Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria.

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-É£). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-É£) responses were measured in whole blood cultures using the IFN-É£ assay and the IFN-É£ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-É£ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-É£ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-É£ immune responses to the 10 R-Mag measured by the IFN-É£ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests.

Detection of bovine tuberculosis in herds with different disease prevalence and influence of paratuberculosis infection on PPDB and ESAT-6/CFP10 specificity.

Bovine tuberculosis (BTB) is a major animal health problem with zoonotic implications. Current control programs are based on test and slaughter strategies utilizing skin tests with tuberculins as antigens. The low specificity and associated operative difficulties of these tests have driven the search for new antigens and diagnostic assays. In this multicenter study, using herds from Argentina, Mexico and Northern Ireland, we selected skin test positive and negative animals from herds with different prevalence's of BTB and compared tuberculin (PPDB) and ESAT-6+CFP10 as antigens ex vivo. In low prevalence herds, crossreactivity of PPDB was apparent since up to 60% of the PPDB skin test and ex vivo positive animals did not responded to ESAT-6+CFP10 ex vivo. The superior specificity of ESAT-6+CFP10 was confirmed in a Mycobacterium avium sp. paratuberculosis infected herd where several of the animals had strong crossreactivity to PPDB and PPDA but not to ESAT-6+CFP10. In high prevalence herds 85% of the skin test-positive animals, were confirmed ex vivo using either PPDB or ESAT-6+CFP10 as antigen. However, within this group 60% of the skin test negative animals were PPDB and ESAT-6+CFP10 positive ex vivo indicating that the skin test can in some herds yield a significant number of false negative results. In conclusion, the ex vivo test is recommended as an ancillary test to accelerate BTB eradication. In high prevalence herds, PPDB or ESAT-6+CFP10 can be used as antigen whereas in low and medium prevalence herds ESAT-6+CFP10 is the preferred choice.

Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis: ESAT6-CFP10 mixture shows optimal sensitivity and specificity.

Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.

Self-assembly of recombinant prion protein of 106 residues.

The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform. In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice.

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.

Archaeal introns: splicing, intercellular mobility and evolution.

Until recently, it appeared that archaeal introns were spliced by a process specific to the archaeal domain in which an endoribonuclease cuts a 'bulge-helix-bulge' motif that forms at exon-intron junctions. Recent results, however, have shown that the endoribonuclease involved in archaeal intron splicing is a homologue of two subunits of the enzyme complex that excises eukaryotic nuclear tRNA introns. Moreover, some archaeal introns encode homing enzymes that are also encoded by group I introns.

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI.

I- Dmo I is a homing enzyme of the LAGLI-DADG type that recognizes up to 20 bp of DNA and is encoded by an archaeal intron of the hyperthermophilic archaeon Desulfurococcus mobilis . A combined mutational and DNA footprinting approach was employed to investigate the specificity of the I- Dmo I-substrate interaction. The results indicate that the enzyme binds primarily to short base paired regions that border the sites of DNA cleavage and intron insertion. The minimal substrate spans no more than 15 bp and while sequence degeneracy is tolerated in the DNA binding regions, the sequence and size of the cleavage region is highly conserved. The enzyme has a slow turnover rate and cuts the coding strand with a slight preference over the non-coding strand. Complex formation produces some distortion of the DNA double helix within the cleavage region. The data are compatible with the two DNA-binding domains of I- Dmo I bridging the minor groove, where cleavage occurs, and interacting within the major groove on either side, thereby stabilizing a distorted DNA double helix. This may provide a general mode of DNA interaction at least for the LAGLIDADG-type homing enzymes.

General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid.

Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two beta-galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O-nitrophenyl beta-D-galactopyranoside as substrate.

Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius.

Some intron-containing rRNA genes of archaea encode homing-type endonucleases, which facilitate intron insertion at homologous sites in intron- alleles. These archaeal rRNA genes, in contrast to their eukaryotic counterparts, are present in single copies per cell, which precludes intron homing within one cell. However, given the highly conserved nature of the sequences flanking the intron, homing may occur in intron- rRNA genes of other archaeal cells. To test whether this occurs, the intron-containing 23S rRNA gene of the archaeal hyperthermophile Desulfurococcus mobilis, carried on nonreplicating bacterial vectors, was electroporated into an intron- culture of Sulfolobus acidocaldarius. PCR experiments demonstrated that the intron underwent homing and spread through the culture. By using a double drug-resistant mutant of S. acidocaldarius, it was shown that spreading resulted partly from a selective advantage of intron+ cells and partly from intercellular mobility of the intron and homing.

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance.

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.

Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA.

A putative base-pairing interaction that determines the folding of the central region of 23S rRNA has been investigated by mutagenesis. Each of the possible base substitutions has been made at the phylogenetically covariant positions adenine-1262 (A1262) and U2017 in Escherichia coli 23S rRNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits to form translationally active ribosomes. The function of 23S rRNA is largely reestablished by introduction of an alternative G1262.C2017 or U1262.A2017 pair, although neither of these supports polysome formation quite as effectively as the wild-type pair. A 23S rRNA with a C1262.G2017 pair is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also indicate that these nucleotides engage in additional interactions within the ribosome and that these interactions determine what base pairs are acceptable there.

Erythromycin binding is reduced in ribosomes with conformational alterations in the 23 S rRNA peptidyl transferase loop.

The antibiotic erythromycin inhibits protein synthesis by binding to the 50 S ribosomal subunit, where the drug interacts with the unpaired bases 2058A and 2059A in the peptidyl transferase loop of 23 S rRNA. We used a chemical modification approach to analyse conformational changes that are induced by mutations in the peptidyl transferase loop, and to determine how these changes affect drug interaction. Mutations at positions 2057 (G-->A) and 2058 (A-->G, or -->U), all of which confer drug resistance, induce a more open conformation in the peptidyl transferase loop. Erythromycin still protects against chemical modification in the mutant peptidyl transferase loops, but the affinity of the drug interaction is reduced 20-fold in the 2057A mutant, 10(3)-fold in the 2058U mutant and 10(4)-fold in the 2058G mutant. Single mutations at position 2032 in the adjacent hairpin loop, which have previously been shown to alter drug tolerances, gave no detectable effects on the structure of the peptidyl transferase loop or on erythromycin binding. Dual mutations at positions 2032 and 2058, however, induce a marked change in the rRNA conformation with opening of the phylogenetically conserved base-pair 2063C.2447G, and confer a slow growth, drug-sensitive phenotype. The data suggest that the target site of erythromycin lies within the peptidyl transferase loop, and that limited disruption of the conformation of this site reduces drug binding, and consequently confers resistance. In addition, there is structurally and functionally important interaction between the drug target site in the peptidyl transferase loop and position 2032.

Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli.

The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous ribosome population, which consequently hinders direct probing of mutant rRNAs. Here, we describe how nonconserved helical regions of plasmid-coded rRNA have been altered in a manner that preserves their secondary structures while creating new sites for primer extension of mutant rRNAs. This facilitates specific biochemical probing of mutagenised rRNA regions despite the background of wild-type molecules. Four priming sites have been made to investigate the structural effects of mutations in the GTPase centre, helix 1200-1250, the peptidyl transferase region and the alpha-sarcin loop of 23S rRNA.