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Differential gene repertoire in Mycobacterium ulcerans identifies candidate genes for patho-adaptation.

Käser M, Pluschke G - PLoS Negl Trop Dis (2008)

Bottom Line: Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression.Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.

View Article: PubMed Central - PubMed

Affiliation: Swiss Tropical Institute, Basel, Switzerland. m.kaeser@unibas.ch

ABSTRACT

Background: Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.

Methodology: Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise approximately 7% of the entire M. ulcerans genome.

Principal findings: Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.

Conclusions/significance: The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.

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Related in: MedlinePlus

Involvement of ISEs in genomic diversity.A) Positions of the RDs and ISEs on the genomes of M. marinum M (top) and M. ulcerans Agy99 (bottom). RDs1-15 are located at different positions upon alignment of the two genomes, and some RDs occur on several loci on the M. marinum sequence due to genomic rearrangement and dislocations that formed the M. ulcerans Agy99 genome. The ISEs IS2404 and IS2606 are indicated as black bars in the marked lane. Sequence comparison was illustrated using the Artemis Comparison Tool software release 5 [31]. B) Test for whole genome abundance of IS2404 and IS2606 throughout a world-wide M. ulcerans strain collection (n = 34) and M. marinum. Indicated are the numbers of observed ISE involvements in the analysed 7% of the genome and the experimentally determined copy numbers. Whereas M. marinum M and water controls were devoid of ISEs, the abundance of IS2404 and IS2606 was measured between the lineages by quantitative real-time PCR. C) CT values were normalized using a unique gene target to account for differences in template input and calculated into copy numbers/genome. *These values reflect approximate numbers calculated from obtained CT values. Note that minor changes in CT value differences result in dramatic changes of determined copy numbers, i.e. the retrieved calculated values for the IS2606 in the classical lineage, for which a genome information is available, deviate by a factor of three.
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pntd-0000353-g003: Involvement of ISEs in genomic diversity.A) Positions of the RDs and ISEs on the genomes of M. marinum M (top) and M. ulcerans Agy99 (bottom). RDs1-15 are located at different positions upon alignment of the two genomes, and some RDs occur on several loci on the M. marinum sequence due to genomic rearrangement and dislocations that formed the M. ulcerans Agy99 genome. The ISEs IS2404 and IS2606 are indicated as black bars in the marked lane. Sequence comparison was illustrated using the Artemis Comparison Tool software release 5 [31]. B) Test for whole genome abundance of IS2404 and IS2606 throughout a world-wide M. ulcerans strain collection (n = 34) and M. marinum. Indicated are the numbers of observed ISE involvements in the analysed 7% of the genome and the experimentally determined copy numbers. Whereas M. marinum M and water controls were devoid of ISEs, the abundance of IS2404 and IS2606 was measured between the lineages by quantitative real-time PCR. C) CT values were normalized using a unique gene target to account for differences in template input and calculated into copy numbers/genome. *These values reflect approximate numbers calculated from obtained CT values. Note that minor changes in CT value differences result in dramatic changes of determined copy numbers, i.e. the retrieved calculated values for the IS2606 in the classical lineage, for which a genome information is available, deviate by a factor of three.

Mentions: RDs 1 through 15 are evenly distributed on the genome as shown in Fig. 2. An overlay of positions of both ISEs and RDs (Fig. 3A) for the whole genome sequences of M. marinum M and M. ulcerans Agy99 shows that most RDs are associated with the presence of ISEs. Comparison of the two M. ulcerans lineages throughout RDs1 to 15 revealed a difference in ISE abundance (Fig. 2 and 3B), and Southern hybridization of representatives of the two lineages already indicated significant differences for IS2606 [32]. We therefore compared the number of whole genome IS2404 and IS2606 copies by quantitative real-time PCR (Fig. 3B, C). The estimated mean difference between the classical and ancestral lineage for IS2404 signals was 1.66 (95% CI = 0.64 to 2.68), indicating that the pronounced difference in abundance of IS2404 between the two lineages was largely restricted to the analysed RDs. However, for IS2606 an elevated CT value (27.24) was measured in the ancestral lineage resulting in an estimated mean difference between the lineages of 6.34 (95% CI = 4.87 to 7.81). This reflects a very low abundance of IS2606 in the whole genome of strains of the ancestral lineage, explaining the observed lack of IS2606 involvement in genome rearrangements in this lineage.


