Mechanisms of the Development and Maintenance of Salmonella Gallbladder Carriage

沙门氏菌属肠杆菌科，可引起人类和动物的一系列疾病，包括伤寒和肠胃炎。伤寒主要由肠沙门氏菌血清型伤寒（S. Typhi）引起，是一种危及生命的全身性疾病，每年在世界范围内造成大量发病率和死亡率。大约5%的个体感染斑疹伤寒沙门氏菌成为慢性携带者，并以胆囊（GB）为持久性部位。伤寒沙门氏菌是一种限制人类传播的病原体，因此无症状携带者是疾病进一步传播的关键宿主。我们已经证明，胆结石（GSs）在小鼠模型（利用鼠伤寒沙门氏菌，在小鼠中引起类似伤寒的疾病）和人类中有助于GB载体的发展和维持，作为沙门氏菌附着的底物并形成保护性生物膜。因此，生物膜的形成是载体建立的关键步骤。传统的抗生素治疗通常无法清除慢性伤寒沙门氏菌感染，因为生物膜表型使细菌对这些药物的机制具有耐受性。因此，鉴定能够靶向伤寒沙门氏菌生物膜的新疗法对于消除慢性携带和根除该疾病是必要的。为此，我们的团队已经确定了四种小分子和两种能够在体外抑制和/或破坏沙门氏菌生物膜的抗体。我们在本提案中提出了两个小分子JG-1和M4，它们在体外抑制和破坏生物膜，并在体内减少GB细菌数量。我们假设，在我们的小鼠慢性携带模型中，与单独使用抗生素治疗相比，这些抗生物膜化合物与抗生素联合使用将更有效地抑制和破坏沙门氏菌生物膜。在Aim 1中，我们将利用我们建立的伤寒慢性携带小鼠模型，与传统抗生素相比，评估这些抗生物膜化合物在预防和治疗慢性感染方面的功效。我们还将测量这些化合物的重要药代动力学和耐受性参数。为了阐明这些化合物拮抗沙门氏菌生物膜的机制，在目标2中，我们将通过富集表现出化合物抗性的突变体和从细菌裂解物中直接拉下靶标来确定每种化合物的特定细菌靶标。具有增强的物理化学和生物特性的结构活性关系和衍生物将在Aim 3中生成。综上所述，我们建议对新型抗生物膜化合物预防和治疗伤寒沙门氏菌慢性感染的安全性、有效性和机制进行研究。据我们所知，这项研究将是第一次尝试（利用抗生物膜药物化学和沙门氏菌慢性感染方面的专家）专门针对体内沙门氏菌生物膜的形成作为对抗慢性携带的手段。

Salmonellae are Enterobacteriaceae that cause a spectrum of diseases in humans and animals, including enteric (typhoid) fever and gastroenteritis. Typhoid fever caused primarily by Salmonella enterica serovar Typhi (S. Typhi), results in a life-threatening systemic disease that is responsible for significant morbidity and mortality annually worldwide. Approximately 5% of individuals infected with S. Typhi become chronic carriers with the gallbladder (GB) as the site of persistence. S. Typhi is a human- restricted pathogen, therefore asymptomatic carriers represent a critical reservoir for further spread of disease. We have demonstrated that gallstones (GSs) aid in the development and maintenance of GB carriage in a mouse model (utilizing S. Typhimurium, which causes a typhoid-fever like disease in mice) and in humans, serving as a substrate to which Salmonellae attach and form a protective biofilm. Thus, biofilm formation is a key step in the establishment of carriers. Traditional antibiotic therapies are usually incapable of clearing chronic S. Typhi infections, as the biofilm phenotype renders the bacteria tolerant to the mechanisms of these drugs. Thus, the identification of novel therapeutics capable of targeting S. Typhi biofilms is necessary in order to eliminate chronic carriage and eradicate the disease. Towards this end, our group has identified four small molecules and two antibodies capable of inhibiting and/or disrupting Salmonella biofilms in vitro. We advance two of the small molecules in this proposal, JG-1 and M4, that both inhibit and disrupt biofilms in vitro and reduce GB bacterial numbers in vivo. We hypothesize that the use of these anti-biofilm compounds in conjunction with an antibiotic will more effectively inhibit and disrupt Salmonella biofilms in vivo in our mouse model of chronic carriage when compared to the administration of antibiotic therapy alone. In Aim 1, we will assess the efficacy of these anti-biofilm compounds at preventing and treating chronic infection compared to traditional antibiotics alone by utilizing our established mouse model of typhoidal chronic carriage. We will also measure important pharmacokinetic and tolerability parameters of these compounds. In order to elucidate the mechanisms by which these compounds antagonize Salmonella biofilms, in Aim 2 we will identify the specific bacterial target(s) of each compound by enriching for mutants exhibiting compound resistance and by performing direct pull-downs of targets from bacterial lysates. Structure activity relationships and derivatives with enhanced physiochemical and biological properties will be generated in Aim 3. In summary, we propose an investigation into the safety, efficacy, and mechanisms of novel anti-biofilm compounds to prevent and treat chronic infections by typhoidal Salmonella. To our knowledge this study will be the first attempt (utilizing subject experts in anti-biofilm medicinal chemistry and Salmonella chronic infection) to specifically target Salmonella biofilm formation in vivo as a means of combating chronic carriage.