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Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

  • Anna C. Llewellyn,

    Affiliations Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, United States of America, Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America

  • Crystal L. Jones ,

    Contributed equally to this work with: Crystal L. Jones, Brooke A. Napier

    Affiliations Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, United States of America, Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America

  • Brooke A. Napier ,

    Contributed equally to this work with: Crystal L. Jones, Brooke A. Napier

    Affiliations Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, United States of America, Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America

  • James E. Bina,

    Affiliation Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America

  • David S. Weiss

    david.weiss@emory.edu

    Affiliations Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America, Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, United States of America

Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

  • Anna C. Llewellyn, 
  • Crystal L. Jones, 
  • Brooke A. Napier, 
  • James E. Bina, 
  • David S. Weiss
PLOS
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Abstract

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.

Introduction

Francisella tularensis is a unique facultative intracellular pathogen that can cause a potentially lethal disease with an infectious dose as low as 10 bacteria [1]. A small Gram-negative coccobacillus, F. tularensis is the causative agent of tularemia, a vector- and water-borne zoonotic disease resulting in non-specific, flu-like symptoms that may culminate in pneumonic, glandular, and systemic infections [1]. When left untreated, pneumonic tularemia can result in a mortality rate as high as 60% [2]. F. tularensis subspecies are endemic across the Northern Hemisphere, with the majority of reported cases of disease in the United States, Europe, Russia, and Japan [3]. Due to its extreme infectivity, high morbidity and mortality rates, history of weaponization, and ease of aerosolization, dissemination, and genetic manipulation, F. tularensis is considered a category A potential bioweapon by the Centers for Disease Control and Prevention (CDC) [4][6].

The virulence mechanisms of F. tularensis subspecies and other Francisella species are still being characterized, including the role of the Francisella pathogenicity island (FPI) which is thought to encode a Type VI secretion system that facilitates the release of virulence proteins into host cells [7][13]. While F. tularensis and F. holarctica are responsible for the majority of disease burden in humans [5], many important virulence determinants are conserved among other Francisella species, including F. novicida. In addition to the FPI, these conserved virulence determinants include the presence of a non-inflammatory lipopolysaccharide (LPS), protective capsule, siderophores, and proteins involved in resistance to oxidative stress [14][21]. F. novicida, which has 98% nucleotide identity with the human pathogenic species, causes disease mainly in immunocompromised individuals but has also been shown to cause disease in healthy individuals [22][24]. The live vaccine strain (LVS) is an attenuated strain of the highly pathogenic species F. holarctica that was originally developed as a vaccine and retains 99.92% identity to its parental species [25], [26]. Though work involving F. tularensis and F. holarctica is restricted to Select Agent Biosafety Level 3 (BSL3) laboratories, both F. novicida and LVS are approved for use in BSL2 laboratories, are readily genetically manipulated, and cause tularemia-like disease in mice, making them both good laboratory models for studying Francisella pathogenesis [27], [28].

Francisella's primary replicative niche is thought to be the cytosolic compartment of both phagocytic and non-phagocytic cells such as macrophages, neutrophils, hepatocytes, alveolar epithelial cells, and fibroblasts [29][32]. After being engulfed by phagocytic host cells, the bacteria are taken up into phagosomes where they are confronted with a myriad of antimicrobial defenses including degradative enzymes, acidic pH, and oxidative stress [17][21], [33][36]. The reactive oxygen species (ROS) which cause oxidative stress can directly damage bacterial macromolecules such as proteins, DNA, and lipids. They can also react with these macromolecules to generate more ROS and toxic oxygen compounds, including the highly toxic organic hydroperoxides that result from the destructive lipid peroxidation of cell membranes [37][40]. Within host cells, ROS are generated by multiple mechanisms including the NADPH oxidase, myeloperoxidase, lipoxygenases, and cellular respiration [41], [42]. The NADPH oxidase, which produces superoxide radicals that lead to ROS formation, has been shown to be important for the host response to infection with Francisella species [43][46]. These bacteria employ numerous strategies to resist oxidative stress including limiting the activation of the NADPH oxidase [35], [36], [47] and using multiple enzyme systems to detoxify reactive oxygen compounds [18], [32], [48], [49].

While a general outline of Francisella's interaction with host cells is known, the specific mechanisms of cell entry, phagosomal escape, cytosolic replication, and some of the ways it evades immune defenses are still unknown. Genome-wide in vivo screens have identified genes required for the virulence of several Francisella species but do not shed light on how the majority of these genes contribute to pathogenesis [50][52]. As replication within host cells is a major part of Francisella's infectious cycle, we set out to determine which of the genes that are known to be required for virulence in vivo are also required for replication in host macrophages.

We performed an intracellular replication screen using transposon mutants representing 224 genes that have previously been shown to be required for virulence in vivo. Fifty-three of the genes tested were required for replication in macrophages including many of the FPI genes, validating the screen. We also identified biotin biosynthetic genes and the fsl/fig siderophore biosynthetic genes [16], [53][55] as well as numerous proteins of unknown function as being required for replication in macrophages. We validated the intracellular and in vivo requirement of two of these novel genes, FTN_1133 and FTN_0096. We then further investigated the importance of FTN_1133, which encodes a protein with sequence similarity to Ohr, a protein involved in oxidative stress resistance [56][64]. Accordingly, we find that FTN_1133 is required for resistance to, and degradation of, organic hydroperoxides. Furthermore, the replication defect of the FTN_1133 mutant is rescued in macrophages lacking a functional NADPH oxidase and partially rescued in mice with the same defect. We further demonstrate that F. holarctica LVS also requires this protein for organic hydroperoxide resistance and replication in macrophages and mice. Taken together, these data highlight the critical role that novel virulence factors play in Francisella pathogenesis and contribute to the elucidation of the requirements for this pathogen's largely uncharacterized intracellular lifecycle.

Materials and Methods

Ethics Statement

All experimental procedures were approved by the Emory University Institutional Animal Care and Use Committee (protocol #069-2008Y).

Bacterial strains and growth conditions

Wild-type F. novicida strain U112, a previously described mglA point mutant, GB2 [65], and the F. holarctica Live Vaccine Strain (LVS) were generous gifts from Dr. Denise Monack (Stanford University, Stanford, CA). F. novicida overnight cultures were grown at 37°C on a rolling drum in tryptic soy broth (TSB; Difco/BD, Sparks, MD) supplemented with 0.02% L-cysteine (Sigma-Aldrich, St. Louis, MO) while LVS cultures were grown in modified Mueller-Hinton broth (mMHB) supplemented with 1 mM CaCl2, 1 mM MgCl2, 0.1% glucose (Sigma-Aldrich), 2% Isovitalex (Difco/BD), and 0.025% ferric pyrophosphate as previously described [66]. Growth in minimal medium was determined using Chamberlain's chemically defined minimal medium, prepared as previously described [67]. For the replication screen, F. novicida was plated for enumeration on tryptic soy agar (TSA; Difco/BD) and supplemented with 0.01% L-cysteine. Bacteria from all other F. novicida experiments were plated on modified Mueller Hinton (mMH) (Difco/BD) plates supplemented with 0.025% ferric pyrophosphate (Sigma-Aldrich), 0.1% glucose, and 0.01% L-cysteine. LVS was plated on mMH supplemented additionally with 2% Isovitalex. When appropriate, kanamycin (Kan; Fisher Scientific, Fair Lawn, NJ) was added to media at a concentration of 30 µg/ml for F. novicida and 10 µg/ml for LVS.

Macrophages

RAW264.7 murine macrophages (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle medium (high glucose, L-glutamine; DMEM; Lonza, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClone, Logan, UT). Bone marrow-derived macrophages (BMM) were isolated from either wild-type C57BL/6 or gp91phox-/- mice (Jackson Laboratories, Bar Harbor, ME) and cultured as described previously [68] in DMEM supplemented with 10% heat-inactivated FCS and 10% macrophage colony-stimulating factor (M-CSF)-conditioned medium (collected from M-CSF-producing L929 cells). Macrophages were incubated before and during infection at 37°C with 5% CO2.

Intracellular replication screen and macrophage infections

The screen library was assembled by inoculation of individual transposon mutants from the F. novicida two-allele transposon library [69] into 96-well plates containing cysteine-supplemented TSB. These were grown overnight at 37°C, glycerol (Fisher Scientific) was added to 20% final volume, and the plates were stored at −80°C. RAW264.7 murine macrophages were seeded at 105 cells/well in 96-well tissue culture plates for the replication screen or 5×105cells/well in 24-well tissue culture plates for subsequent infections and incubated overnight. The medium was then removed and the macrophages were infected with overnight cultures of individual mutants from the screen library that had been diluted in DMEM/10% FCS to achieve a multiplicity of infection (MOI) of twenty bacteria per macrophage. The plates were centrifuged for 15 minutes at 931 x g at room temperature and then incubated for 30 minutes. Next, the macrophages were washed twice with DMEM and incubated for an additional 30 minutes in DMEM/10% FCS containing 100 µg/ml of gentamicin (TekNova, Hollister, CA). The macrophages were again washed twice and DMEM/10% FCS with 10 µg/ml gentamicin was added. At 1 and 24 hours post-infection, the macrophages were washed twice and then lysed with 1% saponin (Alfa Aesar, Heysham, Lancs., UK) in phosphate buffered solution (PBS) without calcium and magnesium (Lonza, Walkersville, MD). Serial dilutions of the resulting macrophage lysates were plated onto cysteine-supplemented TSA in sterile 24-well plates. Finally, the colony forming units (CFU) for each transposon mutant were counted and the fold replication (CFU at 24 hr/ CFU at 1 hr) was calculated and compared to the fold replication of wild-type F. novicida U112. A similar infection procedure was followed for both wild-type and gp91phox-/- BMM infections with the following modifications: 3×105 BMM were plated per well in a 24-well plate, DMEM/10% FCS/10% M-CSF was used throughout and the final time point was 5.5 hours for F. novicida infections or 24 hours for LVS infections. The replication screen was performed twice and the data were averaged to determine the final results. All transposon mutants that replicated less than or equal to 30% of the wild-type value (mutant fold replication/wild-type fold replication ≤0.3) were considered attenuated for replication in vitro. Results for all transposon mutants tested are listed in Table S1.

