The Experts below are selected from a list of 10053 Experts worldwide ranked by ideXlab platform
Jean Celli - One of the best experts on this subject based on the ideXlab platform.
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Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis
2016Co-Authors: Henriette Geier, Jean CelliAbstract:Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the comple-ment receptor CR3, the scavenger receptor A (SRA), and the Fc receptor (FcR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisell
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the Francisella o antigen mediates survival in the macrophage cytosol via autophagy avoidance
Cellular Microbiology, 2014Co-Authors: Elizabeth Di Russo Case, Audrey Chong, Tara D Wehrly, Bryan Hansen, Robert Child, Seungmin Hwang, Herbert W Virgin, Jean CelliAbstract:Summary Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP-LC3-expressing murine macrophages by microscopy for clones localized within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at 6 h post-infection, whose HimarFT insertions clustered to fourgenetic loci involved in lipopolysaccharidic and capsular O-antigen biosynthesis. Consistent with the HimarFT mutants, in-frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O-antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild-type Francisella, the O-antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/SQSTM1 and LC3 prior to cytosolic clearance. Autophagy-deficient macrophages partially supported replication of both mutants, indicating that O-antigen-lacking Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.
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structure function analysis of dipa a Francisella tularensis virulence factor required for intracellular replication
PLOS ONE, 2013Co-Authors: Audrey Chong, Tara D Wehrly, Robert Child, Dedeke Rockxbrouwer, Aiping Qin, Barbara J Mann, Jean CelliAbstract:Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen.
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mechanisms of Francisella tularensis intracellular pathogenesis
Cold Spring Harbor Perspectives in Medicine, 2013Co-Authors: Jean Celli, Thomas C ZahrtAbstract:Francisella tularensis is a zoonotic intracellular pathogen and the causative agent of the debilitating febrile illness tularemia. Although natural infections by F. tularensis are sporadic and generally localized, the low infectious dose, with the ability to be transmitted to humans via multiple routes and the potential to cause life-threatening infections, has led to concerns that this bacterium could be used as an agent of bioterror and released intentionally into the environment. Recent studies of F. tularensis and other closely related Francisella species have greatly increased our understanding of mechanisms used by this organism to infect and cause disease within the host. Here, we review the intracellular life cycle of Francisella and highlight key genetic determinants and/or pathways that contribute to the survival and proliferation of this bacterium within host cells.
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phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis
Infection and Immunity, 2011Co-Authors: Henriette Geier, Jean CelliAbstract:Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.
Bradley D Jones - One of the best experts on this subject based on the ideXlab platform.
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metabolic reprogramming of host cells by virulent Francisella tularensis for optimal replication and modulation of inflammation
Journal of Immunology, 2016Co-Authors: Elliott V Wyatt, Jed A Rasmussen, Bradley D Jones, Karina Diaz, Amanda J Griffin, Deborah D Crane, Catharine M. BosioAbstract:A shift in macrophage metabolism from oxidative phosphorylation to aerobic glycolysis is a requirement for activation to effectively combat invading pathogens. Francisella tularensis is a facultative intracellular bacterium that causes an acute, fatal disease called tularemia. Its primary mechanism of virulence is its ability to evade and suppress inflammatory responses while replicating in the cytosol of macrophages. The means by which F. tularensis modulates macrophage activation are not fully elucidated. In this study, we demonstrate that virulent F. tularensis impairs production of inflammatory cytokines in primary macrophages by preventing their shift to aerobic glycolysis, as evidenced by the downregulation of hypoxia inducible factor 1α and failure to upregulate pfkfb3 We also show that Francisella capsule is required for this process. In addition to modulating inflammatory responses, inhibition of glycolysis in host cells is also required for early replication of virulent Francisella Taken together, our data demonstrate that metabolic reprogramming of host cells by F. tularensis is a key component of both inhibition of host defense mechanisms and replication of the bacterium.
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interactions of Francisella tularensis with alveolar type ii epithelial cells and the murine respiratory epithelium
PLOS ONE, 2015Co-Authors: Matthew Faron, Joshua R Fletcher, Jed A Rasmussen, Michael A Apicella, Bradley D JonesAbstract:Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.
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Francisella tularensis schu s4 o antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages
Infection and Immunity, 2011Co-Authors: Stephen R Lindemann, Denise M Monack, Michael A Apicella, Bradley D Jones, Kaitian Peng, Matthew E Long, Jason Hunt, Leeann H AllenAbstract:Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.
Wolf D Splettstoesser - One of the best experts on this subject based on the ideXlab platform.
