Genetic and mechanistic analysis of carbon dioxide tolerance in Cryptococcus pathogenesis

摘要 隐球菌性脑膜脑炎(Cme)是影响人类的最重要的机会性感染之一。 感染艾滋病毒/艾滋病。这种疾病的重要性不仅是因为它在本地区和地区的高发病率和死亡率 其他患者群体，也与CME症状是导致 艾滋病毒/艾滋病的诊断。因此，许多患者必须首先存活下来，然后才能从 艾滋病治疗的新进展。不幸的是，CME治疗的结果远不能被接受，特别是在 资源有限、疾病负担高的地区。因此，有效和广泛可用的治疗方法 对于CME来说，这是一个具有全球重要性的未得到满足的临床需求。隐球菌属是担子菌酵母菌 其主要的利基是外部环境。因此，只有隐球菌的菌株和物种可以 过渡到人类宿主并在其中复制能够导致疾病。我们的中心前提是 了解隐球菌在人类体内生存所需的生物学机制可能 为治疗提供新的靶点，就像研究肿瘤生物学一样，为设计新的抗肿瘤药物提供信息 抗癌药物。人类宿主和自然生态位之间的一个重要环境差异 隐球菌是二氧化碳(CO2)的浓度。我们假设，对宿主水平的适应 CO2可能是隐球菌致病过程中的关键步骤。为了具体检验这一假设，我们 比较了新生隐孢子虫的生长情况。Grubii菌株在只有浓度变化的条件下 二氧化碳的排放量。与我们的假设一致，生长速度在经历二氧化碳浓度时减慢。 主持人。接下来，我们测试了一组已知毒力特性的环境菌株的二氧化碳耐受性。 在小鼠模型中，在宿主二氧化碳浓度存在时生长减慢的菌株是无毒的。 而那些生长速度与人类患者临床分离株相匹配的菌株是毒力较强的。因此，我们， 已经发现，二氧化碳耐受性是一种以前未被识别的宿主环境相关 新生葡萄球菌的毒力属性。因此，这项提议的目标是识别基因， 允许特定菌株对宿主作出反应的转录和调控反应 二氧化碳的浓度，从而导致了CME。为了实现这些目标，我们提出了以下建议 具体目标：目标1.表征二氧化碳和二氧化碳之间的毒性、转录和基因组差异 耐受和不耐受的新生葡萄球菌；目标2.通过以下途径确定耐二氧化碳所需的基因 有针对性的大规模遗传筛选；以及目标3.确定基因的分子机制 新生假单胞菌二氧化碳反应所需的。成功实现这些目标不仅将推动我们的 了解新生芽孢杆菌的致病机制和宿主存活，也为未来确定新的分子靶点 作为毒品目标的探索。

Abstract Cryptococcal meningoencephalitis (CME) is one of the most important opportunistic infections affecting people with HIV/AIDS. The importance of this disease is due not only to its high incidence and mortality in this and other patient populations but also to the fact that symptoms of CME are a common sentinel event leading to the diagnosis of HIV/AIDS. Thus, many patients must first survive CME before they can benefit from the advances in HIV therapy. Unfortunately, the outcomes for CME therapy are far from acceptable, particularly in resource-limited regions with high burdens of disease. Consequently, effective and widely available therapies for CME are an unmet clinical need of global importance. Cryptococcus spp. are basidiomycetous yeasts whose primary niche is the external environment. As such, only strains and species of Cryptococcus that can transition to, and replicate within, the human host are able to cause disease. Our central premise is that an understanding of the biological mechanisms required for Cryptococcus to survive in human beings could provide new targets for therapy in the same way as studying tumor biology informs the design of new anti- cancer drugs. An important environmental distinction between the human host and the natural niche of Cryptococcus is the concentration of carbon dioxide (CO2). We hypothesized that adaptation to host levels of CO2 may represent a critical step in Cryptococcus pathogenesis. To specifically test this hypothesis, we compared the growth of C. neoformans var. grubii strains under conditions that varied only in the concentration of CO2. Consistent with our hypothesis, the growth rate was reduced at concentrations of CO2 experienced in the host. Next, we tested the CO2 tolerance of a set of environmental strains with known virulence properties in a mouse model; strains with reduced growth in the presence of host concentrations of CO2 were avirulent while those with growth rates that matched clinical isolates from human patients were virulent. We, therefore, have discovered that CO2 tolerance is a previously unrecognized host environment-associated virulence attribute of C. neoformans. Accordingly, the goal of this proposal is to identify the genetic, transcriptional, and regulatory responses that allow specific strains of C. neoformans to respond to host concentrations of CO2 and, thereby, cause CME. To accomplish these goals, we propose the following specific aims: Aim 1. Characterize the virulence, transcriptional, and genomic distinctions between CO2- tolerant and -non-tolerant C. neoformans strains; Aim 2. Identify genes required for CO2 tolerance through targeted and large-scale genetic screening; and Aim 3. Determine the molecular mechanisms of genes required for C. neoformans CO2 response. Successful execution of these aims will not only further our understanding C. neoformans pathogenesis and host survival but also identify new molecular targets for future exploration as drug targets.