Mutagenesis and complementation

To generate F. novicida deletion mutants, PCR was used to amplify flanking DNA regions upstream and downstream of the gene of interest. A Kan-resistance cassette was sewn in between these flanking regions using overlapping PCR reactions. The final linear PCR products were then gel purified and transformed into chemically competent wild-type U112 as previously described [70]. The primers used to create the Kan-resistant deletion mutants contained FRT sites flanking the Kan-resistance cassette, which allowed removal of the cassette using the plasmid pFFlp encoding the Flp-recombinase as previously described [71]. Constructs for the complementation of each mutant were generated by overlapping PCR using PCR-amplified fragments of the wild-type gene of interest, upstream and downstream flanking regions, and a Kan-resistance cassette. These constructs were then transformed into the appropriate chemically competent deletion mutants. Verification of allelic replacement in mutant and complemented strains was performed using check primers in PCR reactions on purified genomic DNA from each strain. PCR products of the correct size were subsequently sequenced (MWG Operon, Huntsville, AL) for final verification of allelic replacement. For LVS mutagenesis, we employed both targeted gene disruption via group II introns as previously described [72] and allelic replacement. Briefly, in order to perform targeted gene disruption, primers for targeting the FTL_0803 allele were generated using the TargeTron system (Sigma-Aldrich) and the resulting PCR product was cloned into the Francisella targeting vector, pKEK1140 (generously provided by Dr. Karl Klose, UTSA, San Antonio, TX) [72]. LVS was then transformed with the resulting vector and FTL_0803 insertion mutants were selected for as previously described [72]. To generate LVS deletion mutants via allelic replacement, PCR was used to amplify flanking DNA regions upstream and downstream of the gene of interest which were then sewn together using overlapping PCR reactions. The final linear PCR products were then gel purified, digested with BamHI restriction enzyme (New England Biolabs, Ipswich, MA), dephosphorylated with Antarctic phosphatase (New England Biolabs), and ligated using T4 ligase (New England Biolabs) into the Francisella suicide vector pXB186 that encodes the sacB enzyme (James Bina, University of Tennessee, Memphis, TN). LVS was then transformed and mutants selected as previously described [73], [74]. Briefly, pXB186 ligations were transformed into LVS via electroporation and plated on chocolate agar with kanamycin (10 ug/ml). Next, kanamycin resistant colonies were plated on 10% sucrose, 1% hemoglobin cysteine heart agar (CHA) and surviving colonies were then patched onto kanamycin plates. Finally, genomic DNA from the kanamycin sensitive colonies was PCR-verified and sequenced to confirm the deletion. The FTL_0803 targeted disruption mutant was used in the RAW264.7 LVS macrophage experiment and the FTL_0803 clean deletion mutant was used in all other LVS experiments. Neither FTN_1133 nor FTL_0803 appear to be in an operon as the genes adjacent to both are transcribed in opposing directions. All primers used in this study are listed in Table S2.

Mouse experiments

Female C57BL/6 and gp91phox-/- mice (Jackson Laboratory, Bar Harbor, ME) between 7 and 10 weeks of age were kept under specific pathogen-free conditions in filter-top cages at Emory University and provided with sterile food and water ad libitum. Experimental studies were performed in accordance with the Emory University Institutional Animal Care and Use Committee guidelines. For competition experiments, mice were inoculated subcutaneously with a 1∶1 ratio of kanamycin-resistant deletion mutant and kanamycin-sensitive wild-type F. novicida for a total of 2×105 CFU in 50 µl sterile PBS. For single infections, mice were infected with 2×105 CFU subcutaneously. After 48 (F. novicida infections) or 72 hours (LVS infections), mice were sacrificed and the spleen, liver, and skin at the site of infection were harvested, homogenized (Tissue Tearor, Cole-Parmer, Vernon Hills, Illinois), plated for CFU on MH plates (with and without kanamycin for competition experiments), and then incubated overnight at 37°C. For single infections, organs were weighed before homogenization and the resulting CFU were divided by the weight of each organ to determine CFU/gram. For survival experiments, mice were infected as described for single strain infections and then observed for illness and sacrificed if they appeared moribund. For collection of RNA, mice were infected intraperitoneally with an infectious dose of 2×106 CFU, four hours after which the mice were sacrificed and the livers collected for RNA isolation. Competitive index (CI) values were determined using the formula: (CFU mutant output/CFU WT output)/(CFU mutant input/CFU WT input).

RNA isolation and quantitative real-time PCR

At various time points post-infection, BMM were lysed and homogenized in trizol reagent (MRC, Cincinnati, Ohio). Similarly, liver samples from intraperitoneally-infected mice (4 hours post-infection) were homogenized in trizol reagent. For both in vitro and in vivo samples, RNA was isolated using the RNeasy Mini kit (QIAGEN, Germantown, MD). Gene-specific primers (Table S2) were used to amplify FTN_1133 transcripts using the Power Sybr Green One Step Kit (Applied Biosystems, Foster City, CA) on an Applied Biosystems StepOnePlus Real Time PCR System per the manufacturers' instructions. Expression of FTN_1133 transcript was calculated relative to the expression of the DNA helicase uvrD (FTN_1594).

Susceptibility assays

Overnight cultures of F. novicida or LVS strains were diluted to an OD600 of 1.0 and 100 μl of each were spread on mMH agar plates. Six mm filter disks (Bel-Art Scienceware, Lake Charles, LA) were then added to the center of each plate and 3 μl of the appropriate dilution of chemical agent was spotted on the disks. The following concentrations of chemical agents were used: 250 mM (F. novicida) and 25 mM (LVS) tert-butyl hydroperoxide, 150 mM cumene hydroperoxide, 3% H2O2, and 200 mg/ml sodium dodecyl sulfate (SDS). Plates were then grown overnight at 37°C and the zones of inhibition measured.

Organic hydroperoxide degradation assay

Degradation of tert-butyl hydroperoxide was measured using a xylenol orange colorimetric assay based on previously described methods [59], [60], [75], [76]. Briefly, overnight cultures of F. novicida were subcultured to an OD600 of 0.01–0.03 and then incubated with shaking at 37°C. Once cultures reached mid-log phase (∼OD600 1.0), they were diluted to OD600 0.5 and a 2 ml sample of each culture or TSB alone were added to a 24 well plate. Tert-butyl hydroperoxide was then added to each sample to a final concentration of 300 µM, after which the plate was incubated with shaking at room temperature for 30 minutes. 20 µl samples were taken every five minutes from each well and immediately added to 80 µl 25 mM H2SO4. Once all samples were collected, 100 µl reaction buffer [200 µM xylenol orange (Alfa Aesar), 200 µM ammonium ferrous sulfate (Ricca Chemical, Arlington, TX), and 25 mM H2SO4 (Fisher Scientific) prepared in 9∶1 methanol to water solution] was added to each well and the OD540 measured. The concentration of tert-butyl hydroperoxide in each sample was calculated based on a standard curve.

Statistical analysis

All macrophage replication, susceptibility, and qRT-PCR data were analyzed for significance using the unpaired Student's t test. The CI values from the mouse experiments were analyzed with the one-sample Student's t test and compared to 1, with the exception of the CI values comparing replication in wild-type versus gp91phox-/- mice which were analyzed using the unpaired Student's t test. The single strain mouse infection data were analyzed for significance using the Mann-Whitney test.

Results

Intracellular replication screen

To further characterize the role in virulence of genes known to be required in animal infection models, we screened a library of corresponding transposon mutants for replication in RAW264.7 macrophages. We screened a total of 451 transposon mutants representing 224 genes and identified 53 of these genes to be required for F. novicida replication in RAW264.7 macrophages (Table 1). The screen results were validated by the identification of genes that have previously been reported to be required for intracellular replication, including genes encoded in the Francisella Pathogenicity Island (FPI) [8], [9], [11], [12], [77][80]. To the best of our knowledge, this study is the first to report the requirement of Francisella's biotin biosynthetic genes and the fsl/fig siderophore biosynthetic genes for replication in mammalian cells [16], [53][55]. A more in-depth review of the genes identified in this screen is included in the Discussion. Interestingly, a large proportion of the genes identified to be required for intracellular replication by this screen encode proteins of unknown function. Two such proteins were chosen for further study: FTN_0096 and FTN_1133. FTN_0096 was selected because of the severe intracellular replication defect of the FTN_0096 transposon mutant (Table 1). FTN_1133 was chosen because, although it is annotated as a hypothetical protein, we found that it has sequence similarity to Ohr, a protein involved in resistance to organic hydroperoxides which can induce oxidative stress, resistance to which is a critical virulence mechanism of Francisella species [17][21], [56][63].

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Table 1. List of genes required for replication in RAW264.7 macrophages.

https://doi.org/10.1371/journal.pone.0024201.t001

Validation of Screen Results

Deletion mutants for FTN_0096 and FTN_1133 were generated using allelic replacement as previously described [70]. The mutants exhibited wild-type replication kinetics when grown in both tryptic soy broth (TSB) supplemented with cysteine (Fig. S1A) and Chamberlain's chemically defined minimal medium (Fig. S1B). To validate the phenotypes of the corresponding transposon mutants in our screen, the replication phenotype of each deletion mutant was determined in RAW264.7 macrophages. Twenty-four hours post-infection, the FTN_0096 mutant was severely attenuated for replication compared to wild-type F. novicida, with a fold replication value similar to that of the replication-deficient control strain, GB2, which we will refer to as mglA (Fig. 1A). This strain harbors a point mutation in mglA, a gene known to be essential for intracellular replication [65]. The FTN_1133 mutant displayed an approximate six-fold replication deficiency compared to wild-type (Fig. 1C). In order to ensure that the observed phenotypes resulted from deletion of the targeted gene and not unintended secondary site mutations, we generated complemented strains of each mutant in which the deleted gene was replaced. All of the complemented strains displayed wild-type levels of replication (Fig. 1A, C). These data further validate our screen and demonstrate the requirement of FTN_0096 and FTN_1133 for F. novicida replication in RAW264.7 macrophages.

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Figure 1. Validation of replication screen results.

RAW264.7 macrophages (A and C) or primary murine bone marrow-derived macrophages (BMM) (B and D) were infected with a 20∶1 MOI of the indicated bacterial strains. Twenty-four (A and C) or 5.5 (B and D) hours post-infection, fold replication was determined for the FTN_0096 (A and B) and FTN_1133 (C and D) deletion mutants (Δ96 and Δ1133) and their respective complemented strains. Wild-type F. novicida and an mglA point mutant strain (GB2) were used as positive and negative replication controls, respectively. For each strain, bars represent the average fold replication and error bars represent the standard deviation (n = 3). Data shown is representative of at least three independent experiments. Asterisks indicate significance as compared to wild-type. (**) p<0.005, (***) p<0.0005.

https://doi.org/10.1371/journal.pone.0024201.g001

To verify that the replication defects of these F. novicida mutants were not specific to RAW264.7 cells, we next infected primary murine bone marrow-derived macrophages (BMM) and measured replication levels. Compared to the wild-type strain, the FTN_0096 mutant again displayed a severe replication deficiency similar to the mglA mutant, and the FTN_1133 mutant had an approximate three-fold replication defect (Fig. 1B, D). Replication was restored to the wild-type level in the complemented strains (Fig. 1B, D). We measured bacterial replication at 5.5 hrs post-infection, before any macrophage cell death occurred, to ensure that the attenuated phenotypes of the mutants were not a consequence of the cell death response. RAW264.7 macrophages are known to be deficient in ASC/caspase-1 inflammasome-mediated cell death [81], an inflammatory host cell death pathway known to be triggered by F. novicida infection [82], explaining why we could measure bacterial replication at later time points in these cells. Taken together, these results demonstrate that in both of the macrophage cell types tested, FTN_0096 and FTN_1133 play a role in replication and that the deletion mutants lacking these genes displayed intracellular replication deficiencies similar to those predicted by our screen.

