Jacqueline Inwald

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 σ-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.

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.

Spoligotype Diversity of Mycobacterium bovis Strains Isolated in France from 1979 to 2000

ABSTRACT The molecular fingerprints of 1,349 isolates ofMycobacterium bovis received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain Mycobacterium bovis BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of M. bovis across national borders.

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.

Molecular Epidemiology of Disease Due to Mycobacterium bovis in Humans in the United Kingdom

ABSTRACT 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.

Molecular Typing of Mycobacterium bovis Isolates from Cameroon

ABSTRACT In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.

Genetic composition of Mycobacterium bovis BCG substrain sofia

Mycobacterium bovis BCG remains the most widely used vaccine in the world, with a range of substrains that are named after their locations of production. More than 40 million doses of the BCG substrain Sofia are distributed annually through the United Nations Children’s Fund (UNICEF) and the Pan American Health Organization (PAHO) to approximately 120 different countries. However, in spite of its widespread use BCG Sofia has never been investigated at the genetic level, raising questions as to its genetic stability and exact provenance. The Bulgarian BCG Laboratory was established in 1949 when Dr. Srebra Rodopska obtained the BCG strain from the Institute Pasteur. However, due to suppurative cervical lymphadenitis in about 1% of orally vaccinated newborns, a few years later it was replaced with the Russian BCG strain. During 1972 lot 222 was approved as a master seed lot, originating the BCG substrain Sofia SL222. A working seed lot was produced from the master in 1993 from which all commercial lots are currently produced. To investigate the genetic properties of BCG Sofia, including its position on the BCG genealogical tree developed by Behr and colleagues (1), we performed genotyping assays on the master and working seed lots and on commercial batches no. 906 and 909 of BCG Sofia. We initially concentrated on genomic loci that are prone to variation. Spoligotyping exploits a polymorphic direct repeat (DR) locus that is composed of multiple 36-bp DR copies interspersed by unique spacers, with strains varying in the presence or absence of spacers (4). No difference among any of the BCG Sofia lots tested was found, with all strains showing patterns identical to those of other BCGs. Variable number of tandem repeat (VNTR) typing targeted six alleles (A through F [2]) that vary in the length and number of repeats at each target; i.e., 7-5-5-4-3-3 would have 7 copies of allele A, 5 of B, etc. (2). The VNTR profile of BCG Sofia was determined to be 5-5-5-2-3-3.1, which is identical to the profiles of all other BCG strains tested. A whole-genome analysis of BCG Sofia was undertaken by using M. tuberculosis microarrays (5). This disclosed a range of deletions and a duplication that places BCG Sofia in the same lineage as BCG Russia, in accordance with the BCG genealogy. However, a novel 1.6-kb deletion that affects the Rv3697c and Rv3698 homologues was revealed. To determine whether this deletion had occurred during in vitro propagation of BCG Sofia, we screened for this region in other BCG strains. The region was also deleted in BCG Russia but not in any other strain. Hence, this deletion occurred prior to the in vitro cultivation of BCG Sofia. Public concern over the safety of various vaccines demands that they should be described in exquisite detail (3). Our analyses show that BCG Sofia and BCG Russia are indistinguishable, although this does not preclude the existence of single nucleotide changes between the strains. We have therefore confirmed the genetic identity and provenance of a BCG vaccine that is given to over 40 million individuals a year.

Genomic Analysis of Mycobacterium tuberculosis Complex Strains Used for Production of Purified Protein Derivative

ABSTRACT 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.

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.

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, Wilks 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.