Host regulation of secreted, bacterial virulence factors

描述(申请人提供)：这项研究的长期目标是了解细胞内病原体如何与其宿主相互作用，以建立病原体可以复制的受保护的生态位。对于单核细胞增生性李斯特氏菌来说，建立和维持其胞内生态位，宿主胞浆，需要对一个重要的毒力因子--李斯特菌溶素O(LLO)进行精确的空间调控。LIO是一种分泌型造孔蛋白，介导细菌从寄主液泡逃逸到寄主细胞质。尽管LLO是由细菌持续分泌的，但它只在宿主液泡中活跃。我们已经在LLO的氨基末端确定了一个顺式作用序列，这是将LLO的活性限制在液泡中所必需的。缺乏该序列的突变体无法正确划分LLO活性，渗透宿主质膜和液泡膜，从而破坏其细胞内生态位。重要的是，这些突变体在体内是无毒的。上述序列含有丰富的氨基酸，如：脯氨酸(P)、谷氨酸(E)、丝氨酸(S)和苏氨酸(T)，因此与真核害虫序列相似。PEST序列可以针对真核蛋白进行磷酸化和/或降解。我们已经证明，缺少PEST区域的突变体LLO在细胞内积累的水平高于野生型蛋白，这表明该区域可能影响LLO的细胞内稳定性。此外，我们还证明了LLO在寄主细胞质溶质中被磷酸化，并且位于害虫序列中的缺乏潜在的磷受体位置的突变体也渗透寄主质膜并降低了毒力。这一建议的具体目标是了解LLO的PEST序列调节宿主细胞质中蛋白质活性的机制。在目标1中，我们将识别LLO害虫序列中对其功能重要的特定残基，这些残基可能代表相互作用的宿主分子的接触部位。在目标2中，我们将定义LLO降解的途径，并确定有害生物序列是否影响这一途径。最后，在目标3中，我们将定义LLO在细胞内磷酸化的作用。控制毒力因子何时何地发挥作用，对于细胞内病原体协调生产性感染并导致疾病至关重要。通过定义LLO活性在宿主细胞内被划分的机制，我们将更多地了解细胞内病原体如何利用宿主细胞机制来调节在宿主细胞质中发挥作用的关键毒力因子的活性。

DESCRIPTION (provided by applicant): The long-term goal of this study is to understand how an intracellular pathogen interacts with its host to establish a protected niche in which the pathogen can replicate. For the bacterial pathogen Listeria monocytogenes establishing and maintaining its intracellular niche, the host cytosol, requires precise spatial regulation of an essential virulence factor, listeriolysin O (LLO). LLO is a secreted pore-forming protein that mediates bacterial escape from the host vacuole to the host cytosol. Despite being continuously secreted by the bacterium, LLO is active only in the host vacuole. We have identified a cis-acting sequence in the amino terminus of LLO that is necessary to restrict the activity of LLO to the vacuole. Mutants that lack this sequence fail to correctly compartmentalize LLO activity, permeabilize the host plasma membrane in addition to the vacuolar membrane, and consequently destroy their intracellular niche. Importantly, these mutants are avirulent in vivo. The above sequence is rich in the amino acids proline (P), glutamate (E), serine (S), and threonine (T) and thus resembles eukaryotic PEST sequences. PEST sequences can target eukaryotic proteins for phosphorylation and/or degradation. We have shown that a mutant LLO lacking the PEST region accumulates to higher intracellular levels than the wild type protein suggesting that this region may influence intracellular stability of LLO. In addition, we have shown that LLO is phosphorylated in the host cytosol and that mutants lacking potential phospho-acceptor sites located within the PEST sequence also permeabilize the host plasma membrane and have decreased virulence. The specific goal of this proposal is to understand the mechanism by which LLO's PEST sequence regulates the protein's activity in the host cytosol. In Aim 1, we will identify specific residues within LLO's PEST sequence that are important for its function and which may represent contact sites for interacting host molecules. In Aim 2, we will define the pathway of LLO degradation and determine whether the PEST sequence affects this pathway. Lastly, in Aim 3, we will define the role of intracellular phosphorylation of LLO. Controlling when and where a virulence factor acts is critical for an intracellular pathogen to orchestrate a productive infection and cause disease. By defining the mechanism by which LLO activity is compartmentalized within a host cell, we will learn more about how intracellular pathogens can take advantage of host cell machinery to regulate the activity of key virulence factors that function within the host cytosol.