Deletion mutants are attenuated in vivo

To test whether the macrophage replication defects correlated with in vivo attenuation levels, competition experiments were performed in mice. Briefly, mice were infected with a 1∶1 ratio of wild-type F. novicida and each mutant strain. Forty-eight hours post-infection, mouse organs were harvested, homogenized, and plated for enumeration of wild-type and mutant CFU. The number of FTN_0096 deletion mutant CFU in the spleen and liver was one log below that of wild-type (Fig. 2B, C), although no attenuation was observed in the skin (Fig. 2A). The FTN_1133 mutant had a one log attenuation in the skin, roughly two log attenuation in the spleen, and nearly three log attenuation in the liver (Fig. 2). Both mutant phenotypes were restored to wild-type levels in the complemented strains (Fig. 2). Taken together, these results demonstrate that FTN_0096 and FTN_1133 are involved in F. novicida pathogenesis in vivo, though FTN_1133 appears to have a more significant role in virulence in mice.

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Figure 2. FTN_0096 and FTN_1133 deletion mutants are attenuated for virulence in vivo.

Mice were subcutaneously infected with a 1∶1 mixture of wild-type F. novicida and either Δ96 or Δ1133 (105 CFU each) or respective complemented strains. Forty-eight hours after infection, organs were harvested, CFU enumerated, and the competitive index (CI) calculated for the skin at the site of infection (A), spleen (B), and liver (C). CI  =  (CFU mutant output/CFU WT output)/(CFU mutant input/CFU WT input). Bars represent the geometric mean CI values from each group of mice (n = 5). CI values below 1 (dashed line) indicate attenuation of the mutant strain. Data shown is representative of two independent experiments. Asterisks indicate significance as compared to a CI value of 1. (***) p<0.0005.

https://doi.org/10.1371/journal.pone.0024201.g002

FTN_1133 is required for virulence in single infections

Since the FTN_1133 mutant was the most severely attenuated in vivo, we chose this gene for further characterization. To ensure that the attenuation of this mutant was not only observed when in competition with wild-type bacteria, we performed single infection experiments. We observed that the FTN_1133 mutant was attenuated in each organ to a similar degree as in competition experiments (Fig. 3A–C), confirming that FTN_1133 is required for full virulence of F. novicida. We further verified this result by monitoring the survival of these mice. By day 10 post-infection, only 40% of mice infected with wild-type bacteria survived while 100% of those infected with the FTN_1133 deletion mutant survived (Fig. 3D).

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Figure 3. FTN_1133 is required for F. novicida pathogenesis in single infections.

Mice were subcutaneously infected with 2×105 CFU of either wild-type F. novicida (WT) or the FTN_1133 mutant (Δ1133). Forty-eight hours after infection, organs were harvested and plated and CFU were enumerated 24 hours later for the skin at the site of infection (A), spleen (B), and liver (C). To test survival, mice were infected as described above and then sacrificed upon display of moribundity (D). Bars represent the geometric mean of each group of mice (n = 5). Data shown is representative of two independent experiments. Asterisks indicate significance. (**) p<0.005, (*)  =  P<0.05.

https://doi.org/10.1371/journal.pone.0024201.g003

FTN_1133 is expressed during infection of macrophages and mice

Given its importance in F. novicida replication and survival in macrophages and mice, we tested whether FTN_1133 was transcribed during infection. Levels of FTN_1133 transcript from samples of wild-type F. novicida-infected BMM at 30 minutes, 2 hours, and 4 hours post-infection were determined using quantitative real-time RT-PCR (qRT-PCR) (Fig. 4A). The expression of FTN_1133 was induced during macrophage infection, as indicated by a moderate but significant increase in expression between 30 minutes and 4 hours (Fig. 4A). We also observed a similar level of expression in livers of infected mice 4 hours after intraperitoneal infection (Fig. 4B). Taken together and consistent with its role in pathogenesis, these results show that FTN_1133 is expressed during infection of both macrophages and mice.

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Figure 4. FTN_1133 is expressed during infection of macrophages and mice.

Relative expression of FTN_1133 was determined in wild-type F. novicida-infected BMM (A) at 30 minutes, 1 hour, and 4 hours post-infection with an MOI of 20∶1 and (B) in the livers of intraperitoneally-infected mice at 4 hours. Relative expression of FTN_1133 transcript was calculated by comparison with the expression levels of the DNA helicase uvrD (FTN_1594). Data shown is representative of two independent experiments. Bars represent the standard deviation from each set of samples (n = 5). Asterisk indicates significance compared to 30 minutes. (*) p<0.05.

https://doi.org/10.1371/journal.pone.0024201.g004

FTN_1133 is involved in resistance to organic hydroperoxides

Bioinformatic analysis revealed that FTN_1133 has sequence similarity to an organic hydroperoxide resistance protein (Ohr) from Bacillus megaterium. Specifically, the C-terminal half of Ohr has significant similarity to residues 18–86 of FTN_1133 (Fig. S2). Based on these findings, we tested whether FTN_1133 is involved in resistance to organic hydroperoxides in F. novicida. We quantified bacterial sensitivity by measuring zones of inhibition upon exposure to organic hydroperoxides via the disk diffusion method. The FTN_1133 mutant showed increased sensitivity to cumene and tert-butyl hydroperoxides compared to the wild-type and complemented strains (Fig. 5A, B). Since Ohr is often required for resistance to organic hydroperoxides but not inorganic hydroperoxides [59], [60], [62], [63], [83], we grew the bacterial strains in the presence of inorganic hydrogen peroxide and found that the wild-type, mutant, and complemented strains showed equal levels of sensitivity, indicating that FTN_1133 is indeed not required for resistance to an inorganic hydroperoxide (Fig. 5C). Finally, to further demonstrate the specificity of FTN_1133 for organic hydroperoxides and rule out a general sensitivity to stresses, we found that the wild-type, mutant and complemented strains were equally susceptible to SDS, a membrane-damaging detergent (Fig. 5D). Taken together, these data demonstrate that FTN_1133, similar to most Ohr proteins, is required for resistance to organic but not inorganic hydroperoxides.

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Figure 5. FTN_1133 confers resistance to organic hydroperoxides.

Two hundred and fifty mM tert-butyl hydroperoxide (A), 150 mM cumene hydroperoxide (B), 3% hydrogen peroxide (C), and 200 mg/ml SDS (D) were spotted on filter disks placed on lawns of wild-type, FTN_1133 mutant (Δ1133), and the complemented strain and the zones of inhibition for each were measured. In each graph, bars represent the mean and error bars represent the standard deviation (n = 3). Data shown is representative of at least three independent experiments. Asterisks indicate significance as compared to wild-type. (***) p<0.0005.

https://doi.org/10.1371/journal.pone.0024201.g005

FTN_1133 is required for degradation of an organic hydroperoxide

To determine if FTN_1133 is important not only for resistance to organic hydroperoxides, but also detoxification of these chemicals, we added tert-butyl hydroperoxide to cultures of wild-type F. novicida or the FTN_1133 deletion mutant and measured its concentration over time. Between 15 minutes and 30 minutes, the FTN_1133 deletion mutant degraded approximately 50% less tert-butyl hydroperoxide than wild-type bacteria (Fig. 6). These data indicate that FTN_1133 is involved in degradation of an organic hydroperoxide.

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Figure 6. FTN_1133 is required for degradation of an organic hydroperoxide.

Three hundred μM tert-butyl hydroperoxide was added to cultures of wild-type F. novicida (circles), the FTN_1133 deletion mutant (Δ1133, squares), or TSB media alone (triangles) and its degradation was measured over time. Data points represent the mean and error bars represent the standard deviation (n = 3). Data shown is representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0024201.g006

FTN_1133 is required for resistance to the action of the NADPH oxidase

To test if the sensitivity of the FTN_1133 mutant to oxidative stress was the cause of its replication defect in macrophages, we infected BMM from both wild-type and gp91phox-/- mice with wild-type F. novicida and the FTN_1133 deletion mutant. gp91 is a subunit of the NADPH oxidase and is required for the generation of reactive oxygen species and the oxidative stress induced by this enzyme. While the FTN_1133 mutant was attenuated for replication in wild-type BMM (Fig. 7A, and similar to Fig. 1), it replicated to the same levels as wild-type F. novicida in the gp91phox-/- BMM (Fig. 7B). In order to determine whether a similar phenotype is observed in vivo, we infected wild-type and gp91phox-/- mice with either wild-type F. novicida or the FTN_1133 deletion mutant. Indeed, the two log attenuation of the FTN_1133 deletion mutant in wild-type mice was rescued by one log in gp91phox-/- mice (Fig. 7C). Together, these data indicate that FTN_1133 is required to resist the oxidative stress generated by the NADPH oxidase and makes an important contribution to F. novicida's intracellular and in vivo pathogenesis.

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Figure 7. FTN_1133 is required for resistance to the action of the NADPH oxidase.

Wild-type (A) or gp91phox-/- (B) bone marrow-derived macrophages were infected with a 20∶1 MOI of the indicated bacterial strains. (A, B) Five and a half hours post-infection, fold replication (CFU at 5.5 hr/ CFU at 30 minutes) was determined for wild-type F. novicida and the FTN_1133 deletion mutant (Δ1133). For each strain, bars represent the average fold replication and error bars represent the standard deviation (n = 3). Data shown are representative of three independent experiments. (C) Wild-type and gp91phox-/- mice were subcutaneously infected with a 1∶1 mixture of wild-type F. novicida and Δ1133 (105 CFU each). Forty-eight hours after infection, organs were harvested, CFU enumerated, and the competitive index (CI) calculated. Data shown include two independent experiments. Asterisks indicate significance as compared to wild-type F. novicida. (*) p<0.05, (***) p<0.0005.

https://doi.org/10.1371/journal.pone.0024201.g007

The FTN_1133 ortholog, FTL_0803, confers resistance to an organic hydroperoxide

In order to determine whether FTN_1133 is also involved in organic hydroperoxide resistance in other Francisella species, we constructed an F. holarctica LVS deletion mutant lacking the FTN_1133 ortholog, FTL_0803. FTN_1133 and FTL_0803 share 98% amino acid identity. To test the requirement of FTL_0803 for LVS resistance to organic hydroperoxides, we performed disk diffusion assays on wild-type LVS and the FTL_0803 deletion mutant. As with FTN_1133, we found that FTL_0803 is required for wild-type resistance to the organic hydroperoxide tert-butyl hydroperoxide (Fig. 8A), but not H2O2 (Fig. 8B) or SDS (Fig. 8C).

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Figure 8. The FTN_1133 ortholog, FTL_0803, confers resistance to tert-butyl hydroperoxide.

