Differences in Gamma Interferon Production Induced by Listeriolysin O and Ivanolysin O Result in Different Levels of Protective Immunity in Mice Infected with Listeria monocytogenes and Listeria ivanovii

Abstract

Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-␥) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD 50 ) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD 50 was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-␥ in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-␥-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-␥-inducing activity than LLO. By comparing the IFN-␥-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-␥-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-␥ production is responsible for the failure of L. ivanovii to generate effective protective immunity.Among the bacterial species belonging to the genus Listeria, two species, Listeria monocytogenes and Listeria ivanovii, are known to be pathogenic (42). L. monocytogenes causes serious infections in newborns, pregnant women, immunocompromised individuals (9,11,24), and animals (25). L. ivanovii is pathogenic to ruminants (25) but rarely causes human disease (6, 23).These two species possess a similar central virulence gene cluster consisting of prfA, plcA, hly, mpl, actA, and plcB, the transcription of which is positively regulated by PrfA (13, 21). Both listeriolysin O (LLO), an hly gene product in L. monocytogenes, and ivanolysin O (ILO), an ilo gene product in L. ivanovii, are 58-kDa secretory proteins that are the major virulence determinants in each (12,20,40). They show 80% homology in amino acid sequence (14, 16) and belong to a family of cholesterol-dependent cytolysins (CDCs) characterized by the presence of a highly conserved undecapeptide sequence (ECTGLAWEWWR) located near the C terminus (7, 42). The CDCs are known to bind cholesterol on the cell surface and form oligomers, resulting in a ring-shaped pore on the cytoplasmic membrane. The cytolytic activity of CDC is easily blocked by treatment with small amounts of free cholesterol (2). Rossjohn et al. analyzed the three-dimensional structure of perfringolysin O, a member of the CDC family produced by Clostridium perfringens, and reported that perfringolysin O consists of four domains (34). The continuous domains 1 to 3 are involved in oligomerization and insertion of the oligomer into th...

Dependency of Caspase-1 Activation Induced in Macrophages by Listeria monocytogenes on Cytolysin, Listeriolysin O, after Evasion from Phagosome into the Cytoplasm

“…It has been reported that LLO is recognized by TLR4 and activates a signaling pathway downstream of TLR4 (35). Moreover, recombinant LLO protein induced the production of various cytokines by splenocytes or PEC cultures in a TLR4-dependent manner, whereas recombinant ILO protein did not induce their production (17,35). However, our present results indicated that TLR4 was not involved in the production of IL-18 and the activation of caspase-1 in macrophages infected with LM (Fig.…”

“…If either of these possibilities is the case, ILO itself must have no or less ability to induce caspase-1 activation compared with LLO. In our previous study using recombinant LLO and ILO, it was shown that ILO is far less capable of inducing cytokines than LLO when added from outside of the cells in vitro (17). Therefore our assumption is that there is some molecular structure in LLO that is exclusively important for caspase-1 activation in the cytosol of macrophages.…”

“…For that, two mechanisms have been proposed: the dependency of cytolytic activity on acidic pH (40) and the presence of an N-terminal Pro-Glu-Ser-Thr (PEST)-like sequence, which is thought to target for phosphorylation and/or degradation in eukaryote cells (41). Although both LLO and ILO exhibited a similar optimal pH for cytolytic activity (42), the hemolytic activity of recombinant ILO was relatively higher than that of recombinant LLO (17). Besides, the sequence analysis of the ilo gene revealed the absence of the PEST-like sequence that is present in LLO (data not shown).…”

“…LI produces ivanolysin O (ILO) encoded by ilo, a cytolysin that shows ϳ80% homology with LLO in amino acid sequence (16). Although LI is capable of evasion into and multiplication inside the macrophage cytoplasm like LM, IFN-␥ responses after infection with LI in vitro and in vivo were very low as compared with those induced by LM (17). It is therefore unlikely that only the bacterial entry into the macrophage cytoplasm is sufficient for the induction of IFN-␥ production.…”

Listeriolysin O as a strong immunogenic molecule for the development of new anti-tumor vaccines

“…[45][46][47][48][49][50][51][52][53][54] To date, three immunodominant epitopes have been determined by different experiments. As shown in Figure 1B, these include one dominant cytotoxic T lymphocyte (CTL) epitope, LLO [91][92][93][94][95][96][97][98][99] (residues 91-99), and two typical CD4 + T cell epitopes, LLO 189-201 (residues 189-201), and LLO 215-226 (residues 215-226). 45,50,54 Although LLO is essential for phagosomal escape and cell-to-cell spread in most cell types, its membrane-perforating activity is potentially cytotoxic and must be tightly regulated to ensure that L. monocytogenes remains in its intracellular replicative niche.…”