Identification ofmigR, a Regulatory Element of theFrancisella tularensisLive Vaccine StrainiglABCDVirulence Operon Required for Normal Replication and Trafficking in Macrophages

Abstract

Francisella 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 igl genes (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 iglABCD operon. We also used a genetic approach to identify and characterize a new regulator of the igl operon, designated migR (macrophage intracellular growth regulator; FTL_1542). Quantitative real-time reverse transcription-PCR in a site-directed migR mutant confirmed the reduction in the number of iglC transcripts in this strain and also demonstrated reduced expression of fevR.Comparison of the migR and fevR mutants in monocyte-derived macrophages (MDMs) and epithelial cell lines revealed a reduced ability for each mutant to grow in MDMs, yet only the fevR mutant exhibited impaired replication in epithelial cell lines. Confocal analysis of infected MDMs revealed that although neither mutant reached the MDM cytosol, the fevR mutant was trapped in lamp-1-positive phagosomes, whereas the migR mutant resided in mature phagolysosomes enriched with both lamp-1 and cathepsin D. Disruption of migR and fevR also impaired the ability of F. tularensis to prevent neutrophil oxidant production. Thus, we have identified migR, a gene that regulates expression of the iglABCD operon and is essential for bacterial growth in MDMs and also contributes to the blockade of neutrophil NADPH oxidase activity.

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…Mutagenesis using this transposon system has allowed identification of genes from F. tularensis LVS that are required for inhibition of the respiratory burst in neutrophils and for intracellular growth or survival within this cell type (179). Additionally, this system was used to identify FevR, a novel regulator of iglB (22). Finally, useful genetic surrogates of F. novicida which lack all of the resident restriction-modification systems that otherwise act as a barrier to gene transfer have now been developed (75).…”

“…A variety of pFNLTP1 variants have been generated, and these include derivatives that carry antibiotic resistance elements amenable for use in type A strains of F. tularensis, multiple cloning sites, reporter genes and counterselectable markers, and temperature-sensitive origins of replication (93,125). In addition to their use as complementation and reporter gene platforms, pFNLTP1-based vectors (or vectors that have been derived from them) have been used as delivery vehicles to carry out transposon mutagenesis, targeted allelic exchange, and promoter-trap library construction (22,125,128).…”

“…Genes present in the FPI are arranged into two major transcriptional units (138) and are induced during intracellular growth of F. tularensis (29,111,212). FPI genes have also been shown to be regulated by other environmental conditions, including iron (22,23,47,105,113,177) MigR (22). One major transcriptional unit encodes genes of the intracellular growth locus (i.e., iglABCD).…”

“…fevR (Francisella effector of virulence regulation) encodes a hypothetical protein and was identified in a screen of genes requiring MglA/SspA for their expression (20). FevR is required for replication of F. novicida and F. tularensis LVS in murine and human macrophages (20,22), in the human epithelial cell lines HEp-2 and A549 (22), and in mice (20). While the mechanism(s) of FevR's activity remains unclear, fevR mutants of F. tularensis LVS remain trapped within a phagosome that retains late endosomal marker LAMP-1 but not lysosomal marker cathepsin D (22).…”

“…FevR is required for replication of F. novicida and F. tularensis LVS in murine and human macrophages (20,22), in the human epithelial cell lines HEp-2 and A549 (22), and in mice (20). While the mechanism(s) of FevR's activity remains unclear, fevR mutants of F. tularensis LVS remain trapped within a phagosome that retains late endosomal marker LAMP-1 but not lysosomal marker cathepsin D (22). fevR mutants are also compromised in their ability to block NADPH oxidase activity following infection into neutrophils (22).…”