Identification of an Attenuated Substrain of Francisella tularensis SCHU S4 by Phenotypic and Genotypic Analyses

Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia.

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Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

Journal of Bacteriology ◽

10.1128/jb.00630-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 8

Author(s):

Philip M. Ireland ◽

Helen L. Bullifent ◽

Nicola J. Senior ◽

Stephanie J. Southern ◽

Zheng Rong Yang ◽

...

Keyword(s):

Francisella Tularensis ◽

Insertion Site ◽

Therapeutic Drug ◽

Content Type ◽

Fischer 344 ◽

A Genome ◽

Fischer 344 Rat ◽

Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

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Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

PLoS ONE ◽

10.1371/journal.pone.0047639 ◽

2012 ◽

Vol 7 (10) ◽

pp. e47639 ◽

Cited By ~ 17

Author(s):

Aimee L. Signarovitz ◽

Heather J. Ray ◽

Jieh-Juen Yu ◽

M. N. Guentzel ◽

James P. Chambers ◽

...

Keyword(s):

Rat Model ◽

Mucosal Immunization ◽

Francisella Novicida ◽

Fischer 344 ◽

Fischer 344 Rat ◽

Schu S4

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Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

PLoS ONE ◽

10.1371/journal.pone.0009007 ◽

2010 ◽

Vol 5 (2) ◽

pp. e9007 ◽

Cited By ~ 8

Author(s):

Ufuk Nalbantoglu ◽

Khalid Sayood ◽

Michael P. Dempsey ◽

Peter C. Iwen ◽

Stephen C. Francesconi ◽

...

Keyword(s):

Clinical Isolate ◽

Francisella Tularensis ◽

Genomic Rearrangements ◽

Direct Repeats ◽

Tularensis Subsp ◽

Schu S4

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An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology Is Protective in an Inhalational Rat Model of Tularemia

Journal of Immunology Research ◽

10.1155/2018/8087916 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Laura E. Marshall ◽

Michelle Nelson ◽

Carwyn H. Davies ◽

Adam O. Whelan ◽

Dominic C. Jenner ◽

...

Keyword(s):

Rat Model ◽

Significant Protection ◽

Lethal Challenge ◽

Subunit Vaccines ◽

Fischer 344 ◽

O Antigen ◽

Fischer 344 Rat ◽

Glycoconjugate Vaccine ◽

Efficacious Vaccine ◽

Schu S4

There is a requirement for an efficacious vaccine to protect people against infection fromFrancisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) ofF. tularensisis suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugateF. tularensisO-antigen glycans to the immunogenic carrier proteinPseudomonas aeruginosaexoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain ofF. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge withF. tularensisSchu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

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The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis Subspecies tularensis Strain SCHU S4

American Journal Of Pathology ◽

10.1016/j.ajpath.2016.09.021 ◽

2017 ◽

Vol 187 (2) ◽

pp. 252-267 ◽

Cited By ~ 11

Author(s):

Julie A. Hutt ◽

Julie A. Lovchik ◽

Alexander Dekonenko ◽

Andrew C. Hahn ◽

Terry H. Wu

Keyword(s):

Natural History ◽

Francisella Tularensis ◽

Fischer 344 Rats ◽

Fischer 344 ◽

History Of ◽

Schu S4

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Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4

Microbiology ◽

10.1099/mic.0.000550 ◽

2017 ◽

Vol 163 (11) ◽

pp. 1664-1679 ◽

Cited By ~ 7

Author(s):

Amber L. Murch ◽

Paul J. Skipp ◽

Peter L. Roach ◽

Petra C. F. Oyston

Keyword(s):

Gene Expression ◽

Francisella Tularensis ◽

Stringent Response ◽

Pathogenicity Island ◽

Whole Genome ◽

Tularensis Subsp ◽

Schu S4 ◽

Highly Virulent

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Identification of an Essential Francisella tularensis subsp. tularensis Virulence Factor

Infection and Immunity ◽

10.1128/iai.01113-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 152-161 ◽

Cited By ~ 76

Author(s):

Aiping Qin ◽

David W. Scott ◽

Jennifer A. Thompson ◽

Barbara J. Mann

Keyword(s):

Francisella Tularensis ◽

Virulence Factor ◽

Etiologic Agent ◽

Intravenous Route ◽

Insertion Mutant ◽

Intranasal Route ◽

Tularensis Subsp ◽

Transposon Insertion ◽

Vaccine Potential ◽

Schu S4

ABSTRACT Francisella tularensis, the highly virulent etiologic agent of tularemia, is a low-dose intracellular pathogen that is able to escape from the phagosome and replicate in the cytosol. Although there has been progress in identifying loci involved in the pathogenicity of this organism, analysis of the genome sequence has revealed few obvious virulence factors. We previously reported isolation of an F. tularensis subsp. tularensis strain Schu S4 transposon insertion mutant with a mutation in a predicted hypothetical lipoprotein, FTT1103, that was deficient in intracellular replication in HepG2 cells. In this study, a mutant with a defined nonpolar deletion in FTT1103 was created, and its phenotype, virulence, and vaccine potential were characterized. A phagosomal integrity assay and lysosome-associated membrane protein 1 colocalization revealed that ΔFTT1103 mutant bacteria were defective in phagosomal escape. FTT1103 mutant bacteria were maximally attenuated in the mouse model; mice survived, without visible signs of illness, challenge by more than 1010 CFU when the intranasal route was used and challenge by 106 CFU when the intraperitoneal, subcutaneous, or intravenous route was used. The FTT1103 mutant bacteria exhibited dissemination defects. Mice that were infected by the intranasal route had low levels of bacteria in their livers and spleens, and these bacteria were cleared by 3 days postinfection. Mutant bacteria inoculated by the subcutaneous route failed to disseminate to the lungs. BALB/c or C57BL/6 mice that were intranasally vaccinated with 108 CFU of FTT1103 mutant bacteria were protected against subsequent challenge with wild-type strain Schu S4. These experiments identified the FTT1103 protein as an essential virulence factor and also demonstrated the feasibility of creating defined attenuated vaccines based on a type A strain.

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