Mechanisms of the Development and Maintenance of Salmonella Gallbladder Carriage

沙门氏菌是肠杆菌科，可引起人类和动物的一系列疾病，包括肠（伤寒）热和胃肠炎。伤寒主要由伤寒沙门氏菌引起。伤寒）导致一种威胁生命的全身性疾病，每年在世界范围内造成显著的发病率和死亡率。大约5%的人感染了S。伤寒成为胆囊（GB）作为持久性部位的慢性携带者。S.伤寒是一种人类限制性病原体，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（GS）的发展和维持的GB运输在小鼠模型（利用S。鼠伤寒沙门氏菌（Typhimurium），在小鼠和人类中引起伤寒样疾病，作为沙门氏菌附着并形成保护性生物膜的底物。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不能清除慢性S。伤寒感染，因为生物膜表型使细菌耐受这些药物的机制。因此，鉴定能够靶向S.伤寒生物膜是必要的，以消除慢性携带和根除疾病。为此，我们的小组已经确定了四个小分子和两个抗体能够抑制和/或破坏沙门氏菌生物膜在体外。我们提出了两个小分子，JG-1和M4，这两个小分子在体外抑制和破坏生物膜，并减少体内GB细菌的数量。我们假设，当与单独施用抗生素治疗相比时，这些抗生物膜化合物与抗生素联合使用将在我们的慢性携带小鼠模型中更有效地抑制和破坏体内沙门氏菌生物膜。在目的1中，我们将通过利用我们建立的伤寒慢性携带小鼠模型，评估这些抗生物膜化合物与单独的传统抗生素相比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，在目的2中，我们将通过富集表现出化合物抗性的突变体和通过从细菌裂解物中直接下拉靶标来鉴定每种化合物的特异性细菌靶标。目标3中将生成具有增强的理化和生物学特性的构效关系和衍生物。总之，我们提出了一个新的抗生物膜化合物的安全性，有效性和机制，以预防和治疗伤寒沙门氏菌的慢性感染的调查。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的学科专家），以专门针对沙门氏菌生物膜形成在体内作为一种手段，打击慢性携带。

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.