Twenty-five mM tert-butyl hydroperoxide (A), 3% hydrogen peroxide (B), and 200 mg/ml SDS (C) were spotted on filter disks placed on lawns of wild-type LVS or the FTL_0803 mutant (Δ803), incubated overnight, and then the zones of inhibition for each were measured. In each experiment, bars represent the mean and error bars represent the standard deviation (n = 3). Data shown is representative of at least three independent experiments. Asterisks indicate significance as compared to wild-type. (**) p<0.005.

https://doi.org/10.1371/journal.pone.0024201.g008

FTL_0803 is required for F. holarctica LVS virulence and resistance to the action of the NADPH oxidase

Next we investigated the importance of FTL_0803 in LVS pathogenesis by determining the replication phenotype of the deletion mutant both in macrophages and mice. First, RAW264.7 macrophages were infected with wild-type LVS and the FTL_0803 mutant, and bacterial levels were measured at 24 hours post-infection. Similar to the phenotype of the FTN_1133 mutant, the FTL_0803 mutant exhibited a five-fold intracellular replication defect (Fig. 9A). Mouse infection experiments revealed that the FTL_0803 deletion mutant was present at one log lower levels than LVS in the organs tested (Fig. 9B–D). Finally, to determine if the replication deficiency of this mutant in macrophages is also rescued in the absence of a functional NADPH oxidase, we infected both wild-type BMM and gp91phox-/- BMM with either LVS or the FTL_0803 deletion mutant. The five-fold replication deficiency of the mutant in wild-type BMM was largely rescued in gp91phox-/- BMM (Fig. 10). These data demonstrate that the importance of FTN_1133/FTL_0803 in resisting oxidative stress, promoting intracellular replication, and contributing to in vivo virulence is conserved in multiple Francisella species.

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Figure 9. FTL_0803 is important for LVS replication in macrophages and mice.

(A) RAW264.7 macrophages were infected with a 20∶1 MOI of wild-type LVS and the FTL_0803 mutant (Δ803). Twenty-four hours post-infection, intracellular CFUs were determined for both strains. Bars represent the mean and error bars represent the standard deviation (n = 3). (B–D) Mice were subcutaneously infected with 2×105 CFU of either wild-type F. holarctica LVS (WT) or the FTL_0803 mutant (Δ803). Seventy-two hours after infection, organs were harvested, plated, and CFU were enumerated 48 hours later for the skin at the site of infection (B), spleen (C), and liver (D). Bars represent the geometric mean from each group of mice (n = 5). Data shown is representative of two independent experiments. Asterisks indicate significance as compared to wild-type. (*) p<0.05, (**) p<0.005.

https://doi.org/10.1371/journal.pone.0024201.g009

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Figure 10. FTL_0803 contributes to resistance against the action of the NADPH oxidase.

Bone marrow-derived macrophages (BMM) were infected with a 20∶1 MOI of wild-type LVS or the FTL_0803 mutant (Δ803). Twenty-four hours post-infection, fold replication was determined for both strains. Bars represent the mean and error bars represent the standard deviation (n = 3). Data shown is representative of two independent experiments. Asterisks indicate significance as compared to wild-type. (**) p<0.005.

https://doi.org/10.1371/journal.pone.0024201.g010

Discussion

Recently, in vivo screens have identified many genes required for Francisella virulence, though the functions of these genes and an understanding of the stage of infection at which they contribute to virulence are largely unknown [50], [51]. In an effort to further characterize these virulence determinants and to begin to answer questions that remain regarding Francisella's intracellular lifecycle, we performed an intracellular replication screen using transposon insertion mutants of genes that were identified in the two mammalian in vivo genome-wide screens that were published at the time of this study: one using F. novicida transposon mutants [50] and one using transposon mutants of the Francisella live vaccine strain (LVS) [51]. Recently, Kraemer et al. published another mammalian in vivo genome-wide screen, the results of which were published after our screen was performed and therefore genes specifically identified in this study were not included [52].

Of the 224 genes screened, 53 were shown to be required for replication in murine macrophages. The 171 genes that were not identified in this screen may be required for replication in other cell types known to be infected by Francisella, such as neutrophils, hepatocytes, alveolar epithelial cells, or fibroblasts [29][32], [84], [85]. Alternatively, these genes may be required for processes other than intracellular replication such as immune evasion or systemic dissemination. To the best of our knowledge, the intracellular replication data from mutants for 140 of the 224 genes that were represented in this screen have not been previously reported in mammalian cells. One gene, feoB (FTN_0066), was included in the screen but was excluded from the results because of a growth defect on cysteine-enriched tryptic soy agar (TSA), despite no apparent growth defect in cysteine-enriched tryptic soy broth (TSB) (data not shown).

The results of our screen were validated by the identification of genes that have previously been shown to be required for replication in macrophages, including most of the Francisella pathogenicity island (FPI) genes [8], [9], [11], [12], [77][80], [86]. Of the 18 genes in the F. novicida FPI, mutants representing 13 genes were identified as attenuated for replication in macrophages. Among the FPI genes not identified in this screen (pdpA, pdpC, pdpD, pdpE, and anmK), pdpD has previously been reported to be unnecessary for replication in mouse macrophages [87]. Both pdpC and pdpD were also shown to be unnecessary for replication in an arthropod cell line [88] and pdpC, pdpD, pdpE, and anmK were all reported to have no role in virulence in a live arthropod model [78]. While pdpA has been shown to be required for intracellular replication within mammalian cells [10], [78], [86], it was not identified in this screen. One of the three pdpA transposon mutants we tested displayed a deficiency in intracellular replication, but it was just below the cut-off value used in this screen (Table S1). This may be due to retention of some protein function in the pdpA mutant strains used in this study, a potential problem when using transposon mutants as compared to deletion mutants.

In addition to the identification of FPI genes, this screen was further validated by the identification of other genes that have previously been shown to be required for intracellular replication, including pyrB [29], [32], [78], carA [29], [32], [78], carB [29], [32], [78], [89], purM [90] and clpB [12], [78], [91], [92]. Genes that have previously been shown to play no role in intracellular replication and were also shown to be unnecessary for replication in our screen include FTN_0757 and FTN_0720, verifying the selectivity of our screen [50]. Though an htpG deletion mutant has previously been reported to be attenuated for replication in BMM macrophages [50], and one of the two transposon mutants with insertions in this gene displayed an approximate two-fold replication attenuation in this screen, the level of attenuation was not sufficient for our cut-off.

Our screen led to numerous novel insights into Francisella's genetic requirements for replication within host cells. To the best of our knowledge, this is the first study to implicate a requirement of biotin for Francisella's replication in mammalian cells since each of the biotin synthetic genes, bioA, bioB, bioC, bioD, and bioF (FTN_0812-FTN_0816) was identified. Biotin is critical for various metabolic pathways and biotin biosynthetic genes are required for Mycobacterium tuberculosis intracellular replication and pathogenesis [93][95]. bioF was also recently reported to be important for Francisella replication in an arthropod cell line [96]. In addition, we report here for the first time Francisella's requirement for replication in mammalian cells of at least three of the six siderophore biosynthetic genes: fslA, fslB, and fslC, also known as figA, figB, and figC (FTN_1682-FTN_1684), confirming that this iron acquisition system is essential for efficient replication within mammalian cells. Pathogens generally encounter iron-limiting conditions within the host, and thus iron acquisition proteins are critical virulence factors in numerous pathogens [97], [98]. fslA and fslB were also recently reported to be important for replication in an arthropod cell line [96]. The other three genes from this group (FTN_1685-FTN_1687) were not included in our screen.

Of the 53 genes identified to be required for replication in this screen, 19 are annotated as hypothetical, indicating that F. novicida encodes novel genes that are required both for virulence in vivo and intracellular replication. To study the role of some of these hypothetical proteins during infection as well as further validate the screen results, we chose two genes to study: FTN_0096 and FTN_1133. FTN_0096 is a member of the DUF1275 superfamily of proteins, which has members in other human pathogens such as Vibrio spp, Acinetobacter spp, Neisseria spp, and Burkholderia spp. Analysis with the Simple Modular Architecture Research Tool (SMART) identified 7 potential transmembrane domains as well as a putative signal peptide, indicating that FTN_0096 is likely a membrane protein (http://smart.embl-heidelberg.de/). Interestingly, bioinformatic analyses revealed that FTN_1133 was found only in Francisella species but had significant similarity to the C-terminal domain of an organic hydroperoxide resistance protein, Ohr, from Bacillus megaterium.

We show that both FTN_0096 and FTN_1133 are required for replication in macrophages and virulence in mice. FTN_0096 was absolutely required for replication in both RAW264.7 macrophages and BMM (Fig. 1A, B). In addition, FTN_0096 was recently reported to be important for replication in an arthropod in vivo model as well as murine macrophage-like J774 cells [78]. The FTN_0096 deletion mutant displayed only a moderate one log attenuation in the spleen and liver of mice, despite having a severe intracellular replication defect (Fig. 2). The disparity in this mutant's in vivo versus in vitro phenotypes could be due to an ability of bacteria lacking FTN_0096 to replicate efficiently in other cells types, particularly non-immune cells that have fewer defenses against intracellular infection. Indeed, Horzempa et al. recently showed that a uracil synthesis mutant unable to replicate in macrophages displayed a virulent phenotype in mice attributable to that mutant's ability to replicate in non-phagocytic host cells [99]. Conversely, the FTN_1133 mutant was moderately attenuated for intracellular replication (3-6 fold) in macrophages (Fig. 1), displayed a marked two to three log attenuation in the spleen and liver following mouse infections (Fig. 2, 3), and did not cause lethal infection in mice (Fig. 3D). Furthermore, we showed that FTN_1133 was expressed during infection of both macrophages and mice (Fig. 4).

Bioinformatic analyses revealed that FTN_1133, a 127 amino acid protein, has significant similarity to the C-terminal domain of an organic hydroperoxide resistance protein (Ohr) from Bacillus megaterium (Fig. S2). The C-terminus of Ohr proteins has been shown to be important for enzymatic function in bacteria such as Xylella fastidiosa and Pseudomonas aeruginosa [83], [100][102]. Originally identified in Xanthomonas campestris, Ohr is a 139 amino acid protein which has homologs in several bacterial species including Bacillus subtilis, Pseudomonas aeruginosa, and Acinetobacter baumannii [56], [57], [59], [61]. Ohr is thought to be a hydroperoxide reductase that converts organic hydroperoxides into less toxic metabolites [101]. This protein contributes to resistance to reactive oxygen species (ROS)-induced damage by degrading the highly toxic organic hydroperoxides that are created during lipid peroxidation when oxygen radicals react with the unsaturated and polyunsaturated lipids of cell membranes [37][40], [100].

