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

单核增生乳杆菌的主要毒力因子李斯特菌溶素（LLO）是一种具有胞外和胞内双重作用的分泌分子。LLO促进细菌的内化，从吞噬体液泡中逃离细菌，并在扩散到邻近细胞后从双膜中逃逸。LLO是唯一已知的胆固醇依赖性细胞溶素（CDC）的成员，由细胞内细菌产生。事实上，LLO的活性不能被其他CDC毒素（如perfringolysin）所取代，并且很可能毒素的活性在细菌的细胞内生长过程中被调节，从而使细菌生长而不会明显破坏真核细胞。这种毒素的活性是由特定的亚细胞特性介导的：它的活性在低pH值（例如在液泡中）时高，在中性pH值（例如在细胞质中）时低。该分子n末端的类pest序列已被认为通过靶向LLO降解来调节其胞内活性。我们在Schwerpunkt项目第一阶段的研究揭示了该序列的另一个特性，即它是将LLO从吞噬溶酶体有效转运到细胞质所必需的。这可以通过(i)去除含有pest样序列的区域来证明，这导致细菌被困在吞噬溶酶体中；（ii）将pest样序列区域添加到相关的cdc毒素，溶肺素中，这导致其转运到细胞质的效率提高。进入细胞质对细胞内生长至关重要，因为只有进入细胞质隔室的细菌才能观察到对随后致命剂量攻击的保护作用。纯化的LLO对真核细胞的胆固醇和脂质代谢有深远的影响。我们认为LLO与宿主细胞蛋白相互作用，从而调节其细胞内活性。在本提案中，我们希望开展细胞内宿主细胞蛋白和LLO相互作用的目录研究，以进一步了解参与这种调节的分子机制。

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.