title: Francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain LVS.
abstract: 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.
authors.affiliation: Inflammation Program, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Grant S
authors.initials: GS
authors.lastname: Schulert
authors.forename: Ramona L
authors.initials: RL
authors.lastname: McCaffrey
authors.forename: Blake W
authors.initials: BW
authors.lastname: Buchan
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Lindemann
authors.forename: Clayton
authors.initials: C
authors.lastname: Hollenback
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Jones
authors.forename: Lee-Ann H
authors.initials: LA
authors.lastname: Allen
mesh_terms: D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003470:Culture Media; D004251:DNA Transposable Elements; D004847:Epithelial Cells / Q000382:microbiology; D005604:Francisella tularensis / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000382:microbiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D018375:Neutrophil Activation / Q000276:immunology*; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology; D016897:Respiratory Burst / Q000276:immunology*; D014406:Tularemia / Q000276:immunology* / Q000382:microbiology / Q000473:pathology; D014498:Uracil / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004251:DNA Transposable Elements; D014498:Uracil; D019255:NADPH Oxidases; D001221:Aspartate Carbamoyltransferase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)