Interestingly, Ohr proteins are usually only involved in resistance to organic hydroperoxides, such as tert-butyl hydroperoxide and cumene hydroperoxide, but not inorganic hydroperoxides, such as hydrogen peroxide [59], [60], [62], [63], [83]. Our disk diffusion analysis indicates that FTN_1133, like many Ohr proteins, is required for resistance to organic hydroperoxides but not hydrogen peroxide (Fig. 5). Also similar to Ohr, we demonstrate that FTN_1133 is required for degradation of tert-butyl hydroperoxide (Fig. 6) [59], [60], [75], [76]. The FTN_1133 deletion mutant displays a moderate amount of degradation compared to the wild-type strain, which indicates that Francisella may encode another system involved in degradation of these chemicals. Potential candidates for this function include Francisella's uncharacterized AhpC homologs. Most proteins involved in oxidative stress resistance specifically detoxify inorganic oxygen species, such as oxide radicals (superoxide dismutases) or hydrogen peroxide (catalase). However, AhpC has been shown to be important for resistance to both inorganic and organic hydroperoxides in other bacterial pathogens such as P. aeruginosa, Brucella abortus, and Salmonella typhimurium [103][105]. Consistent with a role in virulence, Kadzhaev et al. demonstrated that an F. tularensis ahpC transposon mutant showed a marked increase in time to death in a low dose challenge in mice [106].

While expression of ohr genes is often increased in response to organic hydroperoxides, several ohr genes have been identified that are not induced in this manner [57], [62], [64]. Quantitative real-time PCR analysis of FTN_1133 expression revealed that while the gene is induced during infection (Fig. 4), this gene was not significantly induced in broth in our hands in response to organic hydroperoxides (data not shown).

To the best of our knowledge, ohr mutants have not previously been characterized in host cells or animal models, though ohr has been shown to be co-expressed with other virulence factors during Actinobacillus pleuropneumoniae infection of pigs [60]. Further suggestive of a role in pathogenesis, ohr is sometimes encoded on mobile genetic elements, such as a genomic island in Actinobacillus pleuropneumoniae [107] and a plasmid in pathogenic Acinetobacter baumannii [61]. Our in vitro screen and previous in vivo screens [51], [52] identified FTN_1133 as important for virulence. We hypothesized that increased sensitivity of the FTN_1133 mutant to oxidative stress may explain its intracellular and in vivo replication defect. Indeed, we observed restoration of wild-type levels of intracellular replication for the FTN_1133 deletion mutant in BMM from mice lacking gp91phox-/- (Fig. 7B), an essential subunit of NADPH oxidase that is required for the generation of superoxide radicals by this enzyme complex. In addition, the in vivo attenuation of the FTN_1133 mutant was significantly rescued in gp91phox-/- mice (Fig. 7C). Though the mutant was not fully complemented in these mice as it was in the gp91phox-/- BMM, this is not surprising since there are alternate ROS generating pathways that do not exist in macrophages but are present during in vivo infection. For example, myeloperoxidase is present in neutrophils but not macrophages and can initiate lipid peroxidation and organic hydroperoxide generation [41]. Furthermore, we demonstrate that the importance of FTN_1133 in resistance to oxidative stress is conserved in multiple Francisella species by showing the requirement of FTL_0803, the F. holarctica LVS FTN_1133 ortholog, for resistance to organic hydroperoxides (Fig. 8) and wild-type replication both in macrophages and mice ( Fig. 9). Finally, we show that the in vitro attenuation of the FTL_0803 mutant was largely rescued in macrophages deficient in oxidative burst (Fig. 10). The molecular bases for the attenuation of the LVS strain have yet to be fully characterized and as such, there may be underlying deficiencies that explain the incomplete complementation of the FTL_0803 mutant strain in gp91phox-/- BMM [26].

Our data demonstrating a role for FTN_1133 in resistance to oxidative stress and Francisella virulence are consistent with the fact that many oxidative stress resistance genes are transcribed during Francisella infection of macrophages [108] and many of these have been identified in screens as being required for replication in macrophages [78], [89] and virulence in arthropods [20], [78], [96]. Francisella species have been shown to use an array of genes to suppress activation of the NADPH oxidase [32], [35], [36], [45], [109]. Francisella species also use numerous genes to detoxify reactive oxygen compounds and thereby resist oxidative stress. Specifically, the catalase KatG [48], [49], [51], [89] and the superoxide dismutases SodB and SodC [18], [49], [110] have all been shown to be essential for survival of Francisella species in vivo. Also, a novel oxidative stress resistance gene, MoxR, was recently described in LVS [21]. The data presented here identify FTN_1133 as a novel Francisella oxidative stress resistance protein, specific for stress induced by organic hydroperoxides.

In this report, we demonstrate for the first time the importance of an Ohr-like protein in virulence during in vitro and in vivo infections as well as its specific role in resistance to oxidative stress both in macrophages and in mice. Furthermore, we show that the importance of this protein for pathogenesis is conserved in multiple Francisella species. Taken together, the results of this screen highlight the requirement of numerous F. novicida virulence determinants for intracellular replication. The critical importance of resisting oxidative stress suggests that Ohr-like proteins, including FTN_1133, may represent attractive drug targets [111]. In this way, continued characterization of FTN_1133 and other novel proteins and mechanisms used by Francisella could contribute to the development of new therapeutics and vaccines against this potential bio-threat.

Supporting Information

Figure S1.

Selected deletion mutants of genes identified in the replication screen display wild-type growth in rich media and in defined minimal media. Bacterial growth at 37°C in (A) cysteine-enriched tryptic soy broth and (B) Chamberlain's minimal defined media is shown for wild-type F. novicida (circles), FTN_0096 (Δ96, squares), and FTN_1133 (Δ1133 triangles). Data shown is representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0024201.s001

(TIF)

Figure S2.

FTN_1133 has similarity to the organic hydroperoxide resistance protein Ohr. The sequences for F. novicida FTN_1133 (a.a. 18 – 86) and Bacillus megaterium Ohr (a.a. 71 – 139) were aligned using CLUSTALW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Identical residues are highlighted in red and similar residues are highlighted in blue. The sequences have 28.6% identity and 42.9% similarity.

https://doi.org/10.1371/journal.pone.0024201.s002

(TIF)

Table S1.

Full list of transposon mutant replication phenotypes in RAW264.7 macrophages. Strains highlighted in bold were attenuated for intracellular replication. Each transposon mutant was screened 2–3 times and each time the fold replication of the mutant was compared to the fold replication of wild-type (fold Mut/ fold WT). AVG Mut/WT is the average of these ratios from all experiments for each mutant. Strain names, plate number, and well location are as annotated in the two-allele transposon mutant library from Gallagher, et al.

https://doi.org/10.1371/journal.pone.0024201.s003

(DOC)

Acknowledgments

We thank Larry Gallagher and Colin Manoil (University of Washington, Seattle) for generously providing the Tn5-based transposon library and pFFlp plasmid and Karl Klose and Stephen A. Rodriguez for the pKEK1140 targeting vector and cloning advice. We also thank William Shafer, Jyothi Rengarajan, and Timothy Sampson for critical reading of this manuscript.

We have, to the best of our knowledge, included all mammalian in vitro and in vivo references for the genes listed in Table 1. However, we apologize in advance for any omissions that may have occurred.

Author Contributions

Conceived and designed the experiments: ACL DSW. Performed the experiments: ACL CLJ BAN DSW. Analyzed the data: ACL DSW. Contributed reagents/materials/analysis tools: ACL CLJ BAN JEB DSW. Wrote the paper: ACL DSW.

