Abstract：
Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq in vivo is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in Akr1c13 that renders the resulting RECON-/- mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen Listeria monocytogenes in vivo. We identified 135 genes which were required for L. monocytogenes growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for L. monocytogenes survival and investigated the role of the folate enzyme FolD in L. monocytogenes liver pathogenesis. A mutant lacking folD was impaired for growth in murine livers by 2.5-log10 compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in alsR, which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log10 and 3-log10, respectively, but showed modest phenotypes in in vitro models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the in vivo growth defect, as deletion of the operon in the ∆alsR background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for L. monocytogenes host infection, which highlights the utility of the RECON-/- mouse model for future screening efforts.
OBJECTIVE: Listeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via transposon sequencing. We identified the liver-specific gene folD and a repressor, alsR, that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.
OBJECTIVE: Listeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via transposon sequencing. We identified the liver-specific gene folD and a repressor, alsR, that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.
摘要：
转座子测序（Tn-seq）是一种强大的全基因组技术，可在不同的生长条件下评估细菌适应性。然而,在体内通过Tn-seq进行筛选是具有挑战性的。剂量限制和宿主限制产生减少被筛选的转座子突变池的瓶颈。这里,我们已经开发了一种在Akr1c13中破坏的小鼠模型，该模型使所得的RECON-/-小鼠对高剂量感染具有抗性。我们利用该模型在体内进行人病原体单核细胞增生李斯特菌的Tn-seq筛选。我们鉴定了小鼠中单核细胞增生李斯特菌生长所需的135个基因，包括以前未鉴定的宿主存活的新基因。我们确定了单核细胞增生李斯特菌存活的器官特异性要求，并研究了叶酸酶FolD在单核细胞增生李斯特菌肝脏发病机理中的作用。与野生型相比，缺乏folD的突变体在鼠肝脏中的生长受损2.5-log10，并且未能在成纤维细胞中细胞间扩散。相比之下,alsR中的一个突变体,它编码一种转录因子，抑制参与D-阿洛糖分解代谢的操纵子，在小鼠的肝脏和脾脏中分别减毒4-log10和3-log10，但在体外模型中表现出适度的表型。我们证实，D-阿洛糖分解代谢操纵子的失调是体内生长缺陷的原因，由于操纵子在ΔalsR背景中的缺失拯救了毒力。通过无偏见的承诺，小鼠的全基因组屏幕，我们已经确定了单核细胞增生李斯特菌宿主感染的新适应性决定因素，这突出了RECON-/-小鼠模型在未来筛查工作中的实用性。
目的：单核细胞增生李斯特菌是导致食源性李斯特菌病的革兰氏阳性菌。尽管单核细胞增生李斯特菌的感染在健康宿主中是有限的，弱势群体,包括孕妇和老人，可以经历高死亡率。因此，了解单核细胞增生李斯特菌体内存活的遗传需求的广度将为治疗和预防李斯特菌病提供新的机会。我们使用RECON-/-小鼠开发了一种小鼠感染模型，该小鼠对全身性单核细胞增生李斯特菌感染具有限制性。我们利用该模型通过转座子测序筛选体内所需的单核细胞增生李斯特菌基因。我们确定了肝脏特异性基因folD和一个阻遏物,alsR,仅表现出体内生长缺陷。AlsR控制D-allose操纵子的表达，D-allose操纵子是诊断技术中鉴定致病性李斯特菌的标志物。更好地了解D-allose操纵子在人类疾病中的作用可能会进一步为诊断和预防措施提供信息。
目的：单核细胞增生李斯特菌是导致食源性李斯特菌病的革兰氏阳性菌。尽管单核细胞增生李斯特菌的感染在健康宿主中是有限的，弱势群体,包括孕妇和老人，可以经历高死亡率。因此，了解单核细胞增生李斯特菌体内存活的遗传需求的广度将为治疗和预防李斯特菌病提供新的机会。我们使用RECON-/-小鼠开发了一种小鼠感染模型，该小鼠对全身性单核细胞增生李斯特菌感染具有限制性。我们利用该模型通过转座子测序筛选体内所需的单核细胞增生李斯特菌基因。我们确定了肝脏特异性基因folD和一个阻遏物,alsR,仅表现出体内生长缺陷。AlsR控制D-allose操纵子的表达，D-allose操纵子是诊断技术中鉴定致病性李斯特菌的标志物。更好地了解D-allose操纵子在人类疾病中的作用可能会进一步为诊断和预防措施提供信息。
