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generation and characterization of hybridoma antibodies for immunotherapy of tularemia.tularemia is caused by the gram-negative facultative intracellular bacterium francisella tularensis, which has been classified as a category a select agent-a likely bioweapon. the high virulence of f. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. we have characterized 14 anti-francisella hybridoma antibodies derived from mice infected with f. tularensis live vaccine strain (lvs) for potential use as immunoth ...200717764754
attenuation and protective efficacy of an o-antigen-deficient mutant of francisella tularensis lvs.francisella tularensis is a zoonotic, gram-negative coccobacillus that causes tularemia in humans and animals. f. tularensis subspecies tularensis (type a) and f. tularensis subspecies holarctica (type b) are antigenically similar and more virulent than francisella novicida in humans. the genetic locus that encodes the lps o antigen was found to be substantially different between the type b live vaccine strain (lvs) and f. novicida. one lvs-specific gene with homology to a galactosyl transferase ...200717768257
a 55 kda hypothetical membrane protein is an iron-regulated virulence factor of francisella tularensis subsp. novicida u112.iron is an important nutritional requirement for bacteria due to its conserved role in many essential metabolic processes. as a consequence of the lack of freely available iron in the mammalian host, bacteria upregulate a range of virulence factors during infection. transcriptional analysis of francisella tularensis subsp. novicida u112 grown in iron-deficient medium identified 21 genes upregulated in response to this condition, four of which were attributed to a siderophore operon. in addition, ...200717893160
fangia hongkongensis gen. nov., sp. nov., a novel gammaproteobacterium of the order thiotrichales isolated from coastal seawater of hong kong.a gram-negative, coccobacillus-shaped, aerobic bacterium, designated strain ust040201-002t, was isolated in february 2004 from seawater at the outlet of a sandfilter in port shelter, hong kong sar, china. this strain possessed ubiquinone-8; its 16s rrna gene sequence shared only 91% similarity with the sequence from caedibacter taeniospiralis and 89-90% similarity with sequences from francisella tularensis, francisella novicida, francisella philomiragia and wolbachia persica. 16s rrna gene seque ...200717978237
construction of targeted insertion mutations in francisella tularensis subsp. novicida.francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. we have developed an efficient targeted mutagenesis strategy in the model organism f. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (soe). this process enables fast and efficient construction of targeted insertion mutations in f. tularensis subsp. novicida that have cha ...200718019340
genetic elements for selection, deletion mutagenesis and complementation in francisella spp.francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to francisella tularensis at the genomic level. in this work a number of antibiotic marker cassettes that incorporate a strong f. novicida promoter is constructed, which greatly enhances selection in f. novicida and f. tularensis. two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used ...200818021237
periplasmic phosphorylation of lipid a is linked to the synthesis of undecaprenyl phosphate.one-third of the lipid a found in the escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid a 1-diphosphate). we now report an inner membrane enzyme, lpxt (yeiu), which specifically transfers a phosphate group to lipid a, forming the 1-diphosphate species. (32)p-labelled lipid a obtained from lpxt mutants do not produce lipid a 1-diphosphate. in vitro assays with kdo(2)-[4'-(32)p]lipid a as the acceptor shows that lpxt uses undecaprenyl pyrophosphate as ...200818047581
periplasmic phosphorylation of lipid a is linked to the synthesis of undecaprenyl phosphate.one-third of the lipid a found in the escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid a 1-diphosphate). we now report an inner membrane enzyme, lpxt (yeiu), which specifically transfers a phosphate group to lipid a, forming the 1-diphosphate species. (32)p-labelled lipid a obtained from lpxt mutants do not produce lipid a 1-diphosphate. in vitro assays with kdo(2)-[4'-(32)p]lipid a as the acceptor shows that lpxt uses undecaprenyl pyrophosphate as ...200818047581
the presence of infectious extracellular francisella tularensis subsp. novicida in murine plasma after pulmonary challenge. 200818087734
a functional genomic yeast screen to identify pathogenic bacterial proteins.many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. identification of these translocated proteins is essential to understanding pathogenesis. yet, their identification remains limited. this, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. in addition, their absence often does not result in phenotypes in virulenc ...200818208325
inactivation of host akt/protein kinase b signaling by bacterial pore-forming toxins.uropathogenic escherichia coli (upec) are the major cause of urinary tract infections (utis), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. this process can be facilitated by the pore-forming toxin alpha-hemolysin (hlya), which is expressed and secreted by many upec isolates. here, we demonstrate that hlya can potently inhibit activation of akt (protein kinase b), a key regulator of host cell survival, inflammatory responses, proliferation, and met ...200818234841
a francisella mutant in lipid a carbohydrate modification elicits protective immunity.francisella tularensis (ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. ft is designated a class a select agent by the centers for disease control and prevention. human clinical isolates of ft produce lipid a of similar structure to ft subspecies novicida (fn), a pathogen of mice. we identified three enzymes required for fn lipid a carbohydrate modifications, specifically the presence of mannose (flmf1), galactosamine (flmf2), or both ca ...200818266468
the tyrosine kinase syk promotes phagocytosis of francisella through the activation of erk.francisella tularensis is a highly infectious, gram-negative intra-cellular pathogen that can cause the zoonotic disease tularemia. although the receptors critical for internalization of francisella by macrophages are beginning to be defined, the identity of the downstream signaling pathways essential for the engulfment are not yet identified. in this study we have tested the role of syk in the phagocytosis of francisella. we report that syk is activated during francisella infection and is criti ...200818295889
