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Infection and Immunity, July 2009, p. 2887-2895, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Purification of Legiobactin and Importance of This Siderophore in Lung Infection by Legionella pneumophila{triangledown}

Kimberly A. Allard,1 Jenny Dao,1 Prakash Sanjeevaiah,2 Kessler McCoy-Simandle,1 Christa H. Chatfield,1 David S. Crumrine,2 Domenic Castignetti,3 and Nicholas P. Cianciotto1*

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611,1 Departments of Chemistry,2 Biology, Loyola University, Chicago, Illinois 606263

Received 22 January 2009/ Returned for modification 4 March 2009/ Accepted 13 April 2009

When cultured in a low-iron medium, Legionella pneumophila secretes a siderophore (legiobactin) that is both reactive in the chrome azurol S (CAS) assay and capable of stimulating the growth of iron-starved legionellae. Using anion-exchange high-pressure liquid chromatography (HPLC), we purified legiobactin from culture supernatants of a virulent strain of L. pneumophila. In the process, we detected the ferrated form of legiobactin as well as other CAS-reactive substances. Purified legiobactin had a yellow-gold color and absorbed primarily from 220 nm and below. In accordance, nuclear magnetic resonance spectroscopy revealed that legiobactin lacks aromatic carbons, and among the 13 aliphatics present, there were 3 carbonyls. When examined by HPLC, supernatants from L. pneumophila mutants inactivated for lbtA and lbtB completely lacked legiobactin, indicating that the LbtA and LbtB proteins are absolutely required for siderophore activity. Independently derived lbtA mutants, but not a complemented derivative, displayed a reduced ability to infect the lungs of A/J mice after intratracheal inoculation, indicating that legiobactin is required for optimal intrapulmonary survival by L. pneumophila. This defect, however, was not evident when the lbtA mutant and its parental strain were coinoculated into the lung, indicating that legiobactin secreted by the wild type can promote growth of the mutant in trans. Legiobactin mutants grew normally in murine lung macrophages and alveolar epithelial cells, suggesting that legiobactin promotes something other than intracellular infection of resident lung cells. Overall, these data represent the first documentation of a role for siderophore expression in the virulence of L. pneumophila.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University Medical School, 320 East Superior St., Chicago, IL 60611. Phone: (312) 503-0385. Fax: (312) 503-1339. E-mail: n-cianciotto{at}northwestern.edu

{triangledown} Published ahead of print on 27 April 2009.

Editor: J. L. Flynn

Infection and Immunity, July 2009, p. 2887-2895, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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