Evaluation of Protein Channel-Attenuated Salmonella Vaccines

描述(由申请人提供)：该项目的长期目标包括通过利用沙门氏菌中的定居因子抗原I(CFA/I)获得的异源基因介导性灭活来开发活疫苗。我们怀疑这种衰减是由于通道CfaC的过度表达，而不是由于CfaBE菌毛，尽管CfaBE可能会增强通道介导的衰减。由于这种减毒策略以前没有被描述过，我们质疑它是否可以用于沙门氏菌疫苗的开发。由于传统的基于基因失活的活疫苗开发方法遇到了障碍，这项拟议的研究将产生沙门氏菌活疫苗用于人类。因此，我们设定了以下两个特定目标：特定目标1：检查菌毛亚单位的缺失、分泌装置或两者是否都与观察到的野生型沙门氏菌的减弱有关。具体目标2：确定哪些CFA/I操纵子缺失突变体可使野生型沙门氏菌减毒，从而可用作沙门氏菌病的保护性疫苗。为了达到这两个目的，我们将在沙门氏菌细胞外膜上安装CfaC通道。通过原子力显微镜(AFM)、各种条件下的存活能力、场发射扫描电子显微镜(FESEM)的形态、巨噬细胞的体外毒力和小鼠模型的体内毒力来评估通道型沙门氏菌的细胞伸展能力。由于OM细胞中的通道CFA/I允许741道尔顿红霉素进入，它也将允许其他较小的分子，如水、离子和有毒分子通过。因此，细胞OM渗透平衡将部分失去，这将导致细菌病原体的减弱。这项研究可能会开发新的人类使用的沙门氏菌活疫苗。公共卫生相关性：拟议的题为“蛋白质通道减毒沙门氏菌疫苗评价”的项目将构建一种旨在预防人类沙门氏菌病的活疫苗。肠沙门氏菌是一种引起各种食物和水传播疾病的疾病，如胃肠炎和伤寒。它可以感染广泛的动物宿主，也可以感染人类。在三种常见的伤寒中，仅伤寒沙门氏菌一项就估计造成约1600万个病例，全世界每年有60万人死亡，美国最近每年有600人死亡。让情况更糟的是，这种疾病对以前用于治疗它的抗生素产生了抗药性。最近，我们实验室发明了一种新的灭活沙门氏菌的策略。这一新策略与常规使用的毒力基因失活方法完全不同。如果证明对沙门氏菌有效，这一策略也将适用于其他肠道细菌。因此，该项目不仅将使美国受益，也将使其他沙门氏菌疫情国家受益。

DESCRIPTION (provided by applicant): The long-term objective of the project involves developing live vaccines through heterologous gene-mediated inactivation, as obtained using colonization factor antigen I (CFA/I) in Salmonella. We suspect this attenuation is because of the overexpression of channel CfaC and not due to the fimbriae CfaBE, although CfaBE may enhance the channel-mediated attenuation. Since this attenuation strategy was not previously described, we questioned whether it could be utilized for Salmonella vaccine development. Since conventional live vaccine development methods based on gene inactivation have been met with obstacles, this proposed study will generate Salmonella live vaccines for application in humans. As such, we set the following two Specific Aims: Specific Aim 1: Examine whether the deletion of fimbrial subunits, the secretion apparatus, or both are responsible for the observed attenuation of wild-type Salmonella. Specific Aim 2: Determine which of the deletion mutants of the cfa/I operon attenuates wild-type Salmonella and can therefore be used as a protective vaccine against salmonellosis. To achieve these two aims, we will install CfaC channel in the Salmonella cell outer membrane (OM). The channeled Salmonella will be evaluated for cell extension via atomic force microscope (AFM), viability in various conditions, morphology via Field Emission Scanning Electron Microscopy (FESEM), in vitro virulence via macrophages, and in vivo virulence by mouse model. Since the channel CFA/I in cell OM allows 741 dalton erythromycin to enter, it will also permit other smaller molecules, such as water, ions, and toxic molecules to pass. Thus, cell OM osmotic balance will be partially lost, which will result in the attenuation of bacterial pathogens. This study will possibly develop new live vaccines against Salmonella for human usage. PUBLIC HEALTH RELEVANCE: The proposed project entitled, "Evaluation of Protein Channel-Attenuated Salmonella Vaccines" will construct a live vaccine that aims at preventing human salmonellosis. Salmonella enterica is a disease that causes a variety of food and water-borne illnesses, such as gastroenteritis and typhoid fever. It can infect a wide range of animal hosts and also humans. Of the three common Typhoid fever disease, S. Typhi alone is estimated to cause approximately 16 million cases, with 600,000 deaths annually worldwide and 600 fatalities recently occurring each year in the United States. To make the situation even worse, this disease has acquired resistance to the antibiotics previously used to treat it. Recently, our laboratory invented a new strategy for inactivating the Salmonella. This new strategy is totally different from the conventionally used virulence gene inactivation method. If demonstrated effective in Salmonella, this strategy will be also applicable to other enterobacteria, as well. Therefore, this project will benefit not only the USA but also other countries epidemic with salmonellosis.