References

  1. 1. Oyston PC (2008) Francisella tularensis: unravelling the secrets of an intracellular pathogen. J Med Microbiol 57: 921–930.PC Oyston2008Francisella tularensis: unravelling the secrets of an intracellular pathogen.J Med Microbiol57921930
  2. 2. Saslow S, Eigelsbach H, Wilson H, Prior J, Carhart S (1961) Tularemia vaccine study, II: respiratory challenge. Arch Intern Med 107: 702–714.S. SaslowH. EigelsbachH. WilsonJ. PriorS. Carhart1961Tularemia vaccine study, II: respiratory challenge.Arch Intern Med107702714
  3. 3. Keim P, Johansson A, Wagner DM (2007) Molecular epidemiology, evolution, and ecology of Francisella. Ann N Y Acad Sci 1105: 30–66.P. KeimA. JohanssonDM Wagner2007Molecular epidemiology, evolution, and ecology of Francisella.Ann N Y Acad Sci11053066
  4. 4. Nigrovic LE, Wingerter SL (2008) Tularemia. Infect Dis Clin North Am 22: 489–504, ix.LE NigrovicSL Wingerter2008Tularemia.Infect Dis Clin North Am 22489504, ix
  5. 5. Sjostedt A (2007) Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations. Ann N Y Acad Sci 1105: 1–29.A. Sjostedt2007Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations.Ann N Y Acad Sci1105129
  6. 6. Darling RG, Catlett CL, Huebner KD, Jarrett DG (2002) Threats in bioterrorism. I: CDC category A agents. Emerg Med Clin North Am 20: 273–309.RG DarlingCL CatlettKD HuebnerDG Jarrett2002Threats in bioterrorism. I: CDC category A agents.Emerg Med Clin North Am20273309
  7. 7. Chong A, Wehrly TD, Nair V, Fischer ER, Barker JR, et al. (2008) The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect Immun 76: 5488–5499.A. ChongTD WehrlyV. NairER FischerJR Barker2008The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression.Infect Immun7654885499
  8. 8. Golovliov I, Sjostedt A, Mokrievich A, Pavlov V (2003) A method for allelic replacement in Francisella tularensis. FEMS Microbiol Lett 222: 273–280.I. GolovliovA. SjostedtA. MokrievichV. Pavlov2003A method for allelic replacement in Francisella tularensis.FEMS Microbiol Lett222273280
  9. 9. Barker JR, Chong A, Wehrly TD, Yu JJ, Rodriguez SA, et al. (2009) The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence. Mol Microbiol 74: 1459–1470.JR BarkerA. ChongTD WehrlyJJ YuSA Rodriguez2009The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.Mol Microbiol7414591470
  10. 10. Nano FE, Schmerk C (2007) The Francisella pathogenicity island. Ann N Y Acad Sci 1105: 122–137.FE NanoC. Schmerk2007The Francisella pathogenicity island.Ann N Y Acad Sci1105122137
  11. 11. de Bruin OM, Ludu JS, Nano FE (2007) The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth. BMC Microbiol 7: 1.OM de BruinJS LuduFE Nano2007The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth.BMC Microbiol71
  12. 12. Gray CG, Cowley SC, Cheung KK, Nano FE (2002) The identification of five genetic loci of Francisella novicida associated with intracellular growth. FEMS Microbiol Lett 215: 53–56.CG GraySC CowleyKK CheungFE Nano2002The identification of five genetic loci of Francisella novicida associated with intracellular growth.FEMS Microbiol Lett2155356
  13. 13. Santic M, Molmeret M, Klose KE, Jones S, Kwaik YA (2005) The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Cell Microbiol 7: 969–979.M. SanticM. MolmeretKE KloseS. JonesYA Kwaik2005The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm.Cell Microbiol7969979
  14. 14. Raynaud C, Meibom KL, Lety MA, Dubail I, Candela T, et al. (2007) Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity. Infect Immun 75: 536–541.C. RaynaudKL MeibomMA LetyI. DubailT. Candela2007Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity.Infect Immun75536541
  15. 15. Sandstrom G, Lofgren S, Tarnvik A (1988) A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes. Infect Immun 56: 1194–1202.G. SandstromS. LofgrenA. Tarnvik1988A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes.Infect Immun5611941202
  16. 16. Ramakrishnan G, Meeker A, Dragulev B (2008) fslE is necessary for siderophore-mediated iron acquisition in Francisella tularensis Schu S4. J Bacteriol 190: 5353–5361.G. RamakrishnanA. MeekerB. Dragulev2008fslE is necessary for siderophore-mediated iron acquisition in Francisella tularensis Schu S4.J Bacteriol19053535361
  17. 17. Lenco J, Pavkova I, Hubalek M, Stulik J (2005) Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DeltaiglC1+2 by proteomics analysis. FEMS Microbiol Lett 246: 47–54.J. LencoI. PavkovaM. HubalekJ. Stulik2005Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DeltaiglC1+2 by proteomics analysis.FEMS Microbiol Lett2464754
  18. 18. Bakshi CS, Malik M, Regan K, Melendez JA, Metzger DW, et al. (2006) Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence. J Bacteriol 188: 6443–6448.CS BakshiM. MalikK. ReganJA MelendezDW Metzger2006Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence.J Bacteriol18864436448
  19. 19. Guina T, Radulovic D, Bahrami AJ, Bolton DL, Rohmer L, et al. (2007) MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress. J Bacteriol 189: 6580–6586.T. GuinaD. RadulovicAJ BahramiDL BoltonL. Rohmer2007MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress.J Bacteriol18965806586
  20. 20. Moule MG, Monack DM, Schneider DS (2010) Reciprocal analysis of Francisella novicida infections of a Drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response. PLoS Pathog 6: MG MouleDM MonackDS Schneider2010Reciprocal analysis of Francisella novicida infections of a Drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response.PLoS Pathog6
  21. 21. Dieppedale J, Sobral D, Dupuis M, Dubail I, Klimentova J, et al. (2011) Identification of a putative Chaperone Involved in Stress Resistance and Virulence in Francisella tularensis. Infect Immun. J. DieppedaleD. SobralM. DupuisI. DubailJ. Klimentova2011Identification of a putative Chaperone Involved in Stress Resistance and Virulence in Francisella tularensis.Infect Immun
  22. 22. Clarridge JE 3rd, Raich TJ, Sjosted A, Sandstrom G, Darouiche RO, et al. (1996) Characterization of two unusual clinically significant Francisella strains. J Clin Microbiol 34: 1995–2000.JE Clarridge 3rdTJ RaichA. SjostedG. SandstromRO Darouiche1996Characterization of two unusual clinically significant Francisella strains.J Clin Microbiol3419952000
  23. 23. Hollis DG, Weaver RE, Steigerwalt AG, Wenger JD, Moss CW, et al. (1989) Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease. J Clin Microbiol 27: 1601–1608.DG HollisRE WeaverAG SteigerwaltJD WengerCW Moss1989Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease.J Clin Microbiol2716011608
  24. 24. Titball RW, Petrosino JF (2007) Francisella tularensis genomics and proteomics. Ann N Y Acad Sci 1105: 98–121.RW TitballJF Petrosino2007Francisella tularensis genomics and proteomics.Ann N Y Acad Sci110598121
  25. 25. Wayne Conlan J, Oyston PC (2007) Vaccines against Francisella tularensis. Ann N Y Acad Sci 1105: 325–350.J. Wayne ConlanPC Oyston2007Vaccines against Francisella tularensis.Ann N Y Acad Sci1105325350
  26. 26. Rohmer L, Fong C, Abmayr S, Wasnick M, Larson Freeman TJ, et al. (2007) Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains. Genome Biol 8: R102.L. RohmerC. FongS. AbmayrM. WasnickTJ Larson Freeman2007Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.Genome Biol8R102
  27. 27. Rick Lyons C, Wu TH (2007) Animal models of Francisella tularensis infection. Ann N Y Acad Sci 1105: 238–265.C. Rick LyonsTH Wu2007Animal models of Francisella tularensis infection.Ann N Y Acad Sci1105238265
  28. 28. Frank DW, Zahrt TC (2007) Genetics and genetic manipulation in Francisella tularensis. Ann N Y Acad Sci 1105: 67–97.DW FrankTC Zahrt2007Genetics and genetic manipulation in Francisella tularensis.Ann N Y Acad Sci11056797
  29. 29. Qin A, Mann BJ (2006) Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2. BMC Microbiol 6: 69.A. QinBJ Mann2006Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2.BMC Microbiol669
  30. 30. Fujita H, Watanabe Y, Sato T, Ohara Y, Homma M (1993) The entry and intracellular multiplication of Francisella tularensis in cultured cells: its correlation with virulence in experimental mice. Microbiol Immunol 37: 837–842.H. FujitaY. WatanabeT. SatoY. OharaM. Homma1993The entry and intracellular multiplication of Francisella tularensis in cultured cells: its correlation with virulence in experimental mice.Microbiol Immunol37837842
  31. 31. Hall JD, Craven RR, Fuller JR, Pickles RJ, Kawula TH (2007) Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation. Infect Immun 75: 1034–1039.JD HallRR CravenJR FullerRJ PicklesTH Kawula2007Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation.Infect Immun7510341039
  32. 32. Schulert GS, McCaffrey RL, Buchan BW, Lindemann SR, Hollenback C, et al. (2009) Francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain LVS. Infect Immun 77: 1324–1336.GS SchulertRL McCaffreyBW BuchanSR LindemannC. Hollenback2009Francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain LVS.Infect Immun7713241336
  33. 33. Santic M, Molmeret M, Klose KE, Abu Kwaik Y (2006) Francisella tularensis travels a novel, twisted road within macrophages. Trends Microbiol 14: 37–44.M. SanticM. MolmeretKE KloseY. Abu Kwaik2006Francisella tularensis travels a novel, twisted road within macrophages.Trends Microbiol143744
  34. 34. Clemens DL, Horwitz MA (2007) Uptake and intracellular fate of Francisella tularensis in human macrophages. Ann N Y Acad Sci 1105: 160–186.DL ClemensMA Horwitz2007Uptake and intracellular fate of Francisella tularensis in human macrophages.Ann N Y Acad Sci1105160186
  35. 35. McCaffrey RL, Schwartz JT, Lindemann SR, Moreland JG, Buchan BW, et al. (2010) Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis. J Leukoc Biol 88: 791–805.RL McCaffreyJT SchwartzSR LindemannJG MorelandBW Buchan2010Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis.J Leukoc Biol88791805
  36. 36. Mohapatra NP, Soni S, Rajaram MV, Dang PM, Reilly TJ, et al. (2010) Francisella acid phosphatases inactivate the NADPH oxidase in human phagocytes. J Immunol 184: 5141–5150.NP MohapatraS. SoniMV RajaramPM DangTJ Reilly2010Francisella acid phosphatases inactivate the NADPH oxidase in human phagocytes.J Immunol18451415150
  37. 37. Storz G, Tartaglia LA, Farr SB, Ames BN (1990) Bacterial defenses against oxidative stress. Trends Genet 6: 363–368.G. StorzLA TartagliaSB FarrBN Ames1990Bacterial defenses against oxidative stress.Trends Genet6363368
  38. 38. Halliwell B, Gutteridge JM (1984) Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1: 1396–1397.B. HalliwellJM Gutteridge1984Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy.Lancet113961397
  39. 39. Girotti AW (1998) Lipid hydroperoxide generation, turnover, and effector action in biological systems. J Lipid Res 39: 1529–1542.AW Girotti1998Lipid hydroperoxide generation, turnover, and effector action in biological systems.J Lipid Res3915291542
  40. 40. Akaike T, Sato K, Ijiri S, Miyamoto Y, Kohno M, et al. (1992) Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides. Arch Biochem Biophys 294: 55–63.T. AkaikeK. SatoS. IjiriY. MiyamotoM. Kohno1992Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides.Arch Biochem Biophys2945563
  41. 41. Splettstoesser WD, Schuff-Werner P (2002) Oxidative stress in phagocytes--"the enemy within". Microsc Res Tech 57: 441–455.WD SplettstoesserP. Schuff-Werner2002Oxidative stress in phagocytes--"the enemy within".Microsc Res Tech57441455
  42. 42. Moslen MT (1994) Reactive oxygen species in normal physiology, cell injury and phagocytosis. Adv Exp Med Biol 366: 17–27.MT Moslen1994Reactive oxygen species in normal physiology, cell injury and phagocytosis.Adv Exp Med Biol3661727
