1α 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-1α 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 Δhly strain, could induce calcium influx and IL-1α 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.