Host Responses: Salmonella Enterica Serovar Typhimurium

宿主反应：肠沙门氏菌鼠伤寒血清型

• 批准号: 6987029
• 负责人: Olivia Steele-Mortimer
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6987029
• 关键词: Salmonella typhimurium; apoptosis; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; biological signal transduction; confocal scanning microscopy; epithelium; gastroenteritis; gene expression; genetic regulation; host organism interaction; inositol phosphates; intermolecular interaction; laboratory mouse; macrophage; nitric oxide synthase; phosphomonoesterases; protein localization; protein structure function; protein transport; serine threonine protein kinase; tissue /cell culture; transport proteins

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis. Virulence is determined by five Salmonella Pathogenicity Islands (SPI) encoded on the bacterial chromosome. SPI1 and SPI2 encode the structural components of two Type Three Secretion Sytems (TTSS) that translocate effectors into the host cell. The SPI1 effector SigD is an inositol phosphatase that induces the activation of Akt/PKB a mammalian serine threonine kinase. The role of Akt activation in pathogenesis remains unclear, however, in cultured epithelial cells it appears to be involved in preventing the onset of apoptosis in infected cells. More specifically we have found that a sigD deletion mutant induces Caspase 3 activation compared to wild type. Wild type Salmonella, but not the sigD mutant can block the induction of apoptosis by camptothecin. This may be important for the survival and replication of intracellular bacteria. In macrophages activation of Akt by Salmonella is more complex since lipopolysaccharide will activate the kinase in the absence of SigD. Comparison of the two pathways has revealed important differences. In particular, SigD-mediated activation is insensitive to Wortmannin a well described inhibitor of Akt activation via phosphatidylinositol-3-kinase. Thus SigD-dependent Akt activation appears to occur via a novel pathway. We have found that sigD also has effects up to 15 hours post invasion (p.i.). This is an important finding since it indicates that SPI1 effectors are not only involved in very early events. In this study we have investigated the role of SigD in iNOS induction in infected macrophages. We found that iNOS induction is affected by Sigd presumably in an Akt dependent process. Furthermore we found that SigD protein is detectable in infected cells for up to 8 hours p.i. Real time PCR confirmed that SigD continues to be expressed for at least 4 hours p.i. In order to further investigate the interaction between host cell and pathogen we are carrying out gene expression studies. Our initial experiments have concentrated on analyzing the expression of SPI1 and SPI2 genes by real time PCR. The results show that the expression is dependent on the mechansim of entry.

鼠伤寒沙门氏菌是胃肠炎的常见原因。毒力由细菌染色体上编码的五个沙门氏菌致病性岛（SPI）决定。SPI 1和SPI 2编码两个三型分泌系统（TTSS）的结构组分，所述三型分泌系统（TTSS）将效应物转运到宿主细胞中。SPI 1效应子SigD是一种肌醇磷酸酶，可诱导Akt/PKB（一种哺乳动物丝氨酸苏氨酸激酶）的激活。Akt激活在发病机制中的作用仍不清楚，然而，在培养的上皮细胞中，它似乎参与预防感染细胞中细胞凋亡的发生。更具体地说，我们已经发现，与野生型相比，sigD缺失突变体诱导半胱天冬酶3活化。野生型沙门氏菌而非sigD突变体可阻断喜树碱诱导的细胞凋亡。这对于细胞内细菌的存活和复制可能是重要的。在巨噬细胞中，沙门氏菌对Akt的激活更为复杂，因为脂多糖将在不存在SigD的情况下激活激酶。对这两种途径的比较揭示了重要的差异。特别地，SigD介导的活化对渥曼青霉素不敏感，渥曼青霉素是一种充分描述的经由磷脂酰肌醇-3-激酶的Akt活化的抑制剂。因此，SigD依赖性Akt激活似乎通过一种新的途径发生。 我们已经发现sigD在侵袭后（p.i.）也具有长达15小时的作用。这是一个重要的发现，因为它表明SPI 1效应子不仅参与非常早期的事件。在这项研究中，我们研究了SigD在感染的巨噬细胞中诱导iNOS的作用。我们发现Sigd可能在Akt依赖性过程中影响iNOS的诱导。此外，我们发现SigD蛋白在感染细胞中可检测到长达8小时的感染后。真实的时间PCR证实SigD继续表达至少4小时p.i. 为了进一步研究宿主细胞和病原体之间的相互作用，我们正在进行基因表达研究。我们的初步实验集中于通过真实的时间PCR分析SPI 1和SPI 2基因的表达。结果表明，该表达式依赖于进入机制。