  43. 43. Lindgren H, Stenman L, Tarnvik A, Sjostedt A (2005) The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages. Microbes Infect 7: 467–475.H. LindgrenL. StenmanA. TarnvikA. Sjostedt2005The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.Microbes Infect7467475
  44. 44. Lindgren H, Stenmark S, Chen W, Tarnvik A, Sjostedt A (2004) Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis. Infect Immun 72: 7172–7182.H. LindgrenS. StenmarkW. ChenA. TarnvikA. Sjostedt2004Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.Infect Immun7271727182
  45. 45. Lee RK, Harris G, Conlan JW, Chen W (2011) Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice. Microbes Infect. RK LeeG. HarrisJW ConlanW. Chen2011Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice.Microbes Infect
  46. 46. Kuolee R, Harris G, Conlan JW, Chen W (2011) Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice. Microbes Infect. R. KuoleeG. HarrisJW ConlanW. Chen2011Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice.Microbes Infect
  47. 47. McCaffrey RL, Allen LA (2006) Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape. J Leukoc Biol 80: 1224–1230.RL McCaffreyLA Allen2006Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape.J Leukoc Biol8012241230
  48. 48. Lindgren H, Shen H, Zingmark C, Golovliov I, Conlan W, et al. (2007) Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG. Infect Immun 75: 1303–1309.H. LindgrenH. ShenC. ZingmarkI. GolovliovW. Conlan2007Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG.Infect Immun7513031309
  49. 49. Melillo AA, Mahawar M, Sellati TJ, Malik M, Metzger DW, et al. (2009) Identification of Francisella tularensis live vaccine strain CuZn superoxide dismutase as critical for resistance to extracellularly generated reactive oxygen species. J Bacteriol 191: 6447–6456.AA MelilloM. MahawarTJ SellatiM. MalikDW Metzger2009Identification of Francisella tularensis live vaccine strain CuZn superoxide dismutase as critical for resistance to extracellularly generated reactive oxygen species.J Bacteriol19164476456
  50. 50. Weiss DS, Brotcke A, Henry T, Margolis JJ, Chan K, et al. (2007) In vivo negative selection screen identifies genes required for Francisella virulence. Proc Natl Acad Sci U S A 104: 6037–6042.DS WeissA. BrotckeT. HenryJJ MargolisK. Chan2007In vivo negative selection screen identifies genes required for Francisella virulence.Proc Natl Acad Sci U S A10460376042
  51. 51. Su J, Yang J, Zhao D, Kawula TH, Banas JA, et al. (2007) Genome-wide identification of Francisella tularensis virulence determinants. Infect Immun 75: 3089–3101.J. SuJ. YangD. ZhaoTH KawulaJA Banas2007Genome-wide identification of Francisella tularensis virulence determinants.Infect Immun7530893101
  52. 52. Kraemer PS, Mitchell A, Pelletier MR, Gallagher LA, Wasnick M, et al. (2009) Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice. Infect Immun 77: 232–244.PS KraemerA. MitchellMR PelletierLA GallagherM. Wasnick2009Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice.Infect Immun77232244
  53. 53. Sullivan JT, Jeffery EF, Shannon JD, Ramakrishnan G (2006) Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production. J Bacteriol 188: 3785–3795.JT SullivanEF JefferyJD ShannonG. Ramakrishnan2006Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production.J Bacteriol18837853795
  54. 54. Deng K, Blick RJ, Liu W, Hansen EJ (2006) Identification of Francisella tularensis genes affected by iron limitation. Infect Immun 74: 4224–4236.K. DengRJ BlickW. LiuEJ Hansen2006Identification of Francisella tularensis genes affected by iron limitation.Infect Immun7442244236
  55. 55. Kiss K, Liu W, Huntley JF, Norgard MV, Hansen EJ (2008) Characterization of fig operon mutants of Francisella novicida U112. FEMS Microbiol Lett 285: 270–277.K. KissW. LiuJF HuntleyMV NorgardEJ Hansen2008Characterization of fig operon mutants of Francisella novicida U112.FEMS Microbiol Lett285270277
  56. 56. Mongkolsuk S, Praituan W, Loprasert S, Fuangthong M, Chamnongpol S (1998) Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli. J Bacteriol 180: 2636–2643.S. MongkolsukW. PraituanS. LoprasertM. FuangthongS. Chamnongpol1998Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli.J Bacteriol18026362643
  57. 57. Fuangthong M, Atichartpongkul S, Mongkolsuk S, Helmann JD (2001) OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis. J Bacteriol 183: 4134–4141.M. FuangthongS. AtichartpongkulS. MongkolsukJD Helmann2001OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis.J Bacteriol18341344141
  58. 58. Rince A, Giard JC, Pichereau V, Flahaut S, Auffray Y (2001) Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis. J Bacteriol 183: 1482–1488.A. RinceJC GiardV. PichereauS. FlahautY. Auffray2001Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis.J Bacteriol18314821488
  59. 59. Ochsner UA, Hassett DJ, Vasil ML (2001) Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa. J Bacteriol 183: 773–778.UA OchsnerDJ HassettML Vasil2001Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa.J Bacteriol183773778
  60. 60. Shea RJ, Mulks MH (2002) ohr, Encoding an organic hydroperoxide reductase, is an in vivo-induced gene in Actinobacillus pleuropneumoniae. Infect Immun 70: 794–802.RJ SheaMH Mulks2002ohr, Encoding an organic hydroperoxide reductase, is an in vivo-induced gene in Actinobacillus pleuropneumoniae.Infect Immun70794802
  61. 61. Dorsey CW, Tomaras AP, Actis LA (2006) Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance. Plasmid 56: 112–123.CW DorseyAP TomarasLA Actis2006Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance.Plasmid56112123
  62. 62. Jenkins C, Samudrala R, Geary SJ, Djordjevic SP (2008) Structural and functional characterization of an organic hydroperoxide resistance protein from Mycoplasma gallisepticum. J Bacteriol 190: 2206–2216.C. JenkinsR. SamudralaSJ GearySP Djordjevic2008Structural and functional characterization of an organic hydroperoxide resistance protein from Mycoplasma gallisepticum.J Bacteriol19022062216
  63. 63. Cussiol JR, Alegria TG, Szweda LI, Netto LE (2010) Ohr (organic hydroperoxide resistance protein) possesses a previously undescribed activity, lipoyl-dependent peroxidase. J Biol Chem 285: 21943–21950.JR CussiolTG AlegriaLI SzwedaLE Netto2010Ohr (organic hydroperoxide resistance protein) possesses a previously undescribed activity, lipoyl-dependent peroxidase.J Biol Chem2852194321950
  64. 64. Saikolappan S, Sasindran SJ, Yu HD, Baseman JB, Dhandayuthapani S (2009) The Mycoplasma genitalium MG_454 gene product resists killing by organic hydroperoxides. J Bacteriol 191: 6675–6682.S. SaikolappanSJ SasindranHD YuJB BasemanS. Dhandayuthapani2009The Mycoplasma genitalium MG_454 gene product resists killing by organic hydroperoxides.J Bacteriol19166756682
  65. 65. Baron GS, Nano FE (1998) MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol Microbiol 29: 247–259.GS BaronFE Nano1998MglA and MglB are required for the intramacrophage growth of Francisella novicida.Mol Microbiol29247259
  66. 66. Baker CN, Hollis DG, Thornsberry C (1985) Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller-Hinton broth. J Clin Microbiol 22: 212–215.CN BakerDG HollisC. Thornsberry1985Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller-Hinton broth.J Clin Microbiol22212215
  67. 67. Chamberlain RE (1965) Evaluation of Live Tularemia Vaccine Prepared in a Chemically Defined Medium. Appl Microbiol 13: 232–235.RE Chamberlain1965Evaluation of Live Tularemia Vaccine Prepared in a Chemically Defined Medium.Appl Microbiol13232235
  68. 68. Schaible UE, Kaufmann SHE (2002) Studying trafficking of intracellular pathogens in antigen-presenting cells. Methods Microbiol 31: 3434–3360.UE SchaibleSHE Kaufmann2002Studying trafficking of intracellular pathogens in antigen-presenting cells.Methods Microbiol3134343360
  69. 69. Gallagher LA, Ramage E, Jacobs MA, Kaul R, Brittnacher M, et al. (2007) A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A 104: 1009–1014.LA GallagherE. RamageMA JacobsR. KaulM. Brittnacher2007A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate.Proc Natl Acad Sci U S A10410091014
  70. 70. Anthony LS, Gu MZ, Cowley SC, Leung WW, Nano FE (1991) Transformation and allelic replacement in Francisella spp. J Gen Microbiol 137: 2697–2703.LS AnthonyMZ GuSC CowleyWW LeungFE Nano1991Transformation and allelic replacement in Francisella spp.J Gen Microbiol13726972703
  71. 71. Gallagher LA, McKevitt M, Ramage ER, Manoil C (2008) Genetic dissection of the Francisella novicida restriction barrier. J Bacteriol 190: 7830–7837.LA GallagherM. McKevittER RamageC. Manoil2008Genetic dissection of the Francisella novicida restriction barrier.J Bacteriol19078307837
  72. 72. Rodriguez SA, Davis G, Klose KE (2009) Targeted gene disruption in Francisella tularensis by group II introns. Methods 49: 270–274.SA RodriguezG. DavisKE Klose2009Targeted gene disruption in Francisella tularensis by group II introns.Methods49270274
  73. 73. LoVullo ED, Sherrill LA, Perez LL, Pavelka MS Jr (2006) Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis. Microbiology 152: 3425–3435.ED LoVulloLA SherrillLL PerezMS Pavelka Jr2006Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis.Microbiology15234253435
  74. 74. Bina XR, Lavine CL, Miller MA, Bina JE (2008) The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice. FEMS Microbiol Lett 279: 226–233.XR BinaCL LavineMA MillerJE Bina2008The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice.FEMS Microbiol Lett279226233
  75. 75. Dringen R, Kussmaul L, Hamprecht B (1998) Rapid clearance of tertiary butyl hydroperoxide by cultured astroglial cells via oxidation of glutathione. Glia 23: 139–145.R. DringenL. KussmaulB. Hamprecht1998Rapid clearance of tertiary butyl hydroperoxide by cultured astroglial cells via oxidation of glutathione.Glia23139145
  76. 76. Chuchue T, Tanboon W, Prapagdee B, Dubbs JM, Vattanaviboon P, et al. (2006) ohrR and ohr are the primary sensor/regulator and protective genes against organic hydroperoxide stress in Agrobacterium tumefaciens. J Bacteriol 188: 842–851.T. ChuchueW. TanboonB. PrapagdeeJM DubbsP. Vattanaviboon2006ohrR and ohr are the primary sensor/regulator and protective genes against organic hydroperoxide stress in Agrobacterium tumefaciens.J Bacteriol188842851
  77. 77. Lai XH, Golovliov I, Sjostedt A (2004) Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis. Microb Pathog 37: 225–230.XH LaiI. GolovliovA. Sjostedt2004Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis.Microb Pathog37225230
  78. 78. Ahlund MK, Ryden P, Sjostedt A, Stoven S (2010) A directed screen of Francisella novicida virulence determinants using Drosophila melanogaster. Infect Immun. MK AhlundP. RydenA. SjostedtS. Stoven2010A directed screen of Francisella novicida virulence determinants using Drosophila melanogaster.Infect Immun
  79. 79. Brotcke A, Weiss DS, Kim CC, Chain P, Malfatti S, et al. (2006) Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect Immun 74: 6642–6655.A. BrotckeDS WeissCC KimP. ChainS. Malfatti2006Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis.Infect Immun7466426655
  80. 80. Santic M, Molmeret M, Barker JR, Klose KE, Dekanic A, et al. (2007) A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol. Cell Microbiol 9: 2391–2403.M. SanticM. MolmeretJR BarkerKE KloseA. Dekanic2007A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol.Cell Microbiol923912403
  81. 81. Pelegrin P, Barroso-Gutierrez C, Surprenant A (2008) P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage. J Immunol 180: 7147–7157.P. PelegrinC. Barroso-GutierrezA. Surprenant2008P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage.J Immunol18071477157
  82. 82. Mariathasan S, Weiss DS, Dixit VM, Monack DM (2005) Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J Exp Med 202: 1043–1049.S. MariathasanDS WeissVM DixitDM Monack2005Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis.J Exp Med20210431049
