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Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M−1cm−1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1–3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50 = 603 CFU) as compared to mice challenged with the parent (LD50 = 1 CFU) or complemented strain (LD50 = 1 CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis , respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 Δ iclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔ iclR were equally cytotoxic and induced equivalent levels of interleukin-1β expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and Δ iclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.

Four US soldiers admitted to the Brian Allgood Army Community Hospital, Seoul, Korea, were diagnosed with hemorrhagic fever with renal syndrome, an acute febrile disease, from 27 October to 13 November 2005. Epidemiological investigations were conducted and rodents live trapped at military training sites where the soldiers had recently trained. Total RNA was extracted from patient sera and lung tissues of Apodemus agrarius, the primary reservoir of Hantaan virus (HTNV), amplified by RT-PCR, sequenced, and identified as HTNV. Phylogenetic relationships based on comparison of the 281-nt region of the G2 glycoprotein-encoding M segment of the patients and Murinae rodent-borne hantaviruses were conducted. HTNV amplified from A. agrarius captured at Firing Point 60 corresponded to patient 1, while HTNV amplified from A. agrarius captured at Twin Bridges Training Area corresponded to patients 2-4. These results strongly support that transmission sites were linked with patient and reservoir host HTNV strains.

Sensitive, rapid and reproducible detection of staphyloccocal enterotoxin B (SEB) in a range of different biological matrices was achieved using the ORIGEN((R)) Immunoassay System (Igen, Inc). The homologous immunoassay format consisted of a double antibody sandwich in which a biotinylated capture antibody, pre-bound to streptavidin-coated paramagnetic beads, was used to bind antigen from test samples. A detector antibody, labeled with ruthenium (II) tris-bipyridal chelate, was added and, when bound to the bead immunocomplex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the ORIGEN analyzer. The sensitivity of this assay was 1 pg of enterotoxin per ml of serum, urine, tissue, or buffer and was highly reproducible. Concentration curves generated from SEB standards produced consistently wide linear ranges (0.1-100 ng/ml), making quantitation possible with only two dilutions of sample (undiluted and 1:1000). The assay used 50 microl of sample per test and required a 30 min incubation period in addition to a 1 min per tube reading time (50 tubes maximum). This assay was significantly better in terms of sensitivity, linear range, and assay time than the standard microplate enzyme-linked immunosorbent assay and should permit early SEB detection in clinical samples, food, and environmental samples.

The threat of biological warfare and bioterrorism has increased in the last two decades. Classical biological threats that were part of the now-defunct U.S. offensive program (terminated in 1969) included Bacillus anthracis, Botulinum toxin, Francisella tularensis, Coxiella burnetii, Venezuelan equine encephalitis (VEE), Brucella suis(brucellosis), Staphylococcal enterotoxin B (SEB), rice blast, rye stem rust, and wheat stem rust. Most of these agents can be produced cheaply by aggressors and could have a larger impact on both military and civilian populations that other weapons of mass destruction. In the hands of a terrorist, these agents can cause great psychological harm and social disruption as well. Of the agents that can cause human disease, all except VEE and C. burnetii could be effectively transmitted through contaminated food and water. Classical methods for identifying biological agents that cause human disease have been used for more than 100 years and are well established. These methods rely on agent cultivation, taking between 3 and 30 days, and require experienced personnel working in a well-equipped laboratory. In order to achieve agent identification with a high level of confidence, a combination of state-of-the-art immunological and nucleic acid analyses methods, as well as classical microbiological approaches, are needed to identify unknown biological agents. Methods based on the immunomagnetic-electrochemiluminescence assay for antigen detection are among the most sensitive.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

BackgroundFrancisella tularensisis a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved amongFrancisellaspecies and is required for intracellular growth.F. tularensis ripAdeletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm.ResultsFurther analysis ofripAwith respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR,ripA-lacZtranscriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that bothripAtranscription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, includingiglA, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantifiedripAandiglAexpression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns ofripAandiglAand that MglA and SspA are positive regulators ofiglAwe tested the impact ofmglAandsspAdeletions onripAandiglAexpression. In the deletion mutant strainsiglAexpression was reduced dramatically as expected, howeverripAexpression was increased over 2-fold.ConclusionExpression ofripAis required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern ofripAcoincided withiglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact oniglAexpression, MglA and SspA negatively impactedripAexpressionin vitro.

Francisella tularensisis a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of theF. tularensislive vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequencedFrancisellaspecies yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele intrans. Based on the deletion mutant phenotype, FTL_1914 was termedripA(required forintracellularproliferation, factorA). Following uptake by J774.A1 cells,F. tularensisLVSΔripAcolocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of theF. tularensisLVS ΔripAmutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. TheF. tularensisLVSΔripAmutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved amongFrancisellaspecies that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis -specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log 10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories.