Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

Hessian fly-associated bacteria: transmission, essentiality, and composition

Plant-feeding insects have been recently found to use microbes to manipulate host plant physiology and morphology. Gall midges are one of the largest groups of insects that manipulate host plants extensively. Hessian fly (HF, Mayetiola destructor) is an important pest of wheat and a model system for studying gall midges. To examine the role of bacteria in parasitism, a systematic analysis of bacteria associated with HF was performed for the first time. Diverse bacteria were found in different developmental HF stages. Fluorescent in situ hybridization detected a bacteriocyte-like structure in developing eggs. Bacterial DNA was also detected in eggs by PCR using primers targeted to different bacterial groups. These results indicated that HF hosted different types of bacteria that were maternally transmitted to the next generation. Eliminating bacteria from the insect with antibiotics resulted in high mortality of HF larvae, indicating that symbiotic bacteria are essential for the insect to survive on wheat seedlings. A preliminary survey identified various types of bacteria associated with different HF stages, including the genera Enterobacter, Pantoea, Stenotrophomonas, Pseudomonas, Bacillus, Ochrobactrum, Acinetobacter, Alcaligenes, Nitrosomonas, Arcanobacterium, Microbacterium, Paenibacillus, and Klebsiella. Similar bacteria were also found specifically in HF-infested susceptible wheat, suggesting that HF larvae had either transmitted bacteria into plant tissue or brought secondary infection of bacteria to the wheat host. The bacteria associated with wheat seedlings may play an essential role in the wheat-HF interaction.     

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.     

Molecular basis of 1,6-anhydro bond cleavage and phosphoryl transfer by Pseudomonas aeruginosa 1,6-anhydro-N-acetylmuramic acid kinase

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the γ phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.     

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus

Background

The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3–16); hence, the mechanisms responsible for changes in copy number at this locus are unclear.

Results

In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure.

Conclusions

Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-614) contains supplementary material, which is available to authorized users.     

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.     

Within-Subject Interlaboratory Variability of QuantiFERON-TB Gold In-Tube Tests

Background

The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) is a viable alternative to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, within-subject variability may limit test utility. To assess variability, we compared results from the same subjects when QFT-GIT enzyme-linked immunosorbent assays (ELISAs) were performed in different laboratories.

Methods

Subjects were recruited at two sites and blood was tested in three labs. Two labs used the same type of automated ELISA workstation, 8-point calibration curves, and electronic data transfer. The third lab used a different automated ELISA workstation, 4-point calibration curves, and manual data entry. Variability was assessed by interpretation agreement and comparison of interferon-γ (IFN-γ) measurements. Data for subjects with discordant interpretations or discrepancies in TB Response >0.05 IU/mL were verified or corrected, and variability was reassessed using a reconciled dataset.

Results

Ninety-seven subjects had results from three labs. Eleven (11.3%) had discordant interpretations and 72 (74.2%) had discrepancies >0.05 IU/mL using unreconciled results. After correction of manual data entry errors for 9 subjects, and exclusion of 6 subjects due to methodological errors, 7 (7.7%) subjects were discordant. Of these, 6 (85.7%) had all TB Responses within 0.25 IU/mL of the manufacturer''s recommended cutoff. Non-uniform error of measurement was observed, with greater variation in higher IFN-γ measurements. Within-subject standard deviation for TB Response was as high as 0.16 IU/mL, and limits of agreement ranged from −0.46 to 0.43 IU/mL for subjects with mean TB Response within 0.25 IU/mL of the cutoff.

Conclusion

Greater interlaboratory variability was associated with manual data entry and higher IFN-γ measurements. Manual data entry should be avoided. Because variability in measuring TB Response may affect interpretation, especially near the cutoff, consideration should be given to developing a range of values near the cutoff to be interpreted as “borderline,” rather than negative or positive.     

Size scaling of whole‐body metabolic activity in the noble crayfish (Astacus astacus) estimated from measurements on a single leg

1. Use of electron transport system (ETS) activity in a single leg for estimating whole‐body ETS activity was explored in the noble crayfish Astacus astacus. Oxygen consumption and ETS activity of the whole body and of a walking leg were measured in different‐sized animals at 10 °C to compare the size scaling of oxygen consumption, whole‐body ETS activity and the ratio of whole‐body ETS activity to oxygen consumption (ETS/R). 2. Electron transport system activity of a leg and the ratio of ETS activity of a whole crayfish to that of a leg were correlated with wet mass of animals. Therefore, metabolic potential in whole noble crayfish can be estimated on the basis of the measured ETS activity in a single leg and crayfish mass. This approach provides a valuable tool for determining metabolic characteristics of crayfish without killing them. 3. Mass‐specific oxygen consumption decreased with increasing wet mass, while ETS activity of whole crayfish showed no significant correlation with wet mass. Both oxygen consumption and ETS activity correlated significantly with protein mass. 4. The increase in ETS/R with increasing wet mass of the noble crayfish indicates that small organisms exploit a greater proportion of their metabolic potential for standard metabolism than larger ones. This is the first report on ETS/R in crayfish.     

Production of functionally active <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> isopenicillin N synthase in the yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Background  

β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha.