RESUMEN
Francisella tularensis infects wild animals and humans to cause tularemia. This pathogen targets the cytosol of macrophages, where it replicates using the genes in the Francisella pathogenicity island (FPI). Virulence gene regulation in Francisella is complex, but transcriptional regulators MglA and SspA have been shown to regulate the expression of approximately 100 genes, including the entire FPI. We utilized a Francisella novicida transposon mutant library to identify additional regulatory factors and identified five additional genes that are essential for virulence gene expression. One regulatory gene, FTN_0480 (fevR, Francisella effector of virulence regulation), present in all Francisella species, is required for expression of the FPI genes and other genes in the MglA/SspA regulon. The expression of fevR is positively regulated by MglA. However, constitutive expression of fevR in an mglA mutant strain did not restore expression of the MglA/SspA regulon, demonstrating that mglA and fevR act in parallel to positively regulate virulence gene expression. Virulence studies revealed that fevR is essential for bacterial replication in macrophages and in mice, where we additionally show that fevR is required for the expression of genes in the MglA/SspA regulon in vivo. Thus, fevR is a crucial virulence gene in Francisella, required for the expression of virulence factors known to be essential for this pathogen's subversion of host defenses and pathogenesis in vivo.
Asunto(s)
Proteínas Bacterianas/genética , Francisella/genética , Francisella/patogenicidad , Regulación de la Expresión Génica , Factores de Transcripción/genética , Factores de Virulencia/biosíntesis , Animales , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Femenino , Francisella/crecimiento & desarrollo , Perfilación de la Expresión Génica , Islas Genómicas , Infecciones por Bacterias Gramnegativas/microbiología , Inmunoprecipitación , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Unión Proteica , Regulón , Factores de Transcripción/metabolismoRESUMEN
Francisella tularensis is a pathogenic bacterium whose virulence is linked to its 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 interleukin (IL)-1beta and -18 and trigger caspase-1-dependent cell death. In this study, we show that cytosolic F. tularensis subspecies novicida (F. novicida) induces a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome-mediated cell death, and release of IL-1beta and -18. Extensive type I IFN-dependent cell death resulting in macrophage depletion occurs in vivo during F. novicida infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria monocytogenes but not vacuole-localized Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These results show the specific connection between type I IFN signaling and inflammasome activation, which are two sequential events triggered by the recognition of cytosolic bacteria. To our knowledge, this is the first example of the positive regulation of inflammasome activation. This connection underscores the importance of the cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.
Asunto(s)
Francisella , Infecciones por Bacterias Gramnegativas/inmunología , Mediadores de Inflamación/inmunología , Interferón Tipo I/metabolismo , Macrófagos/inmunología , Transducción de Señal/inmunología , Animales , Apoptosis/inmunología , Western Blotting , Citosol/inmunología , Citosol/microbiología , Interferón Tipo I/inmunología , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Francisella tularensis subverts the immune system to rapidly grow within mammalian hosts, often causing tularemia, a fatal disease. This pathogen targets the cytosol of macrophages where it replicates by using the genes encoded in the Francisella pathogenicity island. However, the bacteria are recognized in the cytosol by the host's ASC/caspase-1 pathway, which is essential for host defense, and leads to macrophage cell death and proinflammatory cytokine production. We used a microarray-based negative selection screen to identify Francisella genes that contribute to growth and/or survival in mice. The screen identified many known virulence factors including all of the Francisella pathogenicity island genes, LPS O-antigen synthetic genes, and capsule synthetic genes. We also identified 44 previously unidentified genes that were required for Francisella virulence in vivo, indicating that this pathogen may use uncharacterized mechanisms to cause disease. Among these, we discovered a class of Francisella virulence genes that are essential for growth and survival in vivo but do not play a role in intracellular replication within macrophages. Instead, these genes modulate the host ASC/caspase-1 pathway, a previously unidentified mechanism of Francisella pathogenesis. This finding indicates that the elucidation of the molecular mechanisms used by other uncharacterized genes identified in our screen will increase our understanding of the ways in which bacterial pathogens subvert the immune system.
Asunto(s)
Francisella tularensis/genética , Francisella tularensis/patogenicidad , Genes Bacterianos , Selección Genética , Animales , Células de la Médula Ósea/citología , Caspasa 1/genética , Caspasa 1/metabolismo , Células Cultivadas , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Femenino , Marcación de Gen , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana/genética , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Tularemia/etiología , Tularemia/genética , Tularemia/microbiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.