Project description:To understand differences of gene expression profiles between Francisella strains RNA profiles of Francisella strains were generated by deep sequencing, in triplicate, using NovaSeq6000. qRT–PCR validation was performed using SYBR Green assays. Our study represents the first detailed differential transcriptomic analysis of Francisella strains , with biologic replicates, generated by RNA-seq technology.
Project description:The goal of this study is to determine the host response of human epithelial cells during infection with Francisella Tularensis. For this purpose, A549 human epithelial cell line was infected with Francisella tularensis spp. holarctica strain LVS for different times of infection, in duplicates. At different times post infection (0.5/1/3/6/12/24 hours post infection) cells were harvested and total RNA was extracted. RNA-seq libraries were constructed and sequencing of 100bp paired-end was performed on the Illumina NovaSeq 6000 system. Sequencing yielded about 22M reads per sample that were mapped to the human genome (Human: GRCh38) resulting with the identification of 21,066 transcripts. The expression of the infected samples was compared to mock sample, and RNA ratios were clustered using partitioning clustering. This approach allowed clustering of the cellular transcripts into 5 distinct classes based on similarities in temporal expression profiles. We next carried out GO term enrichment analysis for each of these five cluster. Our study represents the first detailed analysis of human epithelial response to Francisella tularensis infection, and provide a framework for comparative investigations of genes and mechanisms that may contribute to the infection.
Project description:Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. Using a global and site-specific phosphoproteomic analysis of Francisella we identified here a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. Phosphorylation of the sheath may constitute a previously unrecognized mechanism contributing to the dynamics of assembly-disassembly of the T6SS.
Project description:Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. Using a global and site-specific phosphoproteomic analysis of Francisella we identified here a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. Phosphorylation of the sheath may constitute a previously unrecognized mechanism contributing to the dynamics of assembly-disassembly of the T6SS.
Project description:Francisella are pathogenic bacteria whose virulence is linked to their ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host protective multimolecular complex called the inflammasome to release the proinflammatory cytokines IL-1 and IL-18 and trigger caspase-1 dependent cell death. Here we show that cytosolic Francisella induce a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome mediated cell death, and release of IL-1 and IL-18. Extensive type I IFN dependent cell death resulting in macrophage depletion occurs in vivo during Francisella infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria but not vacuole localized Salmonella or extracellular ATP. These results show the specific connection between type I IFN signaling and inflammasome activation, two sequential events triggered by recognition of cytosolic bacteria. To our knowledge, this is the first example of positive regulation of inflammasome activation. This connection underscores the importance of cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses. Keywords: murine macrophage response to Francisella tularensis subspecies novicida infection
Project description:Comparison of expression profiles of strains of Francisella to identify virulence factors We used custom microarrays to detail the global gene expression of four strains of Francisella (Schu4, LVS, OR960246, U112) and identified distinct classes of differentially expressed genes during this process.
Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series
Project description:Francisella are pathogenic bacteria whose virulence is linked to their ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host protective multimolecular complex called the inflammasome to release the proinflammatory cytokines IL-1 and IL-18 and trigger caspase-1 dependent cell death. Here we show that cytosolic Francisella induce a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome mediated cell death, and release of IL-1 and IL-18. Extensive type I IFN dependent cell death resulting in macrophage depletion occurs in vivo during Francisella infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria but not vacuole localized Salmonella or extracellular ATP. These results show the specific connection between type I IFN signaling and inflammasome activation, two sequential events triggered by recognition of cytosolic bacteria. To our knowledge, this is the first example of positive regulation of inflammasome activation. This connection underscores the importance of cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses. Keywords: murine macrophage response to Francisella tularensis subspecies novicida infection We analyzed a series of 18 MEEBO arrays on which were hybed RNA randomly amplified from bone marrow derived macrophages infected or not with WT Francisella tularensis subspecies novicida or a the mglA mutant strain GB2.