a conserved and immunodominant lipoprotein of francisella tularensis is proinflammatory but not essential for virulence.francisella tularensis is a highly virulent bacterium that causes tularemia, a disease that is often fatal if untreated. a live vaccine strain (lvs) of this bacterium is attenuated for virulence in humans but produces lethal disease in mice. f. tularensis has been classified as a category a agent of bioterrorism. despite this categorization, little is known about the components of the organism that are responsible for causing disease in its hosts. here, we report the deletion of a well-character ...200818304778
ifngamma enhances il-23 production during francisella infection of human monocytes.we previously demonstrated that monocytes produce il-23 during francisella infection, and that il-23 induces ifngamma from nk cells. here, we demonstrate that ifngamma-priming of monocytes enhances il-23 production during francisella infection. this effect was seen on the il12/23 p40 subunit. induction of il-12/23 p40 is reported to be enhanced by irf-1 and irf-8. consistently, microarray analysis of ifngamma-treated monocytes revealed a significant induction of the irfs. interestingly, ifngamma ...200818319062
determination and comparison of the francisella tularensis subsp.novicida u112 proteome to other bacterial proteomes.the proteins expressed by francisella tularensis subsp. novicida u112 grown to midexponential phase were surveyed by nanolc-tandem mass spectrometry (lc-ms/ms). to improve annotation of the genome and develop a technology to provide high-throughput analysis of the francisella proteome in multiple conditions, we sought to establish a fast and simple analysis that would reduce as much as possible the false discovery rate. our survey detected expression of 63.0% of the predicted proteome from the s ...200818380474
respiratory francisella tularensis live vaccine strain infection induces th17 cells and prostaglandin e2, which inhibits generation of gamma interferon-positive t cells.two key routes of francisella tularensis infection are through the skin and airway. we wished to understand how the route of inoculation influenced the primary acute adaptive immune response. we show that an intranasal inoculation of the f. tularensis live vaccine strain (lvs) with a 1,000-fold-smaller dose than an intradermal dose results in similar growth kinetics and peak bacterial burdens. in spite of similar bacterial burdens, we demonstrate a difference in the quality, magnitude, and kinet ...200818391003
respiratory infection with francisella novicida induces rapid dystrophic cardiac calcinosis (dcc).francisella tularensis causes pulmonary tularemia and death in humans when left untreated. here, using a novel aerosol infection model, we show that acute pulmonary francisella novicida infection not only causes pneumonia and liver damage, but also induces dystrophic cardiac calcinosis (dcc) in balb/c mice. c57bl/6 mice also develop pneumonia and hepatic damage, but fail to develop dcc. development of dcc in balb/c mice is associated with significant induction of rankl but not osteopontin in the ...200818400010
initial delay in the immune response to francisella tularensis is followed by hypercytokinemia characteristic of severe sepsis and correlating with upregulation and release of damage-associated molecular patterns."francisella tularensis subsp. novicida" intranasal infection causes a rapid pneumonia in mice with mortality at 4 to 6 days with a low dose of bacteria (10(2) bacteria). the short time to death suggests that there is a failure of the innate immune response. as the neutrophil is often the first cell type to infiltrate sites of infection, we focused on the emigration of neutrophils in this infection, as well as cytokines involved in their recruitment. the results indicated that there was a signif ...200818411294
cd4+ t cells are required during priming but not the effector phase of antibody-mediated ifn-gamma-dependent protective immunity against pulmonary francisella novicida infection.we have previously demonstrated the protective efficacy of intranasal vaccination with a defined francisella tularensis subsp. novicida deltaiglc mutant (kkf24) against pulmonary f. novicida u112 challenge. in this study, we further characterized the mechanisms of kkf24-induced immunity. intranasally vaccinated kkf24 c57bl/6 major histocompatibility class (mhc) class ii-/- mice produced minimal antigen-specific interferon (ifn)-gamma and serum antibodies and were highly susceptible (0% survival) ...200818427567
large-scale transposon mutagenesis of mycoplasma pulmonis.to obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon tn4001t was determined for 1700 members of a library of mycoplasma pulmonis mutants. after evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. the dispensable and essential genes of m. pulmonis were compared with those reported for mycoplasma genitalium and bacillus subtilis. perhaps the most surpris ...200818452587
antimicrobial activity of human beta-defensins and induction by francisella.the ability of human beta-defensins hbd-1, hbd-2, and hbd-3 to exert direct in vitro antimicrobial effects was evaluated using francisella tularensis live vaccine strain (lvs) and francisella novicida. while hbd-2 showed some antimicrobial activity in these assays, only hbd-3 demonstrated significant potency against francisella. francisella tularensis lvs infection induced elevated levels of hbd-2 mrna in human airway epithelial (a549) cells, while having no significant impact on the levels of h ...200818452706
macrophage proinflammatory response to francisella tularensis live vaccine strain requires coordination of multiple signaling pathways.the macrophage proinflammatory response to francisella tularensis (ft) live vaccine strain (lvs) was shown previously to be tlr2 dependent. the observation that intracellular ft lvs colocalizes with tlr2 and myd88 inside macrophages suggested that ft lvs might signal from within the phagosome. macrophages infected with lvsdeltaiglc, a ft lvs mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of tlr2-dependent, proinflammatory genes (e.g., tnf) but ...200818453609