Differential gene repertoire in Mycobacterium ulcerans identifies candidate genes for patho-adaptation.

Käser M, Pluschke G - PLoS Negl Trop Dis (2008)

Involvement of ISEs in genomic diversity.A) Positions of the RDs and ISEs on the genomes of M. marinum M (top) and M. ulcerans Agy99 (bottom). RDs1-15 are located at different positions upon alignment of the two genomes, and some RDs occur on several loci on the M. marinum sequence due to genomic rearrangement and dislocations that formed the M. ulcerans Agy99 genome. The ISEs IS2404 and IS2606 are indicated as black bars in the marked lane. Sequence comparison was illustrated using the Artemis Comparison Tool software release 5 [31]. B) Test for whole genome abundance of IS2404 and IS2606 throughout a world-wide M. ulcerans strain collection (n = 34) and M. marinum. Indicated are the numbers of observed ISE involvements in the analysed 7% of the genome and the experimentally determined copy numbers. Whereas M. marinum M and water controls were devoid of ISEs, the abundance of IS2404 and IS2606 was measured between the lineages by quantitative real-time PCR. C) CT values were normalized using a unique gene target to account for differences in template input and calculated into copy numbers/genome. *These values reflect approximate numbers calculated from obtained CT values. Note that minor changes in CT value differences result in dramatic changes of determined copy numbers, i.e. the retrieved calculated values for the IS2606 in the classical lineage, for which a genome information is available, deviate by a factor of three.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2600814&req=5

pntd-0000353-g003: Involvement of ISEs in genomic diversity.A) Positions of the RDs and ISEs on the genomes of M. marinum M (top) and M. ulcerans Agy99 (bottom). RDs1-15 are located at different positions upon alignment of the two genomes, and some RDs occur on several loci on the M. marinum sequence due to genomic rearrangement and dislocations that formed the M. ulcerans Agy99 genome. The ISEs IS2404 and IS2606 are indicated as black bars in the marked lane. Sequence comparison was illustrated using the Artemis Comparison Tool software release 5 [31]. B) Test for whole genome abundance of IS2404 and IS2606 throughout a world-wide M. ulcerans strain collection (n = 34) and M. marinum. Indicated are the numbers of observed ISE involvements in the analysed 7% of the genome and the experimentally determined copy numbers. Whereas M. marinum M and water controls were devoid of ISEs, the abundance of IS2404 and IS2606 was measured between the lineages by quantitative real-time PCR. C) CT values were normalized using a unique gene target to account for differences in template input and calculated into copy numbers/genome. *These values reflect approximate numbers calculated from obtained CT values. Note that minor changes in CT value differences result in dramatic changes of determined copy numbers, i.e. the retrieved calculated values for the IS2606 in the classical lineage, for which a genome information is available, deviate by a factor of three.
Mentions: RDs 1 through 15 are evenly distributed on the genome as shown in Fig. 2. An overlay of positions of both ISEs and RDs (Fig. 3A) for the whole genome sequences of M. marinum M and M. ulcerans Agy99 shows that most RDs are associated with the presence of ISEs. Comparison of the two M. ulcerans lineages throughout RDs1 to 15 revealed a difference in ISE abundance (Fig. 2 and 3B), and Southern hybridization of representatives of the two lineages already indicated significant differences for IS2606 [32]. We therefore compared the number of whole genome IS2404 and IS2606 copies by quantitative real-time PCR (Fig. 3B, C). The estimated mean difference between the classical and ancestral lineage for IS2404 signals was 1.66 (95% CI = 0.64 to 2.68), indicating that the pronounced difference in abundance of IS2404 between the two lineages was largely restricted to the analysed RDs. However, for IS2606 an elevated CT value (27.24) was measured in the ancestral lineage resulting in an estimated mean difference between the lineages of 6.34 (95% CI = 4.87 to 7.81). This reflects a very low abundance of IS2606 in the whole genome of strains of the ancestral lineage, explaining the observed lack of IS2606 involvement in genome rearrangements in this lineage.

Bottom Line: Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression.Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.

View Article: PubMed Central - PubMed

Affiliation: Swiss Tropical Institute, Basel, Switzerland. m.kaeser@unibas.ch

ABSTRACT

Background: Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.

Methodology: Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise approximately 7% of the entire M. ulcerans genome.

Principal findings: Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.

Conclusions/significance: The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.

Show MeSH
Related in: MedlinePlus