Francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain LVS. Infect Immun 2009 Apr;77(4):1324-36
Date
02/11/2009Pubmed ID
19204089Pubmed Central ID
PMC2663180DOI
10.1128/IAI.01318-08Scopus ID
2-s2.0-63149103523 (requires institutional sign-in at Scopus site) 59 CitationsAbstract
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-1beta (IL-1beta) 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.
Author List
Schulert GS, McCaffrey RL, Buchan BW, Lindemann SR, Hollenback C, Jones BD, Allen LAAuthor
Blake W. Buchan PhD Professor in the Pathology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAspartate Carbamoyltransferase
Bacterial Proteins
Carbamoyl-Phosphate Synthase (Ammonia)
Cell Line
Culture Media
DNA Transposable Elements
Epithelial Cells
Francisella tularensis
Humans
Macrophages
Mice
Mutagenesis, Insertional
NADPH Oxidases
Neutrophil Activation
Neutrophils
Respiratory Burst
Tularemia
Uracil