francisella tularensis subsp. tularensis schu s4 disulfide bond formation protein b, but not an rnd-type efflux pump, is required for virulence.francisella tularensis subsp. tularensis is a highly virulent bacterium that is a cdc select agent. despite advancements in the understanding of its biology, details pertaining to virulence are poorly understood. in previous work, we identified a transposon insertion mutant in the ftt0107c locus that was defective in intracellular survival in hepg2 and j77a.1 cells. here, we report that this mutant was also highly attenuated in vivo. the ftt0107c locus is predicted to encode an ortholog of the d ...200818458069
mgla and igl proteins contribute to the modulation of francisella tularensis live vaccine strain-containing phagosomes in murine macrophages.the francisella tularensis live vaccine strain (lvs), in contrast to its iglc mutant, replicates in the cytoplasm of macrophages. we studied the outcome of infection of the murine macrophagelike cell line j774a.1 with lvs and with iglc, igld, and mgla mutants, the latter of which is deficient in a global regulator. compared to lvs, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mgla mutant bacteria even decreased. colocalization with lamp-1 was sig ...200818474647
reduced secretion of yopj by yersinia limits in vivo cell death but enhances bacterial virulence.numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. however, the precise role of host cell death during in vivo infection remains poorly understood. macrophages infected by pathogenic species of yersinia typically undergo an apoptotic cell death. this is due to the activity of a type iii secreted effector protein, designated yopj in y. pseudotuberculosis and y. pestis, and yopp in the closely related y. enterocolitica. it has recently been reported that ...200818483548
combined deletion of four francisella novicida acid phosphatases attenuates virulence and macrophage vacuolar escape.francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. it is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. the goal of this study was to delete the four primary acid phosphatases (acps) from francisella novicida and examine the interactions of mutant strains with macro ...200818490464
administration of a synthetic tlr4 agonist protects mice from pneumonic tularemia.francisella tularensis is a gram-negative intracellular pathogen that causes the zoonosis tularemia. because f. tularensis lps causes weak tlr4 activation, we hypothesized that administration of a synthetic tlr4 agonist, aminoalkyl glucosaminide phosphate (agp), would boost the innate immune system and compensate for reduced tlr4 stimulation. intranasal administration of agps induced intrapulmonary production of proinflammatory cytokines and chemokines. mice treated with agps before and after in ...200818490759
isolation and characterization of a novel francisella sp. from human cerebrospinal fluid and blood.we describe the isolation of a francisella sp. from normally sterile sites in acutely ill patients in two different states within 2 years. microbiologic and molecular analyses indicate that this organism represents a novel francisella sp. clinicians and microbiologists should be aware of this new potential pathogen, as infection may be more common than recognized.200818495864
microfluidic-based cell sorting of francisella tularensis infected macrophages using optical forces.we have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. this micro fluorescence-activated cell sorter (microfacs) uses an infrared laser to laterally deflect cells into a collection channel. green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. to rule out po ...200818510341
francisella gains a survival advantage within mononuclear phagocytes by suppressing the host ifngamma response.tularemia is a zoonotic disease caused by the gram-negative intracellular pathogen francisella tularensis. these bacteria evade phagolysosomal fusion, escape from the phagosome and replicate in the host cell cytoplasm. ifngamma has been shown to suppress the intra-macrophage growth of francisella through both nitric oxide-dependent and -independent pathways. since francisella is known to subvert host immune responses, we hypothesized that this pathogen could interfere with ifngamma signaling. he ...200818514317
inhalation of francisella novicida delta mgla causes replicative infection that elicits innate and adaptive responses but is not protective against invasive pneumonic tularemia.francisella tularensis causes the zoonosis tularemia in humans, and inhaled f. tularensis ssp. novicida induces lethal murine tularemia. transcription of virulence factors in f. novicida is regulated by macrophage growth locus a (mgla), a global regulator required for bacterial replication in macrophages in vitro. we examined the infectivity and immunogenicity of attenuated f. novicida delta mgla in the lung in vivo. aerosolized delta mgla caused replicative pulmonary infection that peaked at 7 ...200818539500
identification of fevr, a novel regulator of virulence gene expression in francisella novicida.francisella tularensis infects wild animals and humans to cause tularemia. this pathogen targets the cytosol of macrophages, where it replicates using the genes in the francisella pathogenicity island (fpi). virulence gene regulation in francisella is complex, but transcriptional regulators mgla and sspa have been shown to regulate the expression of approximately 100 genes, including the entire fpi. we utilized a francisella novicida transposon mutant library to identify additional regulatory fa ...200818559431
characterization of fig operon mutants of francisella novicida u112.francisella species secrete a polycarboxylate siderophore that resembles rhizoferrin to acquire ferric iron. several of the francisella siderophore synthesis genes are contained in a fur-regulated operon (designated fig or fsl) comprised of at least seven orfs including fur. reverse transcriptase-pcr showed transcriptional linkage between figd and fige and between fige and figf. mutations were constructed in four of these orfs (figb, figc, figd, and fige) in francisella novicida u112. all four o ...200818564336
a francisella tularensis schu s4 purine auxotroph is highly attenuated in mice but offers limited protection against homologous intranasal challenge.francisella tularensis is a gram-negative coccobacillus that causes the febrile illness tularemia. subspecies that are pathogenic for humans include those comprising the type a (subspecies tularensis) or type b (subspecies holarctica) biovars. an attenuated live vaccine strain (lvs) developed from a type b isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 cfus) challenge with type a strains delivered by the respiratory route ...200818575611
