Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infec... more Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithe-lial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epi-thelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The FTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The FTT_0924 mutant was also significantly more sensitive to-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining pepti-doglycan integrity and virulence.
The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilo... more The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain...
Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Eva... more Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by L...
Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of... more Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homolo-gous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 panG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 panG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary , we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.
Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disea... more Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA ...
The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well estab... more The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. θ is a tightly bound component of the DNA polymerase III core, which contains the α subunit (polymerase), the ε subunit (3′→5′ exonuclease), and the θ subunit, in the linear order α-ε-θ. Previous studies have shown that the θ subunit is not essential, as strains carrying a deletion of the holE gene (which encodes θ) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3′-exonuclease activity of ε to be modestly enhanced by the presence of θ. Here, we report a series of genetic experiments that suggest that θ has a stabilizing role for the ε proofreading subunit. The observations include (i) defined Δ holE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong Δ holE mu...
ABSTRACTFrancisella tularensisis a highly virulent bacterial pathogen that invades and replicates... more ABSTRACTFrancisella tularensisis a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of theF. tularensislive vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequencedFrancisellaspecies yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele intrans. Based on the deletion mutant phenotype, FTL_1914 was termedripA(required forintracellularproliferation, factorA). Following uptake by J774.A1 cells,F. tularensisLVSΔripAcolocaliz...
ABSTRACTFrancisella tularensiscauses systemic disease in humans and other mammals, with high morb... more ABSTRACTFrancisella tularensiscauses systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection.F. tularensisis a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation ofF. tularensisstrains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11bhighmacrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 ...
Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent ... more Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intra-cellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 iclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSiclR were equally cytotoxic and induced equivalent levels of interleukin-1 expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and iclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.
ABSTRACTFrancisella tularensis, a gram-negative facultative intracellular bacterial pathogen, cau... more ABSTRACTFrancisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site ofF. tularensisreplication in infected animals. However, recently it was reported thatF. tularensisalso invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process ofF. tularensisinvasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixedF. tularensislive vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreasedF. tularensisinvasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase ac...
Summary Recruitment of immune cells to infection sites is a critical component of the host respon... more Summary Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1−/− mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1−/− mice displayed similar organ burdens to wild-type mice. CX3CR1−/− mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects ...
Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infec... more Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithe-lial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epi-thelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The FTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The FTT_0924 mutant was also significantly more sensitive to-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining pepti-doglycan integrity and virulence.
The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilo... more The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain...
Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Eva... more Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by L...
Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of... more Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homolo-gous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 panG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 panG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary , we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.
Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disea... more Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA ...
The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well estab... more The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. θ is a tightly bound component of the DNA polymerase III core, which contains the α subunit (polymerase), the ε subunit (3′→5′ exonuclease), and the θ subunit, in the linear order α-ε-θ. Previous studies have shown that the θ subunit is not essential, as strains carrying a deletion of the holE gene (which encodes θ) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3′-exonuclease activity of ε to be modestly enhanced by the presence of θ. Here, we report a series of genetic experiments that suggest that θ has a stabilizing role for the ε proofreading subunit. The observations include (i) defined Δ holE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong Δ holE mu...
ABSTRACTFrancisella tularensisis a highly virulent bacterial pathogen that invades and replicates... more ABSTRACTFrancisella tularensisis a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of theF. tularensislive vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequencedFrancisellaspecies yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele intrans. Based on the deletion mutant phenotype, FTL_1914 was termedripA(required forintracellularproliferation, factorA). Following uptake by J774.A1 cells,F. tularensisLVSΔripAcolocaliz...
ABSTRACTFrancisella tularensiscauses systemic disease in humans and other mammals, with high morb... more ABSTRACTFrancisella tularensiscauses systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection.F. tularensisis a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation ofF. tularensisstrains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11bhighmacrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 ...
Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent ... more Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intra-cellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 iclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSiclR were equally cytotoxic and induced equivalent levels of interleukin-1 expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and iclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.
ABSTRACTFrancisella tularensis, a gram-negative facultative intracellular bacterial pathogen, cau... more ABSTRACTFrancisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site ofF. tularensisreplication in infected animals. However, recently it was reported thatF. tularensisalso invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process ofF. tularensisinvasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixedF. tularensislive vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreasedF. tularensisinvasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase ac...
Summary Recruitment of immune cells to infection sites is a critical component of the host respon... more Summary Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1−/− mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1−/− mice displayed similar organ burdens to wild-type mice. CX3CR1−/− mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects ...
Uploads
Papers by Sharon Taft-Benz