Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. A Francisella pathogenicity island (FPI) has been identified as required for intracellular growth but how it contributes to Francisella virulence is not understood. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. In a model of murine primary macrophage infection with the F. tularensis type B attenuated LVS strain, we have shown that phagosomal escape occurs rapidly after phagocytosis (~ 20 min) using a fluorescence microscopy-based assay to measure phagosomal integrity. Unexpectedly, we have found that bacterial replication in the cytoplasm is followed by the enclosure of bacteria within a membrane-bound compartment (~ 24 h) that displays properties of mature autophagosomes, indicating that autophagy is involved in Francisella intracellular trafficking. Furthermore, formation of these late Francisella-containing vacuoles (FCV) requires bacterial replication and protein synthesis, suggesting that this process is controlled by the replicating bacteria. Type A or B virulent clinical isolates of F. tularensis showed comparable intracellular trafficking in murine macrophages and formed late FCVs with similar kinetics, indicating a conserved mechanism between type A and B strains. Blocking FCV formation through inhibition of autophagy did not affect intracellular proliferation, suggesting FCVs are required for intracellular persistence or egress. These results also indicate that intracellular Francisella can control the macrophage autophagic response during their intracellular trafficking. This work is in press in PNAS. In our efforts to identify Francisella genes that are important for intracellular pathogenesis, we have worked in collaboration with the RML Genomic Core facility to establish the intracellular transcriptome of the Type A virulent F. tularensis subsp. tularensis Schu-S4 strain. We have now optimized our procedures to isolate quality bacterial RNA in sufficient amounts at all times post infection to obtain kinetic, transcriptional profiles of intracellular bacteria using the Affymetrix RML GeneChip II. The analysis of the DNA microarray data will allow us to identify candidate genes involved in intracellular pathogenesis, based on their expression profiles inside macrophages. To validate this approach, preliminary work has been performed to characterize the expression patterns of the igl and pdp genes from the Francisella Pathogenicity Island (FPI), which are known to be involved in intracellular growth. Differential expression patterns have been observed during the intracellular cycle and between FPI genes, indicating that virulence genes are expressed at particular times of the intracellular cycle. To extend these findings, we have analyzed the intracellular behaviour of deletion mutants of some of these genes in F. novicida, in collaboration with Francis Nano (University of Victoria), and found that their intracellular defects correlate with their expression patterns. These data validate our ongoing transcriptome approach of Francisella pathogenesis and should allow us to identify novel genes required for intracellular virulence of this bacterium. In collaboration with Tom Zahrt (Medical College of Wisconsin), we have generated and characterized a purine auxotroph (?purMCD) of the LVS strain, in order to create a vaccine strain of known genetic background. Such a mutant showed clear attenuation inside murine macrophages, where it was unable to fully replicate. Interestingly, this mutant was fully protective to a lethal challenge with the parental strain in a mouse model of tularemia. This work,which has been published in Infection and Immunity, is an important step towards the generation of attenuated Type A strains that can potentially be used as live vaccine strains.