Genetics and Genetic Manipulation in <i>Francisella Tularensis</i>

Frank, Dara W.; Zahrt, Thomas C.

doi:10.1196/annals.1409.008

Cited by 24 publications

(24 citation statements)

References 65 publications

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“…Plasmid DNA was purified from E. coli using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA). The chemical transformation protocol used to introduce pSC-1423/1422K into F. novicida was based on a combination of previously described methods (Frank and Zahrt, 2007; Gallagher et al, 2008). An overnight culture of F. novicida grown on CDMA was suspended in 1 ml of phosphate buffered saline (PBS), pH 7.6.…”

Section: Methodsmentioning

confidence: 99%

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

et al. 2017

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Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. However, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locus in F. tularensis. Following daily subculture of F. novicida in Chamberlain's defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ1212–1218. The subcultured mutant F. novicida Δ1212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ1212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ1212–1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Therefore, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.

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Section: Methodsmentioning

confidence: 99%

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

et al. 2017

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“…This has led to a surge of studies of this human pathogen in an attempt to better understand the pathogenesis of the bacteria and to design novel approaches for diagnostics, prophylaxis, and treatment strategies. Such studies strongly depend on the availability of genetic tools that enable the examination of individual bacterial proteins in a variety of experimental approaches (e.g., directed disruption of genes and/or controlled expression of heterologous proteins), and the paucity of these tools severely limited F. tularensis research for many years (10). We therefore decided to search for, isolate, and characterize different F. tularensis promoters to increase the number of genetic tools that will allow the modulation of gene expression in the background of F. tularensis.…”

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confidence: 99%

Identification and Characterization of Novel and Potent Transcription Promoters of Francisella tularensis

Zaide

Grosfeld

Ehrlich

et al. 2011

Appl Environ Microbiol

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Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli 70 -related promoter motifs. These motifs include the ؊10 (TATAAT) and ؊35 [TTGA(C/T)A] domains and an AT-rich region upstream from ؊35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

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“…The unique presence of this barrier in F. novicida suggests that restriction may have been lost in the evolution of the more virulent lineages. The barrier is also of practical importance, for it has made genetic manipulations such as plasmid transformation and gene targeting in F. novicida challenging (7). In this study we have identified the principle determinants of restriction in F. novicida and have constructed a multiple mutant in which the restriction barrier is fully eliminated.…”

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Genetic Dissection of the Francisella novicida Restriction Barrier

et al. 2008

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Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10 6 -fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.Restriction-modification (R-M) systems in bacteria limit the acquisition of foreign genes that enter the cell by infecting phage, conjugal transfer systems, or transformation. Incoming DNA is cleaved (restricted) if it has not been modified by methylation at specific sequences (24,35). Almost all bacterial species carry R-M systems, with some species predicted to harbor dozens of R-M genes (11). There are four types of R-M systems (types I to IV), classified according to mechanism of action and the distribution of restriction, modification, and specificity functions among enzyme subunits (24,27).Francisella tularensis is an important human and zoonotic pathogen whose ecology and natural reservoirs are only partially understood. There are at least four subspecies which differ in their infectivity. Two of them, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, the causative agents of tularemia, are among the most infectious pathogens known and are classified as category A select agents due to their high infectivity, ease of dissemination, and severity of disease (5). Two others, "F. tularensis subsp. novicida" (Francisella novicida) and F. tularensis subsp. mediasiatica, do not generally cause disease in humans. The genomes of Francisella species are small (Ͻ2.0 Mbp), yet the bacteria are able to colonize hundreds of different animal and insect species, effectively evade host immune responses, and cause serious disease at e...

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Genetics and Genetic Manipulation in Francisella Tularensis

Cited by 24 publications

References 65 publications

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

Identification and Characterization of Novel and Potent Transcription Promoters of Francisella tularensis

Genetic Dissection of the Francisella novicida Restriction Barrier

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