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Tularemia is a disease characterized by profound neutrophil accumulation and tissue destruction. The causative organism, Francisella tularensis, is a facultative intracellular bacterium that replicates in neutrophil cytosol, inhibits caspase activation and profoundly prolongs cell lifespan. Here, we identify unique features of this infection and provide fundamental insight into the mechanisms of apoptosis inhibition. Mitochondria are critical regulators of neutrophil apoptosis. We demonstrate that F. tularensissignificantly inhibits Bax translocation and Bid processing during 24-48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, X-linked inhibitor of apoptosis protein (XIAP) and proliferating cell nuclear antigen (PCNA) inhibit caspase-9 and caspase-3 by direct binding. Notably, we find that PCNA disappeared rapidly and selectively from infected cells, thereby demonstrating that it is not essential for neutrophil survival, whereas upregulation of calpastatin correlated with diminished calpain activity and reduced XIAP degradation. In addition, R-roscovitine is a cyclin-dependent kinase inhibitor developed for the treatment of cancer; it also induces neutrophil apoptosis and can promote the resolution of several infectious and inflammatory disorders. We confirm the ability of R-roscovitine to induce neutrophil apoptosis, but also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance the understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria.

ABSTRACTFrancisella tularensis, the etiological agent of tularemia, is capable of infecting a wide range of animals and causes a severe, lethal disease in humans. The pathogen evades killing by cells of the innate immune system utilizing genes encoding a pathogenicity island, including iglABCD, and instead utilizes these cells as a niche for replication and dissemination to other organs within the host. Regulators of the iglgenes (e.g., MglA, SspA, FevR and PmrA) have been identified, but environmental stimuli and mechanisms of regulation are as yet unknown and are likely to involve additional gene products. In this work, we more closely examine the roles that environmental iron and the ferric uptake repressor protein (Fur) play in the regulation of the iglABCDoperon. We also used a genetic approach to identify and characterize a new regulator of the igloperon, designated migR(macrophage intracellular growth regulator; FTL_1542). Quantitative real-time reverse transcription-PCR in a site-directed migRmutant confirmed the reduction in the number of iglCtranscripts in this strain and also demonstrated reduced expression of fevR. Comparison of the migRand fevRmutants in monocyte-derived macrophages (MDMs) and epithelial cell lines revealed a reduced ability for each mutant to grow in MDMs, yet only the fevRmutant exhibited impaired replication in epithelial cell lines. Confocal analysis of infected MDMs revealed that although neither mutant reached the MDM cytosol, the fevRmutant was trapped in lamp-1-positive phagosomes, whereas the migRmutant resided in mature phagolysosomes enriched with both lamp-1 and cathepsin D. Disruption of migRand fevRalso impaired the ability of F. tularensisto prevent neutrophil oxidant production. Thus, we have identified migR, a gene that regulates expression of the iglABCDoperon and is essential for bacterial growth in MDMs and also contributes to the blockade of neutrophil NADPH oxidase activity.

ABSTRACTFrancisella tularensisis a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensissubspecies holarcticastrain 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 pyrBon 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 Francisellabacteria.