Host regulation of secreted, bacterial virulence factors

描述（由申请人提供）：本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保护生态位。对于单核增生李斯特菌（Listeria monocytogenes）来说，建立和维持其细胞内生态位（宿主细胞质）需要对一种必需的毒力因子李斯特菌溶素O （listeriolyysin O， LLO）进行精确的空间调控。LLO是一种分泌的成孔蛋白，介导细菌从宿主液泡逃逸到宿主细胞质。尽管LLO由细菌不断分泌，但它只在宿主液泡中有活性。我们已经在LLO的氨基端确定了一个顺式作用序列，这是限制LLO对液泡活性所必需的。缺乏这一序列的突变体不能正确地划分LLO活性，除了空泡膜外，也不能渗透宿主的质膜，从而破坏它们的细胞内生态位。重要的是，这些突变体在体内是无毒的。上述序列富含脯氨酸(P)、谷氨酸(E)、丝氨酸(S)和苏氨酸(T)等氨基酸，与真核PEST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，缺乏PEST区域的突变型LLO在细胞内的积累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，我们已经证明LLO在宿主细胞质中被磷酸化，并且在PEST序列中缺乏潜在磷酸化受体位点的突变体也能渗透宿主质膜并降低毒力。本研究的具体目标是了解LLO的PEST序列在宿主细胞质中调节蛋白活性的机制。在目标1中，我们将在LLO的PEST序列中确定对其功能很重要的特定残基，这些残基可能代表相互作用的宿主分子的接触位点。在Aim 2中，我们将定义LLO降解的途径，并确定PEST序列是否影响该途径。最后，在Aim 3中，我们将定义LLO细胞内磷酸化的作用。控制毒力因子作用的时间和地点对于细胞内病原体协调生产感染并引起疾病至关重要。通过定义LLO活性在宿主细胞内划分的机制，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内起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.