Host regulation of secreted, bacterial virulence factors

分泌的细菌毒力因子的宿主调节

• 批准号: 6803998
• 负责人: AMY L DECATUR
• 金额: $ 27.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6803998
• 关键词: Listeria; Listeria infections; SDS polyacrylamide gel electrophoresis; bacteria infection mechanism; bacterial disease; bacterial proteins; cell membrane; cytolysis; cytoplasm; cytotoxicity; glutamates; host organism interaction; laboratory mouse; mitogen activated protein kinase; phosphorylation; pore forming protein; proline; proteasome; serine; threonine; vesicle /vacuole; virulence

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.

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受保护生态位。对于细菌病原体单核细胞增生李斯特菌建立和维持其细胞内生态位，宿主胞质溶胶，需要精确的空间调控的一个重要的毒力因子，溶血素O（LLO）。LLO是介导细菌从宿主液泡逃逸到宿主胞质溶胶的分泌性成孔蛋白。尽管LLO由细菌持续分泌，但它仅在宿主液泡中有活性。我们已经确定了一个顺式作用序列的氨基末端的LLO是必要的限制活性的LLO的液泡。缺乏该序列的突变体不能正确地区室化LLO活性，除了液泡膜之外还透化宿主质膜，并因此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。上述序列富含氨基酸脯氨酸（P）、谷氨酸（E）、丝氨酸（S）和苏氨酸（T），因此类似于真核PEST序列。PEST序列可以靶向真核蛋白以进行磷酸化和/或降解。我们已经表明，缺乏PEST区域的突变体LLO积累到比野生型蛋白更高的细胞内水平，表明该区域可能影响LLO的细胞内稳定性。此外，我们已经表明，LLO在宿主胞质溶胶中被磷酸化，并且缺乏位于PEST序列内的潜在磷酸受体位点的突变体也使宿主质膜透化并具有降低的毒力。该提案的具体目标是了解LLO的PEST序列调节宿主细胞溶质中蛋白质活性的机制。在目标1中，我们将鉴定LLO的PEST序列内的特定残基，这些残基对其功能很重要，并且可能代表相互作用的宿主分子的接触位点。在目标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，在目标3中，我们将定义LLO的细胞内磷酸化的作用。控制毒力因子何时何地起作用对于细胞内病原体协调生产性感染和引起疾病至关重要。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宿主胞质内发挥作用的关键毒力因子的活性。