Metabolic adaptation of Acinetobacter baumannii - the cellular response to desiccation

导致鲍曼不动杆菌在医疗机构传播的一个重要因素是它在没有营养的情况下能够抵抗干燥和长期存活的能力。然而，调节长期存活和耐脱水的分子因素在很大程度上是未知的。我们通过在高盐介质中生长鲍曼曲霉来模拟低水环境，发现它在低水活性下合成谷氨酸、甘露醇和海藻糖作为相容的溶质。甘氨酸甜菜碱是另一种使用的相容溶质；它可以从环境中提取，也可以从胆碱中提取合成。已确定了溶质形成的生物合成途径、关键酶和编码基因。我们将在此基础上揭示溶质在鲍曼不动杆菌病理生物学中的作用。第一个重点是调节溶质的形成，特别是在宿主的背景下，以及它们在毒力中的作用。为此，我们将使用报告基因分析来寻找触发溶质形成的环境信号。甘露醇生物合成的关键酶--甘露醇-1-磷酸脱氢酶/磷酸酶在盐的作用下被激活的生化和分子基础将被揭开。研究表明，海藻糖在感染和持久性方面发挥着重要作用。海藻糖合成中的一个突变缺陷不再能够杀死米氏革兰氏菌并在高温(45摄氏度)下生长。海藻糖这种明显作用的分子基础仍然难以捉摸。此外，我们将讨论甘露醇在毒力中的作用。第二个重点是溶质在抵御各种压力、长期存活和干燥方面的作用。测试的压力条件将模拟宿主，包括pH、氧化应激和温度。建立了一个在特定条件下研究干燥的实验装置，并将利用野生型和突变菌株来研究外源和内源溶质对干燥的影响。另一个重要的问题是，干燥本身是否触发了溶质的合成。我们进行了令人惊讶的观察，发现细胞内的甘露醇和谷氨酸在细胞静止后期消失。这可能暗示在缺乏营养的条件下将溶质重新用作碳和能源，并可能暗示生产可存活但不可培养的细胞。这一假说将通过生理学实验来解决。总之，我们的目标是了解鲍曼不动杆菌中溶质作用的潜在分子基础，以确定新的靶点来对抗这种新的病原体。

An important factor that contributes to the spread of A. baumannii in health care institutions is its ability to withstand desiccation and survive for along time without nutrients. However, the molecular factors that mediate long-term survival and desiccation resistance are largely unknown. We have mimicked low water environments by growing A. baumannii in high salt media and found that it synthesizes glutamate, mannitol and trehalose as compatible solutes in response to low water activities. Glycine betaine is another compatible solute used; it may be taken up from the environment or synthesized from choline taken up. The biosynthetic pathways for solute formation, the key enzymes and the encoding genes have been identified. We will build on this foundation to unravel the role of solutes in the pathobiology of A. baumannii. The first focus is on regulation of solute formation, especially in the context of the host, and their role in virulence. To this end, we will use reporter gene assays to search for environmental signals that trigger solute formation. The biochemical and molecular basis of the activation of the key enzyme in mannitol biosynthesis, the mannitol-1-phosphate dehydrogenase/phosphatase, by salt will be unravelled. It emerges that trehalose plays a prominent role in infection and persistence. A mutant defect in trehalose synthesis is no longer able to kill G. mellonella and to grow at high temperatures (45°C). The molecular basis for this pronounced role of trehalose remains elusive. Furthermore, we will address the role of mannitol in virulence. A second focus is on the role of solutes in protection against various stresses, in long-term survival and desiccation. Stress conditions to be tested will mimic the host and include pH, oxidative stress and temperature. An experimental setup to study desiccation under defined conditions has been established and will be used to study the effect of exogeneous and endogeneous solutes on desiccation using wild type as well as mutant strains. Another important question is whether desiccation itself triggers the synthesis of solutes. We made the astonishing observation that the solutes mannitol and glutamate disappear from the cells during late stationary phase. This could hint to a re-utilization of solutes as carbon and energy source under nutrient-deprived conditions and could hint to the production of viable but non-culturable cells. This hypothesis will be addressed by physiological experiments. In summary, the goal is to understand the underlying molecular basis of the role of solutes in A. baumannii to identify new targets to combat the emerging pathogen.