Studies on Regulatory Mechanism of Salmonella Pathogenesis by Proteome Analysis

细胞内病原体，包括沙门氏菌，在宿主吞噬细胞内保持长期耐药性，引发各种遗传程序，帮助它们适应吞噬体内遇到的恶劣环境条件。为了阐明沙门氏菌感染后在宿主中表达致病机制的遗传程序，对沙门氏菌被巨噬细胞吞噬后新诱导的蛋白进行了蛋白质组学分析。由于这些程序中最突出的是热休克反应，因此研究了分子伴侣和作为热休克蛋白成员的AAA+蛋白酶作为毒力蛋白的作用。(1)血清型鼠伤寒菌ClpXP或Lon的破坏导致小鼠丧失毒力。这种衰减可能是由于巨噬细胞内生存和复制能力受损，这表明ClpXP和Lon对小鼠的系统性沙门氏菌感染至关重要。我们发现ClpXP突变体和Lon突变体在BALB/c小鼠中持续存在很长一段时间，而不会引起压倒性的全身感染，这表明可能是沙门氏菌活疫苗的候选物。因此，我们研究了用lon突变体或clpXP突变体口服免疫是否能保护小鼠免受随后毒力强的血清型鼠伤寒的口服攻击。结果提示，单次口服免疫lon和clpXP突变体可有效防止野生型血清鼠伤寒杆菌在肠道内的定植。(2)我们证明了鼠伤寒血清中atp依赖的Lon蛋白酶的缺失通过caspase -1和-3参与的机制诱导巨噬细胞快速和大量凋亡。这种过度诱导细胞凋亡被破坏的invF所消除，这是沙门氏菌致病性岛1（SPI1）基因表达所必需的。巨噬细胞吞噬后SPI1转录的中心调控因子hilA的表达被抑制，但当Lon突变体在巨噬细胞内生长时，该基因持续表达，因此SPI1蛋白积累。因此，Lon突变体诱导巨噬细胞凋亡的增加可能是由于SPI1基因在正常被抑制的情况下继续表达。一旦沙门氏菌建立了系统性感染，有机体所依赖的巨噬细胞的过度凋亡将对病原体有害。因此，可能需要Lon蛋白酶来充分抑制细胞凋亡，以便细菌有时间进行复制、逃逸和入侵新的巨噬细胞。(3)缺乏DnaK/ dnaj的突变体在小鼠中失去了引起致命全身性疾病的能力。巨噬细胞存活试验显示，突变体在巨噬细胞内完全不能存活或增殖。更令人感兴趣的是，该突变体既不能侵入培养的上皮细胞，也不能分泌沙门氏菌致病性岛1编码的任何入侵蛋白。少

Intracellular pathogens, including Salmonella, which maintain long-term resistance within host phagocytes elicit a variety of genetic programs to help them adapt to the hostile environmental conditions encountered within the phagosome. To elucidate the genetic programs for Salmonella pathogenesis expressed in host after infection, a proteome analysis of proteins newly induced in Salmonella after phagocytosis by macrophages was performed. Since prominent among these programs was the heat shock response, the, role of molecular chaperones and AAA+ proteases which are members of the heat shock proteins as virulence proteins have been studied.(1)The disruption of ClpXP or Lon of serovar Typhimurium results a loss of virulence to mice. The attenuation could be due to the impaired ability to survive and replicate within macrophage cells, suggesting that ClpXP and Lon are critically important for the systemic Salmonella infection of mice. We found that the ClpXP mutant and Lon mutant persist i … More n the BALB/c mice for long periods of time without causing an overwhelming systemic infection, suggesting a possible candidate of Salmonella live vaccine. We therefore examined whether oral immunization with the lon mutant or clpXP mutant protects mice against subsequent oral challenge with virulent serovar Typhimurium. The results suggested that a single oral immunization of the lon and clpXP mutants should be effective to protect against the colonization of wild-type serovar Typhimurium within the intestinal tract.(2)We demonstrated that depletion of ATP-dependent Lon protease in serovar Typhimurium induces rapid and massive apoptosis in macrophages by a mechanism involving both caspases-1 and -3. This excessive induction of apoptosis was abrogated by disruption of invF, which is required for the expression of the Salmonella pathogenicity island 1(SPI1) genes. Expression of hilA, a central regulator of SPI1 transcription, was repressed in the macrophages after phagocytosis, but this gene was continuously expressed when the Lon mutant grew within the macrophages, so the SPI1 proteins accumulated. Thus, the increase in macrophage apoptosis induced by the Lon mutant could be due to continued expression of SPI1 genes under conditions where they are normally repressed. Once Salmonella has established a systemic infection, excess apoptosis of macrophages cells upon which the organism is reliant would be detrimental to the pathogen. Therefore, the Lon protease may be required to suppress apoptosis sufficiently to allow time for the bacterium to replicate, escape, and invade new macrophages.(3)The DnaK/DnaJ-depleted mutant lost the ability to causes a lethal systemic disease in mice. Macrophage-survival assays revealed that the mutant could not survive or proliferate at all within macrophages. Of further interest are the findings that the mutant could neither invade cultured epithelial cells nor secrete any of the invasion proteins encoded by Salmonella pathogenicity island 1. Less