Role of Listeriolysin (LLO) in Listeria monocytogenes-infected host cells

L.单核细胞增生性单核细胞溶解素（LLO）是一种分泌型分子，其具有细胞外和细胞内的功能。LLO促进细菌的内化，从吞噬体空泡中逃离细菌，并在扩散到邻近细胞后从双膜中逃离。LLO是唯一已知的由细胞内细菌产生的胆固醇依赖性溶细胞素（CDC）的成员。实际上，LLO的活性不能被其他CDC毒素如产气荚膜梭菌溶素取代，并且很可能在细菌的细胞内生长期间调节毒素的活性以使得细菌生长而不明显破坏真核细胞。这种毒素的活性由特定的亚细胞特性介导：其活性在低pH值下高（例如在液泡中），在中性pH值下低（例如在细胞质中）。已经提出在分子的N-末端的PEST样序列通过靶向其降解来调节LLO的胞质内活性。我们在Schwerpunkt计划第一阶段的研究揭示了该序列的另一个性质，即它是LLO从吞噬溶酶体有效转运到细胞质所必需的。这通过（i）去除携带PEST样序列的区域（其导致细菌被捕获在吞噬溶酶体中）和（ii）将PEST样序列区域添加到相关CDC毒素肺炎球菌溶血素（pneumolysin）中（其导致其转运到细胞质的效率增加）来证明。进入细胞质对于细胞内生长是必不可少的，因为只有进入该隔室的细菌才能观察到对随后用致死剂量攻击的保护作用。用纯化的LLO进行的全基因组分析表明对真核细胞的胆固醇和脂质代谢有深远的影响。我们建议，LLO经历与宿主细胞蛋白质的相互作用，调节其细胞内的活动。在这项提案中，我们希望启动研究，目录细胞内宿主细胞蛋白和LLO的相互作用，以进一步了解参与这种调制的分子机制。

The major virulence factor of L. monocytogenes listerionlysin (LLO), is a secreted molecule which has functions both extra- and intracellularly. LLO promotes the internalisation of bacteria, escape from the bacteria from the phagososomal vacuole, and escape from the double membrane following spread to neighbouring cells. LLO is the only known member of the cholesterol-dependent cytolysins (CDC) that is produced by an intracellular bacterium. Indeed the activity of LLO cannot be substituted by other CDC toxins such as perfringolysin, and it is likely that the activity of the toxin is modulated during intracellular growth of the bacterium to enable bachterial growth without overt destruction of the eucaryotic cell. The activity of this toxin is mediated by specific subcellular properties: Its activity is high at low pH values (e.g. in the vacuole) and low at neutral pH values (e.g. in the cytoplasm). The PEST-like sequence at the N-terminal end of the molecule has been suggested to modulate the intracytoplsmic activity of LLO by targeting it for degradation. Our studies during the first phase of the Schwerpunkt program revealed yet another property of this sequence, i.e. it is required for efficient transit of LLO from the phagolysosome to the cytoplasm. This was demonstrated by (i) removal of the region harboring the PEST-like sequence which resulted in trapping of the bacterium in the phagolysosome and (ii) addition of the PEST-like sequence region to a related CDC-toxin, pneumolysin, that resulted in an increase in its efficiency to transit to the cytoplasm. Gaining access to the cytoplasm is essential for intracellular growth as protective effects to subsequent challenge with a lethal dose is only observed for bacteria with access to this compartment. Whole genome profiling with purified LLO demonstrated profound effects on cholesterol and lipid metabolism of the eucaryotic cell. We propose that LLO undergoes interactions with host cellular proteins that modulate its intracellular activity. In this proposal we wish to initiate studies that catalog interactions of intracellular host cell proteins and LLO to further understand the molecular mechanisms involved in this modulation.