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Infection and Immunity, April 2009, p. 1324-1336, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01318-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Francisella tularensis Genes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS{triangledown}

Grant S. Schulert,1,2 Ramona L. McCaffrey,1,3,4 Blake W. Buchan,2 Stephen R. Lindemann,2 Clayton Hollenback,1,3 Bradley D. Jones,2 and Lee-Ann H. Allen1,2,3,4*

Inflammation Program,1 Departments of Microbiology,2 Medicine, University of Iowa,3 VA Medical Center, Iowa City, Iowa 522424

Received 28 October 2008/ Returned for modification 3 December 2008/ Accepted 2 February 2009

Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensis subspecies holarctica strain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identified carA, carB, and pyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1β (IL-1β) and IL-8 secretion, were unaffected. The cultivation of carA, carB, or pyrB on uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate of Francisella bacteria.

* Corresponding author. Mailing address: Inflammation Program, MTF D-154, University of Iowa, 2501 Crosspark Rd., Coralville, IA 52241. Phone: (319) 335-4258. Fax: (319) 335-4194. E-mail: lee-ann-allen{at}uiowa.edu

{triangledown} Published ahead of print on 9 February 2009.

Editor: W. A. Petri, Jr.

Infection and Immunity, April 2009, p. 1324-1336, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01318-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Buchan, B. W., McCaffrey, R. L., Lindemann, S. R., Allen, L.-A. H., Jones, B. D. (2009). Identification of migR, a Regulatory Element of the Francisella tularensis Live Vaccine Strain iglABCD Virulence Operon Required for Normal Replication and Trafficking in Macrophages. Infect. Immun. 77: 2517-2529 [Abstract] [Full Text]  


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