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standardized broth microdilution antimicrobial susceptibility testing of Francisella tularensis subsp holarctica strains from europe and rare Francisella species
Journal of Antimicrobial Chemotherapy, 2012Co-Authors: Enrico Georgi, Holger C Scholz, Erik Schacht, Wolf D SplettstoesserAbstract:Objectives: Tularaemia is a widespread zoonosis in Europe caused by Francisella tularensis subsp. holarctica. Because of a lack of standardized CLSI-approved antibiotic susceptibility data from European Francisella strains, the antibiotic susceptibilities of a selection of F. tularensis subsp. holarctica isolates originating from Germany, Austria, France, Spain and other European countries were determined. Rarely isolated species and subspecies of Francisella such as Francisella philomiragia, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica as well as the type strain of Francisella hispaniensis were included in this study. Methods: MIC data were obtained using cation-adjusted Mueller –Hinton broth with a 2% growth supplement. The broth microdilution testing system comprised 14 antibiotics, including gentamicin, streptomycin, ciprofloxacin and tetracycline. Results: All of the 91 strains tested were susceptible to aminoglycosides, quinolones, tetracycline and chloramphenicol. The antimicrobial susceptibility of rare Francisellae was similar to the antibiotic profile of F. tularensis subsp. holarctica strains. For erythromycin, we detected two geographically distinct groups of F. tularensis subsp. holarctica isolates in western Europe. One group was resistant and the other one was susceptible. Both groups overlapped in a small region in Germany. Conclusions: Being performed in accordance with CLSI criteria, this study provides reliable data on antibiotic susceptibility patterns of European Francisella isolates. The standardized methodology of this study can be used for testing of suspicious colonies from clinical specimens for therapeutic guidance. Based on the results, aminoglycosides or quinolones are recommended as first-choice antibiotics for the therapy of F. hispaniensis, F. philomiragia or F. tularensis subsp. novicida infections in immunocompromised patients.
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description of Francisella hispaniensis sp nov isolated from human blood reclassification of Francisella novicida larson et al 1955 olsufiev et al 1959 as Francisella tularensis subsp novicida comb nov and emended description of the genus Francisella
International Journal of Systematic and Evolutionary Microbiology, 2010Co-Authors: Birgit Huber, Erik Seibold, Raquel Escudero, Hansjurgen Busse, Holger C Scholz, Pedro Anda, Peter Kampfer, Wolf D SplettstoesserAbstract:Strain FhSp1T, isolated from human blood in Spain in 2003, was studied for its taxonomic allocation. By 16S rRNA and recA gene sequencing, the strain was shown to belong to the genus Francisella. In the 16S rRNA gene sequence, Francisella sp. FhSp1T shared similarity of more than 99 % with strains of Francisella tularensis subspecies and Francisella novicida U112T, 98 % with Francisella piscicida GM2212T and 98.4 % with Francisella philomiragia ATCC 25015T. In the recA gene sequence, Francisella sp. FhSp1T exhibited 91.6–91.7 % similarity to strains of F. tularensis subspecies, 91.2 % to F. novicida U112T and 84 % to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C10 : 0 (17.2 %), C14 : 0 (11.2 %), C16 : 0 (13.1 %), C18 : 0 3-OH (14.2 %) and C18 : 1 ω9c (7.1 %). DNA–DNA hybridization, which showed unambiguously that FhSp1T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hitherto-described Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1T (=FnSp1T =FSC454T =F62T =DSM 22475T =CCUG 58020T). Furthermore, we formally propose the transfer of the species Francisella novicida to the species Francisella tularensis as Francisella tularensis subsp. novicida comb. nov. (type strain ATCC 15482T =CCUG 33449T =CIP 56.12T). We also present an emended description of the genus Francisella.
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identification of Francisella tularensis by whole cell matrix assisted laser desorption ionization time of flight mass spectrometry fast reliable robust and cost effective differentiation on species and subspecies levels
Journal of Clinical Microbiology, 2010Co-Authors: Erik Seibold, Thomas Maier, Markus Kostrzewa, E Zeman, Wolf D SplettstoesserAbstract:Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.
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detection of Francisella tularensis in biological specimens using a capture enzyme linked immunosorbent assay an immunochromatographic handheld assay and a pcr
Clinical and Vaccine Immunology, 2000Co-Authors: Roland Grunow, Wolf D Splettstoesser, Sahra Mcdonald, Christian Otterbein, Tom Obrien, Cecilia Morgan, Jennifer L Aldrich, Erwin Hofer, Ernstjurgen Finke, Hermann MeyerAbstract:The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 103 to 104 bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.