  83. 83. Atichartpongkul S, Loprasert S, Vattanaviboon P, Whangsuk W, Helmann JD, et al. (2001) Bacterial Ohr and OsmC paralogues define two protein families with distinct functions and patterns of expression. Microbiology 147: 1775–1782.S. AtichartpongkulS. LoprasertP. VattanaviboonW. WhangsukJD Helmann2001Bacterial Ohr and OsmC paralogues define two protein families with distinct functions and patterns of expression.Microbiology14717751782
  84. 84. Hall JD, Woolard MD, Gunn BM, Craven RR, Taft-Benz S, et al. (2008) Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112. Infect Immun 76: 5843–5852.JD HallMD WoolardBM GunnRR CravenS. Taft-Benz2008Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112.Infect Immun7658435852
  85. 85. Craven RR, Hall JD, Fuller JR, Taft-Benz S, Kawula TH (2008) Francisella tularensis invasion of lung epithelial cells. Infect Immun 76: 2833–2842.RR CravenJD HallJR FullerS. Taft-BenzTH Kawula2008Francisella tularensis invasion of lung epithelial cells.Infect Immun7628332842
  86. 86. Schmerk CL, Duplantis BN, Howard PL, Nano FE (2009) A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1. Microbiology 155: 1498–1504.CL SchmerkBN DuplantisPL HowardFE Nano2009A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.Microbiology15514981504
  87. 87. Ludu JS, de Bruin OM, Duplantis BN, Schmerk CL, Chou AY, et al. (2008) The Francisella pathogenicity island protein PdpD is required for full virulence and associates with homologues of the type VI secretion system. J Bacteriol 190: 4584–4595.JS LuduOM de BruinBN DuplantisCL SchmerkAY Chou2008The Francisella pathogenicity island protein PdpD is required for full virulence and associates with homologues of the type VI secretion system.J Bacteriol19045844595
  88. 88. Read A, Vogl SJ, Hueffer K, Gallagher LA, Happ GM (2008) Francisella genes required for replication in mosquito cells. J Med Entomol 45: 1108–1116.A. ReadSJ VoglK. HuefferLA GallagherGM Happ2008Francisella genes required for replication in mosquito cells.J Med Entomol4511081116
  89. 89. Asare R, Abu Kwaik Y (2010) Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis. Environ Microbiol. R. AsareY. Abu Kwaik2010Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis.Environ Microbiol
  90. 90. Pechous R, Celli J, Penoske R, Hayes SF, Frank DW, et al. (2006) Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain. Infect Immun 74: 4452–4461.R. PechousJ. CelliR. PenoskeSF HayesDW Frank2006Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain.Infect Immun7444524461
  91. 91. Maier TM, Casey MS, Becker RH, Dorsey CW, Glass EM, et al. (2007) Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages. Infect Immun 75: 5376–5389.TM MaierMS CaseyRH BeckerCW DorseyEM Glass2007Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages.Infect Immun7553765389
  92. 92. Meibom KL, Dubail I, Dupuis M, Barel M, Lenco J, et al. (2008) The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice. Mol Microbiol 67: 1384–1401.KL MeibomI. DubailM. DupuisM. BarelJ. Lenco2008The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice.Mol Microbiol6713841401
  93. 93. Rengarajan J, Murphy E, Park A, Krone CL, Hett EC, et al. (2008) Mycobacterium tuberculosis Rv2224c modulates innate immune responses. Proc Natl Acad Sci U S A 105: 264–269.J. RengarajanE. MurphyA. ParkCL KroneEC Hett2008Mycobacterium tuberculosis Rv2224c modulates innate immune responses.Proc Natl Acad Sci U S A105264269
  94. 94. Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100: 12989–12994.CM SassettiEJ Rubin2003Genetic requirements for mycobacterial survival during infection.Proc Natl Acad Sci U S A1001298912994
  95. 95. Beckett D (2007) Biotin sensing: universal influence of biotin status on transcription. Annu Rev Genet 41: 443–464.D. Beckett2007Biotin sensing: universal influence of biotin status on transcription.Annu Rev Genet41443464
  96. 96. Asare R, Akimana C, Jones S, Abu Kwaik Y (2010) Molecular bases of proliferation of Francisella tularensis in arthropod vectors. Environ Microbiol. R. AsareC. AkimanaS. JonesY. Abu Kwaik2010Molecular bases of proliferation of Francisella tularensis in arthropod vectors.Environ Microbiol
  97. 97. Payne SM (1993) Iron acquisition in microbial pathogenesis. Trends Microbiol 1: 66–69.SM Payne1993Iron acquisition in microbial pathogenesis.Trends Microbiol16669
  98. 98. Schaible UE, Kaufmann SH (2004) Iron and microbial infection. Nat Rev Microbiol 2: 946–953.UE SchaibleSH Kaufmann2004Iron and microbial infection.Nat Rev Microbiol2946953
  99. 99. Horzempa J, O'Dee DM, Shanks RM, Nau GJ (2010) Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo. Infect Immun 78: 2607–2619.J. HorzempaDM O'DeeRM ShanksGJ Nau2010Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo.Infect Immun7826072619
  100. 100. Oliveira MA, Guimaraes BG, Cussiol JR, Medrano FJ, Gozzo FC, et al. (2006) Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa. J Mol Biol 359: 433–445.MA OliveiraBG GuimaraesJR CussiolFJ MedranoFC Gozzo2006Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.J Mol Biol359433445
  101. 101. Lesniak J, Barton WA, Nikolov DB (2002) Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr. EMBO J 21: 6649–6659.J. LesniakWA BartonDB Nikolov2002Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr.EMBO J2166496659
  102. 102. Cussiol JR, Alves SV, de Oliveira MA, Netto LE (2003) Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase. J Biol Chem 278: 11570–11578.JR CussiolSV AlvesMA de OliveiraLE Netto2003Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase.J Biol Chem2781157011578
  103. 103. Ochsner UA, Vasil ML, Alsabbagh E, Parvatiyar K, Hassett DJ (2000) Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J Bacteriol 182: 4533–4544.UA OchsnerML VasilE. AlsabbaghK. ParvatiyarDJ Hassett2000Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF.J Bacteriol18245334544
  104. 104. Steele KH, Baumgartner JE, Valderas MW, Roop RM, 2nd (2010) Comparative study of the roles of AhpC and KatE as respiratory antioxidants in Brucella abortus 2308. J Bacteriol 192: 4912–4922.KH SteeleJE BaumgartnerMW ValderasRM Roop2nd2010Comparative study of the roles of AhpC and KatE as respiratory antioxidants in Brucella abortus 2308.J Bacteriol19249124922
  105. 105. Parsonage D, Karplus PA, Poole LB (2008) Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin. Proc Natl Acad Sci U S A 105: 8209–8214.D. ParsonagePA KarplusLB Poole2008Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin.Proc Natl Acad Sci U S A10582098214
  106. 106. Kadzhaev K, Zingmark C, Golovliov I, Bolanowski M, Shen H, et al. (2009) Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model. PLoS One 4: e5463.K. KadzhaevC. ZingmarkI. GolovliovM. BolanowskiH. Shen2009Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model.PLoS One4e5463
  107. 107. Wolfram TJ, Leveque RM, Kastenmayer RJ, Mulks MH (2009) Ohr, an in vivo-induced gene in Actinobacillus pleuropneumoniae, is located on a genomic island and requires glutathione-S-transferase for activity. FEMS Immunol Med Microbiol 57: 59–68.TJ WolframRM LevequeRJ KastenmayerMH Mulks2009Ohr, an in vivo-induced gene in Actinobacillus pleuropneumoniae, is located on a genomic island and requires glutathione-S-transferase for activity.FEMS Immunol Med Microbiol575968
  108. 108. Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Child R, et al. (2009) Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages. Cell Microbiol 11: 1128–1150.TD WehrlyA. ChongK. VirtanevaDE SturdevantR. Child2009Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages.Cell Microbiol1111281150
  109. 109. Allen LA, McCaffrey RL (2007) To activate or not to activate: distinct strategies used by Helicobacter pylori and Francisella tularensis to modulate the NADPH oxidase and survive in human neutrophils. Immunol Rev 219: 103–117.LA AllenRL McCaffrey2007To activate or not to activate: distinct strategies used by Helicobacter pylori and Francisella tularensis to modulate the NADPH oxidase and survive in human neutrophils.Immunol Rev219103117
  110. 110. Bakshi CS, Malik M, Mahawar M, Kirimanjeswara GS, Hazlett KR, et al. (2008) An improved vaccine for prevention of respiratory tularemia caused by Francisella tularensis SchuS4 strain. Vaccine 26: 5276–5288.CS BakshiM. MalikM. MahawarGS KirimanjeswaraKR Hazlett2008An improved vaccine for prevention of respiratory tularemia caused by Francisella tularensis SchuS4 strain.Vaccine2652765288
  111. 111. Dubbs JM, Mongkolsuk S (2007) Peroxiredoxins in bacterial antioxidant defense. Subcell Biochem 44: 143–193.JM DubbsS. Mongkolsuk2007Peroxiredoxins in bacterial antioxidant defense.Subcell Biochem44143193
  112. 112. Tempel R, Lai XH, Crosa L, Kozlowicz B, Heffron F (2006) Attenuated Francisella novicida transposon mutants protect mice against wild-type challenge. Infect Immun 74: 5095–5105.R. TempelXH LaiL. CrosaB. KozlowiczF. Heffron2006Attenuated Francisella novicida transposon mutants protect mice against wild-type challenge.Infect Immun7450955105
  113. 113. Quarry JE, Isherwood KE, Michell SL, Diaper H, Titball RW, et al. (2007) A Francisella tularensis subspecies novicida purF mutant, but not a purA mutant, induces protective immunity to tularemia in mice. Vaccine 25: 2011–2018.JE QuarryKE IsherwoodSL MichellH. DiaperRW Titball2007A Francisella tularensis subspecies novicida purF mutant, but not a purA mutant, induces protective immunity to tularemia in mice.Vaccine2520112018
  114. 114. Lindemann SR, Peng K, Long ME, Hunt JR, Apicella MA, et al. (2011) Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages. Infect Immun 79: 581–594.SR LindemannK. PengME LongJR HuntMA Apicella2011Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.Infect Immun79581594
  115. 115. Kanistanon D, Hajjar AM, Pelletier MR, Gallagher LA, Kalhorn T, et al. (2008) A Francisella mutant in lipid A carbohydrate modification elicits protective immunity. PLoS Pathog 4: e24.D. KanistanonAM HajjarMR PelletierLA GallagherT. Kalhorn2008A Francisella mutant in lipid A carbohydrate modification elicits protective immunity.PLoS Pathog4e24
  116. 116. Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP, et al. (2004) MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci U S A 101: 4246–4249.CM LaurianoJR BarkerSS YoonFE NanoBP Arulanandam2004MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival.Proc Natl Acad Sci U S A10142464249
  117. 117. Cong Y, Yu JJ, Guentzel MN, Berton MT, Seshu J, et al. (2009) Vaccination with a defined Francisella tularensis subsp. novicida pathogenicity island mutant (DeltaiglB) induces protective immunity against homotypic and heterotypic challenge. Vaccine 27: 5554–5561.Y. CongJJ YuMN GuentzelMT BertonJ. Seshu2009Vaccination with a defined Francisella tularensis subsp. novicida pathogenicity island mutant (DeltaiglB) induces protective immunity against homotypic and heterotypic challenge.Vaccine2755545561
  118. 118. Bell BL, Mohapatra NP, Gunn JS (2010) Regulation of Virulence Gene Transcripts by the Francisella Orphan Response Regulator PmrA: Role of Phosphorylation and Evidence of MglA/SspA Interaction. Infect Immun. BL BellNP MohapatraJS Gunn2010Regulation of Virulence Gene Transcripts by the Francisella Orphan Response Regulator PmrA: Role of Phosphorylation and Evidence of MglA/SspA Interaction.Infect Immun
  119. 119. Conlan JW, Shen H, Golovliov I, Zingmark C, Oyston PC, et al. (2010) Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization. Vaccine 28: 1824–1831.JW ConlanH. ShenI. GolovliovC. ZingmarkPC Oyston2010Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization.Vaccine2818241831