Neubauer Cajetan, Sessions Alex L, Booth Ian R, Bowen Benjamin P, Kopf Sebastian H, Newman Dianne K, Dalleska Nathan F
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.
Division of Geological and Planetary Sciences, California Institute of Technology, CA, 91125, USA.
Rapid Commun Mass Spectrom. 2018 Dec 30;32(24):2129-2140. doi: 10.1002/rcm.8288.
Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids.
Lipid extracts of D O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.
Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day.
This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D O.
微生物生长速率是一个重要的生理参数，原位测量具有挑战性，部分原因是微生物在许多环境中生长缓慢。最近，已证明通过对主动合成的脂肪酸进行D O标记可以确定囊性纤维化（CF）感染中金黄色葡萄球菌的代时。为了提高物种特异性，并在感染治疗期间对更广泛的病原体进行生长速率监测，准确量化氘在磷脂中的微量掺入是很有必要的。
对经D O处理的大肠杆菌培养物的脂质提取物在配备飞行时间（TOF）和轨道阱质量分析仪的液相色谱/电喷雾电离质谱（LC/ESI-MS）仪器上进行测量，并用于与通过同位素比气相色谱（GC）/MS分析脂肪酸进行比较。然后，我们开发了一种方法，通过跟踪纯培养物中从稳定期到指数生长期的转变来实现对脂质标记的追踪。最后，我们将D O标记脂质组学应用于患有慢性肺部感染的CF患者的临床样本。
脂质组学有助于在脂质中进行氘定量，其水平对许多标记应用有用（>0.03原子%D）。在大肠杆菌培养物中，磷脂的标记动态很大程度上取决于其酰基链，并且在磷脂之间，我们注意到仅从绝对浓度来看不明显的差异。例如，含环丙基的脂质反映了环丙烷脂肪酸合酶的调节，该酶主要在稳定期开始时表达。CF痰液中金黄色葡萄球菌特有的一种脂质中的氘掺入表明该病原体的平均代时约为每天一个细胞倍增。
本研究展示了如何利用完整脂质中稳定同位素的痕量水平测量来量化纯培养物中的脂质代谢，并提供了在与低百分比的D O孵育后能够在微生物组样本中进行生长速率测量的指导方针。
