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Towards measuring growth rates of pathogens during infections by D_2O-labeling lipidomics

Neubauer, Cajetan and Sessions, Alex L. and Booth, Ian R. and Bowen, Benjamin P. and Kopf, Sebastian H. and Newman, Dianne K. and Dalleska, Nathan F. (2018) Towards measuring growth rates of pathogens during infections by D_2O-labeling lipidomics. Rapid Communications in Mass Spectrometry, 32 (24). pp. 2129-2140. ISSN 0951-4198. doi:10.1002/rcm.8288.

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Rationale: 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_2O‐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. Methods: Lipid extracts of D_2O‐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_2O‐labeling lipidomics to clinical samples from CF patients with chronic lung infections. Results: 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. Conclusions: 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_2O.

Item Type:Article
Related URLs:
URLURL TypeDescription ItemBioRxiv preprint
Neubauer, Cajetan0000-0002-5348-5609
Sessions, Alex L.0000-0001-6120-2763
Bowen, Benjamin P.0000-0003-1368-3958
Kopf, Sebastian H.0000-0002-2044-0201
Newman, Dianne K.0000-0003-1647-1918
Dalleska, Nathan F.0000-0002-2059-1587
Additional Information:© 2018 John Wiley & Sons. Version of Record online: 11 November 2018; Accepted manuscript online: 25 September 2018; Manuscript accepted: 18 September 2018; Manuscript revised: 10 August 2018; Manuscript received: 23 May 2018. We thank all reviewers for comments. We are grateful to Drs. Fenfang Wu, Reto Wijker and Jesse Allen for technical advice on the use of instrumentation, and to Dr. Ajay Kasai for CF sputum collection. LC/MS data was collected at the Caltech Environmental Analysis Center (Pasadena) and with the assistance of Dr. Anastasia Kalli at Thermo Fisher Scientific (San Jose, CA, USA). This work was made possible in parts by grants from National Aeronautics and Space Administration (NNX12AD93G), the National Science Foundation (1224158), and the National Institutes of Health (R01HL117328). IRB was funded by CEMI (Caltech) and by the Leverhulme Trust.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Leverhulme TrustUNSPECIFIED
Issue or Number:24
Record Number:CaltechAUTHORS:20181003-105017642
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Official Citation:Neubauer C, Sessions AL, Booth IR, et al. Towards measuring growth rates of pathogens during infections by D2O‐labeling lipidomics. Rapid Commun Mass Spectrom. 2018;32:2129‐2140.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90094
Deposited By: George Porter
Deposited On:05 Oct 2018 21:33
Last Modified:16 Nov 2021 00:40

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