Functional analysis of a clonal deletion in an epidemic strain of Mycobacterium bovis reveals a role in lipid metabolism.

Previous work on the population structure of Mycobacterium bovis strains in Great Britain has identified highly successful clones which are expanding across the country. One such clone, designated M. bovis type 17, differs from all other members of the Mycobacterium tuberculosis complex in having a region of deletion, termed RDbovis(d)_0173, of seven genes between Mb1963c and Mb1971. Three of these genes have functions annotated in lipid metabolism. To explore the molecular basis for the success of this clone, we examined the impact of this deletion on lipid metabolism. While type 17 isolates had similar lipid composition to other M. bovis strains, their ability to incorporate propanoate into mycolic acids was remarkably low. When expressed as a reciprocal (the ratio of incorporation of label from acetate : propanoate into mycolic acids) the ratio was higher for all three type 17 field strains tested (mean: 18.90) than the values of 7.30 to 7.61 for other field strains (P < 0.002) and values < 6.50 for all other strains in the M. tuberculosis complex tested. The label from propanoate was diverted to pyruvate, at significantly higher levels in M. bovis type 17 than all other strains (P < 0.021). Complementation of M. bovis type 17 with an integrating cosmid, IE471, carrying the M. tuberculosis orthologues of Mb1963c-Mb1971 resulted in the ability of the recombinant strain to incorporate label from propanoate into mycolic acids in a manner similar to other strains. M. bovis type 17 : : IE471 labelled pyruvate from propanoate about four times more slowly than the parent strain. Thus, RDbovis(d)_0173 results in a profound effect on carbon metabolism, providing the ability to compensate for the inactivation of the ald and pykA genes, involved in pyruvate metabolism, that is seen in M. bovis (but not in M. tuberculosis). This shift in carbon metabolism may be a factor in the extraordinary clonal expansion reported for M. bovis type 17.

Genome plasticity of BCG and impact on vaccine efficacy.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Ecotypes of the Mycobacterium tuberculosis complex.

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth.

Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.

Microarray analysis of Mycobacterium microti reveals deletion of genes encoding PE-PPE proteins and ESAT-6 family antigens.

Mycobacterium microti is the agent of tuberculosis in wild voles and has been used as a live vaccine against tuberculosis in man and cattle. To explore the M. microti genome in greater detail, we used a M. tuberculosis H37Rv genomic DNA microarray to detect gene deletions among M. microti isolates. A number of deletions were identified that correlated with those described previously (Infect. Immun. 70 (2002) 5568) but a novel M. microti deletion was also found (MiD4) which removes 5 genes that code for ESAT-6 family antigens and PE-PPE proteins. Southern blot experiments showed that this region was also deleted from M. pinnipedii, a mycobacterium isolated from seals that is closely related to M. microti. Genes encoding ESAT-6 antigens and PE-PPE proteins appear to be frequently deleted from M. microti, and the implications of this are discussed.

Advanced significance analysis of microarray data based on weighted resampling: a comparative study and application to gene deletions in Mycobacterium bovis.

MOTIVATION: When analyzing microarray data, non-biological variation introduces uncertainty in the analysis and interpretation. In this paper we focus on the validation of significant differences in gene expression levels, or normalized channel intensity levels with respect to different experimental conditions and with replicated measurements. A myriad of methods have been proposed to study differences in gene expression levels and to assign significance values as a measure of confidence. In this paper we compare several methods, including SAM, regularized t-test, mixture modeling, Wilk's lambda score and variance stabilization. From this comparison we developed a weighted resampling approach and applied it to gene deletions in Mycobacterium bovis. RESULTS: We discuss the assumptions, model structure, computational complexity and applicability to microarray data. The results of our study justified the theoretical basis of the weighted resampling approach, which clearly outperforms the others.

Molecular epidemiology of disease due to Mycobacterium bovis in humans in the United Kingdom.

Mycobacterium bovis is the causative agent of bovine tuberculosis, with a wide host range. Fifty human M. bovis isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.

The population structure of Mycobacterium bovis in Great Britain: clonal expansion.

We have analyzed 11,500 isolates of Mycobacterium bovis (the cause of tuberculosis in cattle and other mammals) isolated in Great Britain (England, Wales and Scotland)] and characterized by spoligotype. Genetic exchange between cells is rare or absent in strains of the Mycobacterium tuberculosis complex so that, by using spoligotypes, it is possible to recognize "clones" with a recent common ancestor. The distribution of variable numbers of tandem repeats types in the most common clone in the data set is incompatible with random mutation and drift. The most plausible explanation is a series of "clonal expansions," and this interpretation is supported by the geographical distribution of different genotypes. We suggest that the clonal expansion of a genotype is caused either by the spread of a favorable mutation, together with all other genes present in the ancestral cell in which the mutation occurred, or by the invasion of a novel geographical region by a limited number of genotypes. A similar pattern is observed in M. tuberculosis (the main cause of tuberculosis in humans). The significance of clonal expansion in other bacteria that have recombination is discussed.

Inactivation of the Mycobacterium bovis homologue of the polymorphic RD1 gene Rv3879c (Mb3909c) does not affect virulence.

The RD1 locus is deleted from all strains of Mycobacterium bovis BCG but present in virulent isolates of M. bovis and Mycobacterium tuberculosis. The RD1 gene Rv3879c encodes a proline- and alanine-rich protein that shows sequence polymorphism across members of the M. tuberculosis complex. The role of this protein in virulence was investigated by deleting the Rv3879c homologue from M. bovis (Mb3909c) and testing the virulence of the mutant in the guinea pig model. The M. bovis Delta Mb3909c mutant was not attenuated in the guinea pig model, showing that this gene does not encode a virulence factor and plays no role in the attenuation caused by loss of RD1.

Genomic analysis of Mycobacterium tuberculosis complex strains used for production of purified protein derivative.

The genomes of the tuberculin production strains Mycobacterium bovis AN5 and Mycobacterium tuberculosis DT were compared to genome-sequenced tubercle bacilli by using DNA microarrays. Neither the AN5 nor DT strain suffered extensive gene deletions during in vitro passage. This suggests that bovine tuberculin made from M. bovis AN5 is suitable to detect infection with presently prevalent M. bovis strains.