Towards dual SPECT/optical bioimaging with a mitochondrial targeting, 99mTc(i) radiolabelled 1,8-naphthalimide conjugate.

A series of six different 1,8-naphthalimide conjugated dipicolylamine ligands (L1-6) have been synthesised and characterised. The ligands possess a range of different linker units between the napthalimide fluorophore and dipcolylamine chelator which allow the overall lipophilicity to be tuned. A corresponding series of Re(i) complexes have been synthesised of the form fac-[Re(CO)3(L1-6)]BF4. The absorption and luminescence properties of the ligands and Re(i) complexes were dominated by the intramolecular charge transfer character of the substituted fluorophore (typically absorption ca. 425 nm and emission ca. 520 nm). Photophysical assessments show that some of the variants are moderately bright. Radiolabelling experiments using a water soluble ligand variant (L5) were successfully undertaken and optimised with fac-[99mTc(CO)3(H2O)3]+. Confocal fluorescence microscopy showed that fac-[Re(CO)3(L5)]+ localises in the mitochondria of MCF-7 cells. SPECT/CT imaging experiments on naïve mice showed that fac-[99mTc(CO)3(L5)]+ has a relatively high stability in vivo but did not show any cardiac uptake, demonstrating rapid clearance, predominantly via the biliary system along with a moderate amount cleared renally.

Predatory bacteria can protect SKH-1 mice from a lethal plague challenge.

With the rise of antimicrobial resistance, novel ways to treat bacterial infections are required and the use of predatory bacteria may be one such approach. Bdellovibrio species have been shown in vitro to predate on a wide range of other Gram-negative bacteria, including CDC category A/B pathogens such as Yersinia pestis. The data reported here show that treatment of SKH-1 mice with Bdellovibrio bacteriovorus HD100 provided significant protection from a lethal challenge of Yersinia pestis CO92. This is the first report of protection conferred by predation in vivo against a systemic pathogen challenge. However, this protective effect was not observed in a preliminary study with Balb/c mice. Therefore the effects of the predatory bacteria are complex and may be dependent on immune status/genetics of the host. Overall, predatory bacteria may have utility as a therapeutic modality but further work is required to understand the predator-host interaction.

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of ß-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

Substrate recognition and mechanism revealed by ligand-bound polyphosphate kinase 2 structures.

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.

Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4.

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction.

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel ß-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.

Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis.

The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 µM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.

Evaluating the role of phage-shock protein A in Burkholderia pseudomallei.

The phage-shock protein (Psp) response is an extracytoplasmic response system that is vital for maintenance of the cytoplasmic membrane when the cell encounters stressful conditions. The paradigm of the Psp response has been established in Escherichia coli. The response has been shown to be important for survival during the stationary phase, maintenance of the proton motive force across membranes and implicated in virulence. In this study, we identified a putative PspA homologue in Burkholderia pseudomallei, annotated as BPSL2105. Similar to the induction of PspA in E. coli, the expression of B. pseudomallei BPSL2105 was induced by heat shock. Deletion of BPSL2105 resulted in a survival defect in the late stationary phase coincident with dramatic changes in the pH of the culture medium. The B. pseudomallei BPSL2105 deletion mutant also displayed reduced survival in macrophage infection - the first indication that the Psp response plays a role during intracellular pathogenesis in this species. The purified protein formed large oligomeric structures similar to those observed for the PspA protein of E. coli, and PspA homologues in Bacillus, cyanobacteria and higher plants, providing further evidence to support the identification of BPSL2105 as a PspA-like protein in B. pseudomallei.

The importance of the magnesium transporter MgtB for virulence of Yersinia pseudotuberculosis and Yersinia pestis.

