Production of avirulent Mycobacterium bovis strains by illegitimate recombination with deoxyribonucleic acid fragments containing an interrupted ahpC gene

Wilson, Theresa; Wards, Barry J.; White, Stefan J.; Skou, B.; Lisle, Geoffrey W. de; Collins, Desmond M.

doi:10.1016/s0962-8479(97)90003-4

Cited by 20 publications

(13 citation statements)

References 25 publications

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“…This signature tag mutagenesis study differed from the two previous studies in using illegitimate recombination instead of transposon mutagenesis and an 8-week guinea pig model of M. bovis infection instead of a 3-week mouse model of M. tuberculosis infection. Many of the attenuating mutations that we identified were different from those found in the mouse studies, and all of them were different from attenuating mutations that we identified previously when employing illegitimate recombination with an in vitro screening method rather than signature tag mutagenesis (8,39). A different approach to signature tag mutagenesis based on determining the importance for virulence in mice of virtually all the nonessential genes in M. tuberculosis has also been employed (32).…”

Section: Discussionmentioning

confidence: 73%

“…The finding that almost half the attenuated mutants had mutations in such a small region of the chromosome was unexpected, as mutations in the other eight mutants were widely scattered through the chromosome. In addition, we had previously used other methods to identify a range of attenuated illegitimate mutants of M. bovis, none of which had mutations in the esxA region (8,39). An explanation for this disparity is that in earlier work we did not test all random mutants for virulence.…”

Section: Discussionmentioning

confidence: 99%

“…We have utilized this system extensively to produce illegitimate recombinants of M. bovis that were subsequently screened using surrogate markers of avirulence before testing the selected strains in a guinea pig model of virulence (8,39). Moderate numbers of avirulent strains were identified by this approach, and some of these strains, when tested for vaccine efficacy in a guinea pig model with aerosol challenge, were found to produce protection against virulent M. bovis that was at least as good as that provided by M. bovis BCG (8,13).…”

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Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

et al. 2005

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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.Tuberculosis exacts enormous human suffering and death on a global basis and is also a widespread cause of animal morbidity and mortality. The primary human pathogen is Mycobacterium tuberculosis, although in many regions a significant amount of human disease results from infection with the very closely related organisms Mycobacterium africanum and the major animal pathogen Mycobacterium bovis. Together with a few less important species, these organisms are collectively referred to as the M. tuberculosis complex. The high degree of similarity in the pathogenesis of disease caused by these pathogens and the recent findings from genomic studies that the vast majority of genes within these species are identical or nearly so (16,22) clearly indicate that the majority of their virulence genes are shared in common. There is particular interest in discovering the identity of these genes, so that new strategies including improved vaccines and antibacterials can be developed for combating tuberculosis.Over the last 15 years, there has been continual development of molecular genetic techniques that can be applied to the M. tuberculosis complex (2). This enabled the discovery of individual tuberculosis virulence genes from 1995 onwards (7, 38) and led to the application over the last 5 years of a number of global techniques for identifying large numbers of genes that are essential for virulence or whose expression is altered during different stages of infection and disease. Among these global techniques, signature tag mutagenesis (18) is widely recognized as a powerful tool for investigating pathogenic organisms because of its ability to directly identify mutants that have reduced virulence (24). This identific...

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

et al. 2005

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“…Attempts to achieve homologous recombination in M. tuberculosis and Mycobacterium bovis BCG revealed a high rate of illegitimate recombination, with random insertion of linear DNA fragments into the chromosome (23,35). Despite this finding, intraplasmic (8,26) and interplasmic (8) recombination experiments have demonstrated that homologous recombination does occur in slowgrowing mycobacteria, although at a significantly lower rate than illegitimate recombination.…”

Section: Discussionmentioning

confidence: 99%

Disruption of the Genes Encoding Antigen 85A and Antigen 85B of Mycobacterium tuberculosis H37Rv: Effect on Growth in Culture and in Macrophages

Armitige¹,

Jagannath²,

Wanger³

et al. 2000

Infect Immun

186

173

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“…In addition to its extremely slow growth, which makes it timeconsuming to do the standard types of gene inactivation (it takes 3 weeks for a single M. tuberculosis cell to become a visible colony on solid media), this bacterium was thought to have lower rates of homologous recombination and higher rates of illegitimate recombination than other mycobacteria, which would complicate gene disruption by standard gene replacement techniques. In fact, earlier attempts to inactivate M. tuberculosis genes by allelic exchange resulted in illegitimate recombination (148,316). The original observation that the M. tuberculosis RecA protein was synthesized with an intein (protein intron) that had to be spliced out (65) led to speculation that this process contributed to the reported low levels of homologous recombination in this species (65,187).…”

Section: Methods Of Genetic Analysis In Mycobacteriamentioning

confidence: 99%

Mycobacterium tuberculosisPathogenesis and Molecular Determinants of Virulence

2003

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Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents

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Production of avirulent Mycobacterium bovis strains by illegitimate recombination with deoxyribonucleic acid fragments containing an interrupted ahpC gene

Cited by 20 publications

References 25 publications

Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

Disruption of the Genes Encoding Antigen 85A and Antigen 85B of Mycobacterium tuberculosis H37Rv: Effect on Growth in Culture and in Macrophages

Mycobacterium tuberculosisPathogenesis and Molecular Determinants of Virulence

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