characterization of the francisella tularensis subsp. novicida type iv pilus.francisella tularensis causes the disease tularaemia. type iv pili (tfp) genes are present in the genomes of all f. tularensis subspecies. we show that the wild-type f. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the tfp genes pilf and pilt disrupt pilus biogenesis. mutations in other tfp genes (pilq and pilg) do not eliminate pilus expression. a mutation in pile4 eliminates pilus expression, whereas mutations in the other pilin subunits pile1-3 and pile5 d ...200818599841
adaptation of francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro.the intracellular bacterium francisella tularensis survives in mammals, arthropods, and freshwater amoeba. it was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted francisella is unknown. as a foundation for our goal of identifying f. tularensis outer membrane proteins which are expressed during mam ...200818644878
construction of signature-tagged mutant library in mesorhizobium loti as a powerful tool for functional genomics.rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. the completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. in order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, mesorhizobium loti, by transposon mutagenesis. transposon insertion mutants were generated u ...200818658183
an improved vaccine for prevention of respiratory tularemia caused by francisella tularensis schus4 strain.vaccination of mice with francisella tularensis live vaccine strain (lvs) mutants described so far have failed to induce protection in c57bl/6 mice against challenge with the virulent strain f. tularensis schus4. we have previously reported that a mutant of f. tularensis lvs deficient in iron superoxide dismutase (sodb(ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tulare ...200818692537
microarray analysis of human monocytes infected with francisella tularensis identifies new targets of host response subversion.francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. one critical characteristic of francisella is its ability to dampen or subvert the host immune response. in order to help understand the mechanisms by which this occurs, we performed affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent type a schu s4 strain. results showed that expression of several host respons ...200818698339
periplasmic orientation of nascent lipid a in the inner membrane of an escherichia coli lpta mutant.the core-lipid a domain of escherichia coli lipopolysaccharide (lps) is synthesized on the inner surface of the inner membrane (im) and flipped to its outer surface by the abc transporter msba. recent studies with deletion mutants implicate the periplasmic protein lpta, the cytosolic protein lptb, and the im proteins lptc, lptf, and lptg in the subsequent transport of nascent lps to the outer membrane (om), where the lptd/lpte complex flips lps to the outer surface. we have isolated a temperatur ...200818768814
a novel receptor - ligand pathway for entry of francisella tularensis in monocyte-like thp-1 cells: interaction between surface nucleolin and bacterial elongation factor tu.francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. it is phagocytosed by immune cells, such as monocytes and macrophages. the precise mechanisms that initiate bacterial uptake have not yet been elucidated. participation of c3, cr3, class a scavenger receptors and mannose receptor in bacterial uptake have been already reported. however, contribution of an additional, as-yet-unidentified receptor for f. tularensis internalization has ...200818789156
effective host response to francisella tularensis requires functional mast cells.evaluation of: ketavarapu jm, rodriguez ar, yu j et al.: mast cells inhibit intramacrophage francisella tularensis replication via contact and secreted products including il-4. proc. natl acad. sci. usa 105(27), 9313-9318 (2008). the intracellular pathogen francisella tularensis is a highly infectious organism that infects cells of the immune system. mast cells have been known for their role in anaphylaxis, although they are also important for their ability to aid in the defense against pathogen ...200818811234
evasion of complement-mediated lysis and complement c3 deposition are regulated by francisella tularensis lipopolysaccharide o antigen.the bacterium francisella tularensis (ft) is a potential weapon of bioterrorism when aerosolized. macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. the nature of complement component c3 deposition, i.e., c3b (opsonization and lysis) or c3bi (opsonization onl ...200818832715
genetic dissection of the francisella novicida restriction barrier.francisella tularensis is the causative agent of tularemia and is a category a select agent. francisella novicida, considered by some to be one of four subspecies of f. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. f. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign dna up to 10(6)-fold. to identify the genetic basis of this barrier, we carried ou ...200818835994
global transcriptional response to mammalian temperature provides new insight into francisella tularensis pathogenesis.after infecting a mammalian host, the facultative intracellular bacterium, francisella tularensis, encounters an elevated environmental temperature. we hypothesized that this temperature change may regulate genes essential for infection.200818842136
rapid dissemination of francisella tularensis and the effect of route of infection.francisella tularensis subsp. tularensis is classified as a category a bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. however, the virulence mechanisms of this organism are not well characterized. francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a micropet scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such disseminatio ...200819068128
inhibition of expression in escherichia coli of a virulence regulator mglb of francisella tularensis using external guide sequence technology.external guide sequences (egss) have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. we previously reported that egs accessible and cleavable sites in the target rnas can rapidly be identified by screening random egs (regs) libraries. here the method of screening regs libraries and a partial rnase t1 digestion assay were used to identify sites accessible to egss in the mrna of a global virulence regulator mglb fro ...200819005569
interrelationship between dendritic cell trafficking and francisella tularensis dissemination following airway infection.francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. nevertheless, evidence for its in vivo intracellular residence is less conclusive. dendritic cells (dc) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. here, we focus on the in vivo interactions of f. tularensis with dc following airway infection of mice. lethal airw ...200819023422
francisella novicida bacteremia, thailand.we report isolation of francisella novicida-causing bacteremia in a woman from thailand who was receiving chemotherapy for ovarian cancer. the organism was isolated from blood cultures and identified by 16s rdna and ppiase gene analyses. diagnosis and treatment were delayed due to unawareness of the disease in this region.200819046526
francisella genes required for replication in mosquito cells.francisella tularensis, a potential bioterrorism agent, is transmitted by arthropod vectors and causes tularemia in many mammals, including humans. francisella novicida causes disease with similar pathology in mice. we show that f. novicida invades hemocyte-like cells of the sualb cell line derived from anopheles gambiae and replicates vigorously within these cells. we used transposon knockouts of single genes of f. novicida to show that bacterial growth within these insect cells is dependent on ...200819058636
comparison of a label-free quantitative proteomic method based on peptide ion current area to the isotope coded affinity tag method.recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. these so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. ...200819259412
an inhibitor of gram-negative bacterial virulence protein secretion.bacterial virulence mechanisms are attractive targets for antibiotic development because they are required for the pathogenesis of numerous global infectious disease agents. the bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. in this study, we developed and performed a high-throughput screen of small molecule libraries and identified one compound, a 2-imino ...200818854237
genomes of model organisms: know thy tools. 200818474084
a subset of the diverse cog0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life.cog0523 proteins are, like the nickel chaperones of the ureg family, part of the g3e family of gtpases linking them to metallocenter biosynthesis. even though the first cog0523-encoding gene, cobw, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family.200919822009
improved shuttle vectors for francisella tularensis genetics.we previously described the construction and characterization of escherichia coli-francisella tularensis shuttle vectors, derived from the cryptic francisella plasmid pfnl10, for the genetic manipulation of f. tularensis ssp. tularensis. we now report further characterization of the biology of these shuttle vectors and the development of a new generation of francisella plasmids. we show that the addition of orf3 from pfnl10 can convert an unstable shuttle vector into a stable one, and that this ...200919067747
improved shuttle vectors for francisella tularensis genetics.we previously described the construction and characterization of escherichia coli-francisella tularensis shuttle vectors, derived from the cryptic francisella plasmid pfnl10, for the genetic manipulation of f. tularensis ssp. tularensis. we now report further characterization of the biology of these shuttle vectors and the development of a new generation of francisella plasmids. we show that the addition of orf3 from pfnl10 can convert an unstable shuttle vector into a stable one, and that this ...200919067747
francisella tularensis subsp. novicida isolated from a human in arizona.francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the centers for disease control and prevention. currently four known subspecies of f. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type a), holarctica (type b), mediasiatica, and novicida. because of the select agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular ...200919895698
eradication of intracellular francisella tularensis in thp-1 human macrophages with a novel autophagy inducing agent.autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. in this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, ar-12, on the survival of francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.200920003180
porphyromonas gingivalis resistance to polymyxin b is determined by the lipid a 4'-phosphatase, pgn_0524.to elucidate the genetic basis for the pronounced resistance that the oral pathogen, porphyromonas gingivalis (p. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin b.200920657724
the francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.francisella tularensis causes the human disease tularemia. f. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. f. tularensis virulence is dependent upon the francisella pathogenicity island (fpi), a cluster of genes that we show here shares homology with type vi secretion gene clusters in vibrio cholerae and pseudo ...200920054881
comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus coxiella.genetically distinct isolates of coxiella burnetii, the cause of human q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. moreover, correlations between c. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. to provide a more-complete understanding of c. burnetii's genetic diversity, evolution, and pathoge ...200919047403
comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus coxiella.genetically distinct isolates of coxiella burnetii, the cause of human q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. moreover, correlations between c. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. to provide a more-complete understanding of c. burnetii's genetic diversity, evolution, and pathoge ...200919047403
francisella tularensis regulates autophagy-related host cell signaling pathways.the gram-negative intracellular pathogen francisella tularensis is known for its ability to dampen host immune responses. we recently performed a microarray analysis comparing human monocyte responses to the highly virulent f. tularensis tularensis schu s4 strain (f.t.) versus the less virulent f. tularensis novicida (f.n.).(1) many groups of genes were affected, including those involved with autophagy and with the regulation of autophagy. here, we discuss the implications in the context of fran ...200919029814