Mg(2+) has been shown to be an important signal controlling gene regulation via the PhoPQ two-component regulatory system for a range of Gram-negative bacteria, including Yersinia pestis and Yersinia pseudotuberculosis. The magnesium ion transporter MgtB is part of the complex PhoPQ regulon, being upregulated in response to low Mg(2+). Despite the presence of other Mg(2+) transport systems in Yersinia, inactivation of mgtB had a significant effect on the ability of the bacteria to scavenge this crucial ion. Whereas inactivation of PhoPQ is reported to adversely affect intracellular survival, we show that Y. pestis and Y. pseudotuberculosis ΔmgtB mutants survived equally as well as the respective parent strain within macrophages, although they were more sensitive to killing in the Galleria model of infection. Surprisingly, despite MgtB being only one member of the Mg(2+) stimulon and PhoPQ controlling the expression levels of a range of genes including mgtB, the Yersinia ΔmgtB mutants were more highly attenuated than the equivalent Yersinia ΔphoP mutants in mouse models of infection. MgtB may be a suitable target for development of novel antimicrobials, and investigation of its role may help elucidate the contribution of this component of the PhoPQ regulon to pathogenesis.

Development of rationally designed DNA N6 adenine methyltransferase inhibitors.

A series of bisubstrate inhibitors for DNA N6 adenine methyltransferase (Dam) have been synthesized by linking an amine analogue of S-adenosylmethionine to an aryl moiety designed to probe the binding pocket of the DNA adenine base. An initial structure-activity relationship study has identified substituents that increase inhibitor potency to the ∼10 µM range and improve selectivity against the human cytosine methyltransferase Dnmt1.

An assessment of common marmoset (Callithrix jacchus) γ9(+) T cells and their response to phosphoantigen in vitro.

γ9δ2 T cells are a primate-specific γδ T cell subtype that expand and become activated during infection, responding directly to phosphoantigens which are by-products of essential metabolic pathways in both bacteria and mammals. Analogues of natural phosphoantigens have been developed as potential immunotherapeutics for treatment of tumours and infectious diseases. Several non-human primate models have been used in preclinical studies, however, little is known about marmoset γ9δ2 T cell responses. We identified γ9(+) T cells in various tissues in the marmoset and determined that these cells respond to phosphoantigen in a similar manner to human γ9δ2 T cells in vitro. Both human γ9δ2 T cells and marmoset γ9(+) T cells were able to reduce growth of the intracellular bacterium Burkholderia pseudomallei in vitro following expansion with phosphoantigen. This suggests that the marmoset is an appropriate model for examining the immunotherapeutic potential of compounds which target γ9δ2 T cells.

A Francisella tularensis SCHU S4 mutant deficient in γ-glutamyltransferase activity induces protective immunity: characterization of an attenuated vaccine candidate.

Francisella tularensis is an intracellular pathogen which causes tularaemia. There is no licensed vaccine currently available for prophylaxis. The γ-glutamyl transpeptidase (GGT) encoded by the ggt gene has been shown to be important for the intracellular survival of F. tularensis. In this study we have constructed a ggt deletion mutant in the highly virulent F. tularensis strain SCHU S4. Characterization of the mutant strain confirmed the function of ggt, and confirmed the role of GGT in cysteine acquisition. The mutant strain was highly attenuated both in vitro and in vivo using murine models of infection. Moreover, we have demonstrated that the attenuated mutant is able to induce protective immunity against an F. tularensis SCHU S4 challenge, and thus may be a candidate for the development of an attenuated vaccine.

Yersinia pseudotuberculosis mntH functions in intracellular manganese accumulation, which is essential for virulence and survival in cells expressing functional Nramp1.

Manganese has an important yet undefined role in the virulence of many bacterial pathogens. In this study we confirm that a null mutation in Yersinia pseudotuberculosis mntH reduces intracellular manganese accumulation. An mntH mutant was susceptible to killing by reactive oxygen species when grown under manganese-limited conditions. The mntH mutant was defective in survival and growth in macrophages expressing functional Nramp1, but in macrophages deficient in Nramp the bacteria were able to survive and replicate. In Galleria mellonella, the mntH mutant was attenuated. Taken together, these data suggest a role for manganese in Y. pseudotuberculosis during macrophage intracellular survival, protecting the bacteria from the antimicrobial products released during the respiratory burst.

Potential of the beta-glucans to enhance innate resistance to biological agents.

The use of numerous mushroom species in traditional medicine has been widely documented, with their observed immunomodulatory effects now attributed, in part, to bioactive components called beta-glucans. The beta-glucans are of particular interest since they are naturally occurring polymers of glucose, are orally active when taken as food supplements and have a long track record of safe use. Due to their immunomodulatory properties, purified beta-glucans have been used clinically as part of a combination therapy for a variety of cancers and their potential anti-infective properties have received attention. This review relates the structure of beta-glucans to their function, with a particular focus on their documented immunomodulatory effects and the mechanisms by which they affect inter- and intracellular function, resulting in potential antimicrobial benefits. Overall, the benefits of dietary supplementation with beta-glucans in order to enhance innate resistance to biological agents are evaluated.

An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella.

Francisella tularensis is an intracellular pathogen capable of multiplying to high levels in macrophages. By protein analysis, only a few proteins have been shown previously to be expressed at high levels in macrophages relative to bacteria grown in culture media. To identify additional genes that show increased expression during intracellular growth, we developed a plasmid for use in Francisella based on the induction of expression of green fluorescent protein. Clones of F. tularensis subsp. novicida were identified that were fluorescent only intracellularly and not when grown in vitro. Sequencing identified a range of genes comprising some such as dnaK that are already known to be expressed intracellularly and some novel targets. One of these newly identified regulated genes, FTN1472/FTT1564, was selected for further study. Isogenic mutants were generated in F. tularensis subsp. novicida and subsp. tularensis by allelic replacement. Inactivation of the gene resulted in abolition of polyphosphate production by F. novicida, strongly supporting the bioinformatic analysis, which had suggested that the gene may encode a polyphosphate kinase. The mutants exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the putative polyphosphate kinase in the virulence of Francisella.

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, a novel iron-regulated gene, FTT0025, was identified which is part of this operon and encodes a 55 kDa hypothetical membrane protein. When grown on chrome azurol S agar, the F. tularensis subsp. novicida U112deltaFTT0025 mutant produced an increased reaction zone compared with the wild-type, suggesting that siderophore production was unaffected but that the bacteria may have a deficiency in their ability to re-sequester this iron-binding molecule. Furthermore, the deltaFTT0025 mutant was attenuated in a BALB/c mouse model of infection relative to wild-type F. tularensis subsp. novicida U112.

Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system.

Recently, a number of attenuated mutants of Yersinia pseudotuberculosis have been identified using a bioinformatics approach. One of the target genes identified in that study was vagH, which the authors now characterized further. VagH shows homology to HemK of Escherichia coli, possessing methyltransferase activity similar to that of HemK, and targeting release factors 1 and 2. Microarray studies comparing the wild-type and the vagH mutant revealed that the mRNA levels of only a few genes were altered in the mutant. By proteome analysis, expression of the virulence determinant YopD was found to be increased, indicating a possible connection between VagH and the virulence plasmid-encoded type III secretion system (T3SS). Further analysis showed that Yop expression and secretion were repressed in a vagH mutant. This phenotype could be suppressed by trans-complementation with the wild-type vagH gene or by deletion of the negative regulator yopD. Also, in a similar manner to a T3SS-negative mutant, the avirulent vagH mutant was rapidly cleared from Peyer's patches and could not reach the spleen after oral infection of mice. In a manner analogous to that of T3SS mutants, the vagH mutant could not block phagocytosis by macrophages. However, a vagH mutant showed no defects in the T3SS-independent ability to proliferate intracellularly and replicated to levels similar to those of the wild-type in macrophages. In conclusion, the vagH mutant exhibits a virulence phenotype similar to that of a T3SS-negative mutant, indicating a tight link between VagH and type III secretion in Y. pseudotuberculosis.

Pathogenesis of Yersinia pestis infection in BALB/c mice: effects on host macrophages and neutrophils.

The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 10(3) to 10(4) CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.