Abstract
Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of Klebsiella pneumoniae (hvKP). In contrast to classical opportunistic strains of K. pneumoniae, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent “super bug.” Despite hvKP harboring four distinct siderophore operons, knocking out production of only aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. In vivo heterologous production and in vitro reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7841-7852 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 20 |
| DOIs | |
| State | Published - May 18 2018 |
Bibliographical note
Funding Information:This work was supported in part by the National Institutes of Health Grant AI116998 (to A. M. G.) and pilot studies supported by The Buffalo Clinical and Translational Research Center, National Institutes of Health Grant UL1TR001412 (to Dr. Timothy F. Murphy). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1 Supported by National Institutes of Health Training Grant T32-AI007614 (to Dr. Laurie K. Read). Dr. Edward Snell for collecting the SAXS data on IucC, and Dr. Thomas Grant for guidance analyzing it. We thank Dr. Artem Evdokimov for invaluable assistance in SER mutant design. We thank Dr. Thomas Russo for insightful discussions regarding hvKP. The compact mass spectrometer was purchased with funds from the Richard W. and Mae Stone Goode Foundation. Diffraction data were collected at the GM/CA ID-B beamline of APS, which is supported by the National Cancer Institute Grant ACB-12002, National Institute of General Medical Sciences Grant AGM-12006 under Department of Energy Contract Number DE-AC02-06CH11357 to APS, and the National Institutes of Health Office of Research Infrastructure Program Grant 1S10OD012289-01A1.
Funding Information:
Acknowledgments—We thank Dr. Edward Snell for collecting the SAXS data on IucC, and Dr. Thomas Grant for guidance analyzing it. We thank Dr. Artem Evdokimov for invaluable assistance in SER mutant design. We thank Dr. Thomas Russo for insightful discussions regarding hvKP. The compact mass spectrometer was purchased with funds from the Richard W. and Mae Stone Goode Foundation. Diffraction data were collected at the GM/CA ID-B beamline of APS, which is supported by the National Cancer Institute Grant ACB-12002, National Institute of General Medical Sciences Grant AGM-12006 under Department of Energy Contract Number DE-AC02-06CH11357 to APS, and the National Institutes of Health Office of Research Infrastructure Program Grant 1S10OD012289-01A1.
Funding Information:
This work was supported in part by the National Institutes of Health Grant AI116998 (to A. M. G.) and pilot studies supported by The Buffalo Clini-cal and Translational Research Center, National Institutes of Health Grant UL1TR001412 (to Dr. Timothy F. Murphy). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
1 Supported by National Institutes of Health Training Grant T32-AI007614 (to Dr. Laurie K. Read).
Publisher Copyright:
© 2018 Bailey et al.