replacement of lipopolysaccharide with free lipid a molecules in escherichia coli mutants lacking all core sugars.escherichia coli mutants deficient in 2-keto-3-deoxy-d-manno-octulosonic acid (kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by overexpression of msba, the inner membrane flippase for core-lipid a. these strains grow on broth with the tetraacylated precursor lipid iv(a) replacing lipopolysaccharide [meredith, t. c., et al. (2006) acs chem. biol. 1, 33-42]. deletion of kdta, which encodes the kdo transferase, is possible under the ...200919754149
differentiation of gram-negative bacterial aerosol exposure using detected markers in bronchial-alveolar lavage fluid.the identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. in particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. we examine a mouse model of aerosol exposure to known gram negative bacterial pathogens, francisella tularensis novicida and pseudomonas aeruginosa. mice were subjected to either a pathogen or control exposure and ...200919756149
rela regulates virulence and intracellular survival of francisella novicida.analysis of the genome of francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. the stringent response is a global stress response mediated by (p)ppgpp. the enzyme rela has been shown to be involved in generation of this signal molecule in a range of bacterial species. we investigated the effect of inactivation of the rela gene in francisella by generating a mutant in francisella novicida. under amino acid ...200919762448
francisella tularensis novicida proteomic and transcriptomic data integration and annotation based on semantic web technologies.this paper summarises the lessons and experiences gained from a case study of the application of semantic web technologies to the integration of data from the bacterial species francisella tularensis novicida (fn). fn data sources are disparate and heterogeneous, as multiple laboratories across the world, using multiple technologies, perform experiments to understand the mechanism of virulence. it is hard to integrate these data sources in a flexible manner that allows new experimental data to b ...200919796400
nemesys: a biological resource for narrowing the gap between sequence and function in the human pathogen neisseria meningitidis.genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. however, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. this holds true for neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia.200919818133
crystal structures of the histidine acid phosphatase from francisella tularensis provide insight into substrate recognition.histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic ph using an active-site histidine. the histidine acid phosphatase from the category a pathogen francisella tularensis (fthap) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. here, we report a structure-based study of ligand recognition by fthap. the 1.70-a-resolution structure of fthap com ...200919836403
computational prediction of essential genes in an unculturable endosymbiotic bacterium, wolbachia of brugia malayi.wolbachia (wbm) is an obligate endosymbiotic bacterium of brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. there is a pressing need for new drugs against filarial parasites, such as b. malayi. as wbm is required for b. malayi development and fertility, targeting wbm is a promising approach. however, the lifecycle of neither b. malayi nor wbm can be maintained in vitro. to facilitate selection of potential drug targets we computationa ...200919943957
working toward the future: insights into francisella tularensis pathogenesis and vaccine development.francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. recent advances in the field of francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. in this review, we summarize the major properties of several francisella species, including f. tularensis ...200919946137
recombinant attenuated listeria monocytogenes vaccine expressing francisella tularensis iglc induces protection in mice against aerosolized type a f. tularensis.fransicella tularensis, the causative agent of tularemia, is in the top category (category a) of potential agents of bioterrorism. to develop a safer vaccine against aerosolized f. tularensis, we have employed an attenuated listeria monocytogenes, which shares with f. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for f. tularensis antigens. we constructed recombinant l. monocytogenes (rlm) vaccines stably expressing seven f. tularensis proteins including iglc ( ...200919126421
a sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid a.a novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, glcn), 2-amino-2-deoxygalactose (galactosamine, galn), and 4-amino-4-deoxyarabinose (aminoarabinose, aran), and for ethanolamine (etn), present in lipid a. this assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid a from a single sample. the method was applied to ...200919130491
remodelling of vipa/vipb tubules by clpv-mediated threading is crucial for type vi protein secretion.the recently identified type vi secretion systems (t6ss) have a crucial function in the virulence of various proteobacteria, including the human pathogen vibrio cholerae. t6ss are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of hcp and vgrg proteins in cell culture supernatants. here, we analysed the function of the v. cholerae t6ss member clpv, a specialized aaa+ protein. clpv is crucial for a functional t6ss and interacts through ...200919131969
inflammasome recognition of influenza virus is essential for adaptive immune responses.influenza virus infection is recognized by the innate immune system through toll like receptor (tlr) 7 and retinoic acid inducible gene i. these two recognition pathways lead to the activation of type i interferons and resistance to infection. in addition, tlr signals are required for the cd4 t cell and igg2a, but not cytotoxic t lymphocyte, responses to influenza virus infection. in contrast, the role of nod-like receptors (nlrs) in viral recognition and induction of adaptive immunity to influe ...200919139171
coxiella type iv secretion and cellular microbiology.coxiella burnetii is a widespread zoonotic bacterial pathogen that causes human q fever. in vivo, coxiella displays a tropism for mononuclear phagocytes where it participates in biogenesis of a lysosome-like replication compartment to conduct its obligate intracellular lifestyle. coxiella actively regulates multiple events during infection, presumably via proteins with effector functions that are delivered to the host cytosol by a dot/icm type iv secretion system. because the organism is current ...200919144560
biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in francisella novicida.lipid a of francisella tularensis subsp. novicida contains a galactosamine (galn) residue linked to its 1-phosphate group. as shown in the preceding paper, this galn unit is transferred to lipid a from the precursor undecaprenyl phosphate-beta-d-galn. a small portion of the free lipid a of francisella novicida is further modified with a glucose residue at position-6'. we now demonstrate that the two f. novicida homologues of escherichia coli arnc, designated flmf1 and flmf2, are essential for li ...200919166326
identification of undecaprenyl phosphate-beta-d-galactosamine in francisella novicida and its function in lipid a modification.francisella tularensis is a highly infectious pathogen that causes tularemia. francisella lipid a contains an unusual galactosamine (galn) unit, attached to its 1-phosphate moiety. two genes, flmf2 and flmk, are required for the addition of galn to francisella lipid a, but the relevant enzymes and the galn donor substrate have not been characterized. we now report the purification and identification of a novel minor lipid from francisella novicida that functions as the galn donor. on the basis o ...200919166327
a conserved alpha-helix essential for a type vi secretion-like system of francisella tularensis.francisella tularensis harbors genes with similarity to genes encoding components of a type vi secretion system (t6ss) recently identified in several gram-negative bacteria. these genes include igla and iglb encoding igla and iglb, homologues of which are conserved in most t6sss. we used a yeast two-hybrid system to study the interaction of the igl proteins of f. tularensis lvs. we identified a region of igla, encompassing residues 33 to 132, necessary for efficient binding to iglb, as well as f ...200919201795
francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain lvs.francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. we have shown that f. tularensis subspecies holarctica strain lvs prevents nadph oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. herein, we used random transposon mutagenesis to identify lvs genes that affect neutrophil activation. our initial screen identified cara, carb, and pyrb, which encode the small and large subunits of carbamoylphosphate synthase ...200919204089
a bayesian integration model of high-throughput proteomics and metabolomics data for improved early detection of microbial infections.high-throughput (htp) technologies offer the capability to evaluate the genome, proteome, and metabolome of an organism at a global scale. this opens up new opportunities to define complex signatures of disease that involve signals from multiple types of biomolecules. however, integrating these data types is difficult due to the heterogeneity of the data. we present a bayesian approach to integration that uses posterior probabilities to assign class memberships to samples using individual and mu ...200919209722
phylogeography of francisella tularensis: global expansion of a highly fit clone.francisella tularensis contains several highly pathogenic subspecies, including francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. the phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (snp) analysis, high-density microarray snp genotyping, and real-time-pcr-based canonical snp (cansnp) assays. almost 30,000 snps were identified among 13 whole genomes for phylogenetic analysis ...200919251856
single-copy chromosomal integration systems for francisella tularensis.francisella tularensis is a fastidious gram-negative bacterium responsible for the zoonotic disease tularemia. investigation of the biology and molecular pathogenesis of f. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. however, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. to expand the molecular toolbox we have developed two systems to stably inte ...200919332817
differential requirements for protection against mucosal challenge with francisella tularensis in the presence versus absence of cholera toxin b and inactivated f. tularensis.francisella tularensis is a category a biothreat agent for which there is no approved vaccine and the correlates of protection are not well understood. in particular, the relationship between the humoral and cellular immune response to f. tularensis and the relative importance of each in protection is controversial. yet, understanding this relationship will be crucial to the development of an effective vaccine against this organism. we demonstrate, for the first time, a differential requirement ...200919342669
identification of migr, a regulatory element of the francisella tularensis live vaccine strain iglabcd virulence operon required for normal replication and trafficking in macrophages.francisella tularensis, the etiological agent of tularemia, is capable of infecting a wide range of animals and causes a severe, lethal disease in humans. the pathogen evades killing by cells of the innate immune system utilizing genes encoding a pathogenicity island, including iglabcd, and instead utilizes these cells as a niche for replication and dissemination to other organs within the host. regulators of the igl genes (e.g., mgla, sspa, fevr and pmra) have been identified, but environmental ...200919349423
lethal pulmonary infection with francisella novicida causes depletion of alphabeta t cells from lungs.respiratory francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. in this study, mice infected intranasally with francisella novicida showed a depletion of alphabeta t cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. the depleted t cells were predominantly cd4(+). the alphabeta t cells in infected mice showed significantly higher levels of annexin v binding than those in mock control mi ...200919356746
characterization of rationally attenuated francisella tularensis vaccine strains that harbor deletions in the guaa and guab genes.francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10 -- 100cfu. f. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. vaccination with the live vaccine strain (lvs), developed from a type b strain, confers partial protection against aerosal exposure to the more virulent type a strains and provides proof of principle that a live attenuated vaccine strain may be efficacio ...200919368784
characterization of the pathogenicity island protein pdpa and its role in the virulence of francisella novicida.francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. a research surrogate for f. tularensis is francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. both francisella species contain a cluster of genes referred to as the francisella pathogenicity island (fpi). pathogenicity determinant protein a (pdpa), encoded by the pdpa gene, is located within the fpi ...200919372153
a francisella novicida pdpa mutant exhibits limited intracellular replication and remains associated with the lysosomal marker lamp-1.several genes contained in the francisella pathogenicity island (fpi) encode proteins needed for intracellular growth and virulence of francisella tularensis. the pdpa gene is the first cistron in the larger of the two operons found in the fpi. in this work we studied the intracellular growth phenotype of a francisella novicida mutant in the pdpa gene. the deltapdpa strain was capable of a small amount of intracellular replication but, unlike wild-type f. novicida, remained associated with the l ...200919372155
intracellular biology and virulence determinants of francisella tularensis revealed by transcriptional profiling inside macrophages.summary the highly infectious bacterium francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. here we have performed a global transcriptional profiling of the highly virulent f. tularensis ssp. tularensis schu s4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression prof ...200919388904
lethal pulmonary infection with francisella novicida is associated with severe sepsis.the bacterial or host determinants of lethality associated with respiratory francisella infections are currently unknown. no exo- or endotoxins that contribute to the severity of this disease have been identified. however, a deregulated host immune response upon infection is characterized by an initial 36- to 48-h delay followed by a rapid and excessive inflammatory response prior to death at 72-120 h. here, we extend these findings by comparing host immune responses between sublethal and lethal ...200919401387
the role of complement opsonization in interactions between f. tularensis subsp. novicida and human neutrophils.the remarkable infectiousness of francisella tularensis suggests that the bacterium efficiently evades innate immune responses that typically protect the host during its continuous exposure to environmental and commensal microbes. in our studies of the innate immune response to f. tularensis, we have observed that, unlike the live vaccine strain (lvs) of f. tularensis subsp. holarctica, f. tularensis subsp. novicida u112 opsonized in pooled human serum activated the nadph oxidase when incubated ...200919409509
identification of genes contributing to the virulence of francisella tularensis schu s4 in a mouse intradermal infection model.francisella tularensis is a highly virulent human pathogen. the most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other f. tularensis strains and that explain their high virulence. here we report the use of targeted mutagenesis to investigate the roles of various ...200919424499
comparative genomic characterization of francisella tularensis strains belonging to low and high virulence subspecies.tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. it is caused by infection by a gram-negative bacterium, francisella tularensis. in order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different francisella subspecies (subsp.): the f. tularensis sub ...200919478886
pyrin critical to macrophage il-1beta response to francisella challenge.relative to monocytes, human macrophages are deficient in their ability to process and release il-1beta. in an effort to explain this difference, we used a model of il-1beta processing and release that is dependent upon bacterial escape into the cytosol. fresh human blood monocytes were compared with monocyte-derived macrophages (mdm) for their il-1beta release in response to challenge with francisella novicida. although both cell types produced similar levels of il-1beta mrna and intracellular ...200919494323
environmental adaptation of francisella tularensis.concerns over weaponizable bacteria have recently prompted considerable interest in francisella tularensis (ft). in addition to its potential illicit use, ft occurs naturally in diverse ecological niches including mammals, arthropods, and fresh water protozoans. here we review the current knowledge of ft adaptation which has ramifications for both basic and applied research.200919524059
rationally designed tularemia vaccines.tularemia, caused by the gram-negative bacterium francisella tularensis, can be contracted by the bite of an arthropod vector or by inhalation. this disease occurs relatively infrequently but can be severe and even life-threatening if untreated. until recently, there were few laboratories studying this organism; however, concerns over its potential use as a biological weapon have led to renewed attention to f. tularensis research, particularly in the area of vaccine development. advances in the ...200919538114
waaa of the hyperthermophilic bacterium aquifex aeolicus is a monofunctional 3-deoxy-d-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.the hyperthermophile aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. although it has long been recognized that this unique gram-negative bacterium carries genes for different steps of lipopolysaccharide (lps) formation, data on the lps itself or detailed knowledge of the lps pathway beyond the first committed steps of lipid a and 3-deoxy-d-manno-oct-2-ulosonic acid (kdo) synthesis are still lacking. we now report the functional characterization of the thermostable kdo ...200919546212
human toll-like receptor 4 responses to p. gingivalis are regulated by lipid a 1- and 4'-phosphatase activities.signal transduction following binding of lipopolysaccharide (lps) to toll-like receptor 4 (tlr4) is an essential aspect of host innate immune responses to infection by gram-negative pathogens. here, we describe a novel molecular mechanism used by a prevalent human bacterial pathogen to evade and subvert the human innate immune system. we show that the oral pathogen, porphyromonas gingivalis, uses endogenous lipid a 1- and 4'-phosphatase activities to modify its lps, creating immunologically sile ...200919552698
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