Thomas H Kawula - One of the best experts on this subject based on the ideXlab platform.
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a phosphatidylinositol 3 kinase effector alters phagosomal maturation to promote intracellular growth of Francisella
Cell Host & Microbe, 2018Co-Authors: Hannah E Ledvina, Thomas H Kawula, Aria Eshraghi, Brook S Peterson, Katherine A Kelly, Rachael L Plemel, Brian H Y Lee, Shaun Steele, Marlen Adler, Alexey J MerzAbstract:Many pathogenic intracellular bacteria manipulate the host phago-endosomal system to establish and maintain a permissive niche. The fate and identity of these intracellular compartments is controlled by phosphoinositide lipids. By mechanisms that have remained undefined, a Francisella pathogenicity island-encoded secretion system allows phagosomal escape and replication of bacteria within host cell cytoplasm. Here we report the discovery that a substrate of this system, outside pathogenicity island A (OpiA), represents a family of wortmannin-resistant bacterial phosphatidylinositol (PI) 3-kinase enzymes with members found in a wide range of intracellular pathogens, including Rickettsia and Legionella spp. We show that OpiA acts on the Francisella-containing phagosome and promotes bacterial escape into the cytoplasm. Furthermore, we demonstrate that the phenotypic consequences of OpiA inactivation are mitigated by endosomal maturation arrest. Our findings suggest that Francisella, and likely other intracellular bacteria, override the finely tuned dynamics of phagosomal PI(3)P in order to promote intracellular survival and pathogenesis.
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Francisella tularensis replicates within alveolar type ii epithelial cells in vitro and in vivo following inhalation
Infection and Immunity, 2007Co-Authors: Joshua D Hall, Robin R Craven, James R Fuller, Raymond J Pickles, Thomas H KawulaAbstract:Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells.
Denise M Monack - One of the best experts on this subject based on the ideXlab platform.
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Francisella tularensis schu s4 o antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages
Infection and Immunity, 2011Co-Authors: Stephen R Lindemann, Denise M Monack, Michael A Apicella, Bradley D Jones, Kaitian Peng, Matthew E Long, Jason Hunt, Leeann H AllenAbstract:Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.
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indoleamine 2 3 dioxygenase 1 is a lung specific innate immune defense mechanism that inhibits growth of Francisella tularensis tryptophan auxotrophs
Infection and Immunity, 2010Co-Authors: Kaitian Peng, Denise M MonackAbstract:Organs at different anatomic sites of the body have different physiological functions and are exposed to vastly different microbial and environmental challenges on a daily basis (32). As a result, depending on its relative sterility, each organ senses impending danger of infection differently and employs unique innate immune responses in an organ-specific homeostatic manner to effectively fight off any potential infection without compromising organ physiology and function. For instance, a sterile organ like the spleen is likely to induce a strong proinflammatory and bactericidal defense to maintain organ sterility, while such a defense would not be induced in a nonsterile organ like the colon. Instead, innate immune responses in the colon support a peaceful coexistence with the gut microflora rather than achieving sterility. To gain a better understanding of tissue-specific innate immune defense mechanisms, we have used Francisella tularensis as a model bacterial pathogen that is capable of entering and surviving in many different host tissues and organs. Francisella tularensis is a facultative, intracellular Gram-negative bacterium that causes the highly debilitating zoonotic disease tularemia (28). There are currently 4 known subspecies of F. tularensis that cause disease of different severities. The most virulent subspecies of Francisella is F. tularensis subsp. tularensis, which is found predominantly in North America and has an infectious dose of less than 10 CFU in humans (33, 34, 37). It is also associated with lethal pulmonary infections. Francisella novicida is also found primarily in North America but rarely causes disease in immunocompetent individuals (16). However, F. novicida shares the same families of virulence genes as F. tularensis, causes a similar disease in mice (27), and is more genetically amenable than F. tularensis, thus making F. novicida infection of mice a good experimental model for the study of Francisella pathogenesis. Francisella infects mammalian hosts via extremely diverse routes of entry, colonizing and replicating in different organs. Tularemia occurs in several forms, depending on the initial route of infection. The most common form of tularemia is ulceroglandular tularemia, which occurs when the bacterium enters the skin subcutaneously (9). Other infection routes include inoculation via the conjunctiva, which leads to oculoglandular tularemia (9, 38), and the ingestion of contaminated food and water, which leads to oropharyngeal or gastrointestinal tularemia (9). The most acute and fatal form of tularemia is pneumonic tularemia, which is caused by inhalation of the bacterium into the lungs. Pneumonic tularemia can also occur as a result of complications from the above forms of tularemia (9, 12), when the bacteria spread systemically from the initial peripheral site of infection to the lungs. From the perspective of the fitness of a microbe, it is advantageous for a microbe to infect its host via multiple entry routes and infect multiple organs. Thus, it is plausible that F. tularensis, a microbe that infects a range of different organs, has to deal with a diverse repertoire of different innate immune responses launched in these various organs during infection. We utilized a genome-wide genetic screen in F. novicida as a tool to identify tissue-specific interactions between the host and pathogen. Genetic screens are very effective tools that have been successfully used for the large-scale identification of virulence factors of many bacterial pathogens in vivo (6, 19, 44). Here, we utilize a microarray-based, negative-selection technique called transposon site hybridization (TraSH) (44) in an intranasal (i.n.) model of Francisella infection to identify Francisella factors important for bacterial growth and/or survival in the lungs. The screen identified almost all of the known Francisella virulence genes, including the Francisella pathogenicity island (FPI) genes. We also identified novel genes not previously known to be important for bacterial growth and/or survival in the lungs. By comparing the genes that are important for Francisella growth and/or survival in the lungs with the genes identified previously for spleen colonization (44), we identified the Francisella tryptophan biosynthetic pathway as being important for bacterial survival and/or growth specifically in the lungs. We demonstrated that the host enzyme that is responsible for catalyzing the first rate-limiting step of tryptophan degradation, indoleamine 2,3-dioxygenase 1 (IDO1), is induced specifically in the lungs during Francisella infection, suggesting that the host innate immune response to F. novicida restricts the availability of this essential amino acid. Indeed, the F. novicida tryptophan mutant bacteria survived better in the lungs of mice that lack IDO1 compared to wild-type mice. These findings suggest that IDO1 acts as an organ-specific, host innate immune mechanism that defends against a highly virulent bacterial pathogen.
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in vivo negative selection screen identifies genes required for Francisella virulence
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: David Weiss, Anna Brotcke, Thomas Henry, Jeffrey J Margolis, Kaman Chan, Denise M MonackAbstract:Francisella tularensis subverts the immune system to rapidly grow within mammalian hosts, often causing tularemia, a fatal disease. This pathogen targets the cytosol of macrophages where it replicates by using the genes encoded in the Francisella pathogenicity island. However, the bacteria are recognized in the cytosol by the host's ASC/caspase-1 pathway, which is essential for host defense, and leads to macrophage cell death and proinflammatory cytokine production. We used a microarray-based negative selection screen to identify Francisella genes that contribute to growth and/or survival in mice. The screen identified many known virulence factors including all of the Francisella pathogenicity island genes, LPS O-antigen synthetic genes, and capsule synthetic genes. We also identified 44 previously unidentified genes that were required for Francisella virulence in vivo, indicating that this pathogen may use uncharacterized mechanisms to cause disease. Among these, we discovered a class of Francisella virulence genes that are essential for growth and survival in vivo but do not play a role in intracellular replication within macrophages. Instead, these genes modulate the host ASC/caspase-1 pathway, a previously unidentified mechanism of Francisella pathogenesis. This finding indicates that the elucidation of the molecular mechanisms used by other uncharacterized genes identified in our screen will increase our understanding of the ways in which bacterial pathogens subvert the immune system.
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identification of mgla regulated genes reveals novel virulence factors in Francisella tularensis
Infection and Immunity, 2006Co-Authors: Anna Brotcke, David Weiss, Charles C Kim, Patrick S G Chain, Stephanie Malfatti, Emilio Garcia, Denise M MonackAbstract:The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.
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innate immunity against Francisella tularensis is dependent on the asc caspase 1 axis
Journal of Experimental Medicine, 2005Co-Authors: Sanjeev Mariathasan, David Weiss, Vishva M Dixit, Denise M MonackAbstract:Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia. This bacterial pathogen causes a plague-like disease in humans after exposure to as few as 10 cells. Many of the mechanisms by which the innate immune system fights Francisella are unknown. Here we show that wild-type Francisella, which reach the cytosol, but not Francisella mutants that remain localized to the vacuole, induced a host defense response in macrophages, which is dependent on caspase-1 and the death-fold containing adaptor protein ASC. Caspase-1 and ASC signaling resulted in host cell death and the release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. F. tularensis–infected caspase-1– and ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for caspase-1 and ASC in innate defense against infection by this pathogen.
