Biomedical subjects

Desmond M Collins

Publications and source records attributed to Desmond M Collins.

10 recordsLinked to original sources

PubMed2006

Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex.

The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distinguishing among members of the M. avium complex. Therefore, in this study, we have targeted the more variable 3' region of the hsp65 gene to determine whether it can effectively discriminate M. avium complex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified as M. avium complex. Sequences obtained indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were obtained for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains of M. avium subsp. paratuberculosis. A unique profile for M. avium subsp. silvaticum was not obtained. These results indicate that sequencing the 3' region of the hsp65 gene can simply and unambiguously distinguish species and subspecies of the M. avium complex.

Animals↗

PubMed2006

Differentiating host-associated variants of Mycobacterium avium by PCR for detection of large sequence polymorphisms.

The Mycobacterium avium species consists of a group of organisms that are genetically related but phenotypically diverse, with certain variants presenting clear differences in terms of their host association and disease manifestations. The ability to distinguish between these subtypes is of relevance for accurate diagnosis and for control programs. Using a comparative genomics approach, we have uncovered large sequence polymorphisms that are, respectively, absent from bird-type M. avium isolates and from cattle types and sheep types of M. avium subsp. paratuberculosis. By evaluating the distribution of these genomic polymorphisms across a panel of strains, we were able to assign unique genomic signatures to these host-associated variants. We propose a simple PCR-based strategy based on these polymorphisms that can rapidly type M. avium isolates into these subgroups.

Animals↗

PubMed2005

Generation of attenuated Mycobacterium bovis strains by signature-tagged mutagenesis for discovery of novel vaccine candidates.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

Animals↗

PubMed2005

Transposon mutagenesis of Mb0100 at the ppe1-nrp locus in Mycobacterium bovis disrupts phthiocerol dimycocerosate (PDIM) and glycosylphenol-PDIM biosynthesis, producing an avirulent strain with vaccine properties at least equal to those of M. bovis BCG.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium bovis, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.

Animals↗

PubMed2004

Extensive genomic polymorphism within Mycobacterium avium.

We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms (LSPs) comprising over 700 kb that distinguish M. avium subsp. avium from M. avium subsp. paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains the in vitro mycobactin dependence of M. avium subsp. paratuberculosis.

Chromosomes, Bacterial↗

PubMed2003

Different susceptibility of two animal species infected with isogenic mutants of Mycobacterium bovis identifies phoT as having roles in tuberculosis virulence and phosphate transport.

The Mycobacterium tuberculosis complex includes Mycobacterium bovis, which causes tuberculosis in most mammals, including humans. In previous work, it was shown that M. bovis ATCC 35721 has a mutation in its principal sigma factor gene, sigA, causing a single amino acid change affecting binding of SigA with the accessory transcription factor WhiB3. ATCC 35721 is avirulent when inoculated subcutaneously into guinea pigs but can be restored to virulence by integration of wild-type sigA to produce M. bovis WAg320. Subsequently, it was surprising to discover that WAg320 was not virulent when inoculated intratracheally into the Australian brushtail possum (Trichosurus vulpecula), a marsupial that is normally very susceptible to infection with M. bovis. In this study, an in vivo complementation approach was used with ATCC 35721 to produce M. bovis WAg322, which was virulent in possums, and to identify the virulence-restoring gene, phoT. There are two point deletions in the phoT gene of ATCC 35721 causing frameshift inactivation, one of which is also in the phoT of BCG. Knockout of phoT from ATCC 35723, a virulent strain of M. bovis, produced M. bovis WAg758, which was avirulent in both guinea pigs and possums, confirming that phoT is a virulence gene. The effect on virulence of mode of infection versus animal species susceptibility was investigated by inoculating all the above strains by aerosol into guinea pigs and mice and comparing these to the earlier results. Characterization of PhoT indicated that it plays a role in phosphate uptake at low phosphate concentrations. At least in vitro, this role requires the presence of a wild-type sigA gene and appears separate from the ability of phoT to restore virulence to ATCC 35721. This study shows the advantages of using different animal models as tools for the molecular biological investigation of tuberculosis virulence.

Animals↗

PubMed2002

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth.

Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-515-->His) in the 4.2 domain of RpoV. We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M. tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-515-->His) allele. Infection of mice and guinea pigs with a M. tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model. In contrast, an M. bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs. However, we found that immunocompetent mice infected with the M. tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M. tuberculosis. Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection. Our results imply that M. tuberculosis replication per se is not a sufficient condition for virulence in vivo. They also indicate a different role for M. bovis and M. tuberculosis whiB3 genes in pathogenesis generated in different animal models. We propose that M. tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host.

Amino Acid Sequence↗

PubMed2002

Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis.

Bacille Calmette-Guerin (BCG) is the world's most widely used vaccine, but there are concerns that it provides little protection against pulmonary tuberculosis of humans in countries that have a high prevalence of environmental mycobacteria. Experiments in cattle provide a model to investigate this situation and to develop an improved tuberculosis vaccine. In the third of a series of BCG vaccination trials, calves had high interferon-gamma (IFN-gamma) responses to purified protein derivative (PPD) from Mycobacterium avium prior to vaccination, indicating that infection with environmental mycobacteria had occurred. The calves vaccinated with BCG had minimal protection against an experimental intratracheal challenge with virulent Mycobacterium bovis. In comparison, calves vaccinated with either of two newly-derived attenuated M. bovis strains had significantly better but not complete protection against the development of tuberculous lesions compared to both BCG-vaccinated and non-vaccinated animals. Vaccination with the newly-derived attenuated M. bovis strains induced strong IFN-gamma and interleukin-2 (IL-2) responses to PPD from M. bovis at 2 weeks after vaccination, while BCG vaccination induced only a weak response at this time. In association with the previous two trials, the results suggest that sensitisation of the calves to environmental mycobacteria adversely affected subsequent protective efficacy of BCG. However, the results of vaccination with the other two attenuated M. bovis strains indicated that improved tuberculosis vaccines could be developed for such sensitised animals.

Animals↗

PubMed2002

Mycobacterium avium subsp. paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a novel DNA sequence difference.

A DNA sequence differing between sheep and cattle types of Mycobacterium avium subsp. paratuberculosis was identified and used to develop a PCR test. The test unequivocally distinguished all sheep types from cattle types and was negative for a wide range of other strains from the Mycobacterium avium-Mycobacterium intracellulare complex. The test will be useful for epidemiological purposes, particularly in hosts such as deer that can be easily infected with either type.

Animals↗

PubMed1998

Antisense RNA to ahpC, an oxidative stress defence gene involved in isoniazid resistance, indicates that AhpC of Mycobacterium bovis has virulence properties.

Antisense RNA is a versatile tool for reducing gene expression. It was used to determine if ahpC, a gene that is involved in defence against oxidative stress and isoniazid (INH) resistance, is important for virulence of Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. Antisense RNA constructs of ahpC were made using different strength promoters in front of a reversed coding sequence of ahpC. These constructs were electroporated into a virulent wild-type M. bovis strain and a moderately virulent INH-resistant M. bovis strain that was catalase/peroxidase-negative. Down-regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains containing antisense RNA were markedly less virulent than their parent strains in guinea pigs. M. bovis with an up-regulated ahpC gene was more resistant to cumene hydroperoxide than its parent strain, which had a wild-type ahpC promoter. These results agree with a model of INH resistance in which overexpression of AhpC compensates in some INH-resistant strains for loss of catalase/peroxidase by maintaining the ability to defend against oxidative stress mediated through organic peroxides. In addition, normal expression of AhpC is crucial for maintaining the virulence of wild-type M. bovis, which has normal catalase/peroxidase levels.

Animals↗