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Phenotypic and transcriptomic response of auxotrophic Mycobacterium avium subsp. paratuberculosis leuD mutant under environmental stress.

Chen JW, Scaria J, Chang YF - PLoS ONE (2012)

Bottom Line: Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response.The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses.Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

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Expression profile of virulence associated MAP genes under various stress conditions.Virulence associated MAP gene definitions were obtained by phylogenomics comparison with other mycobacteria in VFDB. MAP transcriptional expression profiles were then combined with each genes definition. Each column represents a stress condition and each row represents a gene. The stress conditions given as numbers at the top of columns are as follows; 1-Control, 2-Anaerobic, 3-Minimal medium, 4- Temperature, 5-pH 9.0 and 6-pH 5.5.
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pone-0037884-g005: Expression profile of virulence associated MAP genes under various stress conditions.Virulence associated MAP gene definitions were obtained by phylogenomics comparison with other mycobacteria in VFDB. MAP transcriptional expression profiles were then combined with each genes definition. Each column represents a stress condition and each row represents a gene. The stress conditions given as numbers at the top of columns are as follows; 1-Control, 2-Anaerobic, 3-Minimal medium, 4- Temperature, 5-pH 9.0 and 6-pH 5.5.

Mentions: Comparative phylogenomics is a very useful method for deducing the functions of less studied bacterial genomes by extrapolating data from related species. Although there are not many functional genomics studies in MAP, many related species such as M. tuberculosis, M. smegamatis, M. bovis, M. avium, and M. leprae have been studied in more detail. We used the curated phylogenomics data on other mycobacteria from Virulence Factor Database (VFDB) [28] to identify virulence associated genes in MAP(Table S6). We then combined this virulence gene list with microarray expression data from different stress conditions (Fig. 5). As Fig. 5 shows, leuD deletion influenced the expression profile of most of the virulence associated genes. It is interesting to note that leuD gene itself is annotated as a virulence associated gene in VFDB. Some of the virulence genes in VFDB have also been studied extensively in MAP. For example, the functions of MAP_0216 (fbpA), MAP_0187c (sodA) and MAP_3921(sodC) which were found to be modulated in this study are well defined.


Phenotypic and transcriptomic response of auxotrophic Mycobacterium avium subsp. paratuberculosis leuD mutant under environmental stress.

Chen JW, Scaria J, Chang YF - PLoS ONE (2012)

Expression profile of virulence associated MAP genes under various stress conditions.Virulence associated MAP gene definitions were obtained by phylogenomics comparison with other mycobacteria in VFDB. MAP transcriptional expression profiles were then combined with each genes definition. Each column represents a stress condition and each row represents a gene. The stress conditions given as numbers at the top of columns are as follows; 1-Control, 2-Anaerobic, 3-Minimal medium, 4- Temperature, 5-pH 9.0 and 6-pH 5.5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366959&req=5

pone-0037884-g005: Expression profile of virulence associated MAP genes under various stress conditions.Virulence associated MAP gene definitions were obtained by phylogenomics comparison with other mycobacteria in VFDB. MAP transcriptional expression profiles were then combined with each genes definition. Each column represents a stress condition and each row represents a gene. The stress conditions given as numbers at the top of columns are as follows; 1-Control, 2-Anaerobic, 3-Minimal medium, 4- Temperature, 5-pH 9.0 and 6-pH 5.5.
Mentions: Comparative phylogenomics is a very useful method for deducing the functions of less studied bacterial genomes by extrapolating data from related species. Although there are not many functional genomics studies in MAP, many related species such as M. tuberculosis, M. smegamatis, M. bovis, M. avium, and M. leprae have been studied in more detail. We used the curated phylogenomics data on other mycobacteria from Virulence Factor Database (VFDB) [28] to identify virulence associated genes in MAP(Table S6). We then combined this virulence gene list with microarray expression data from different stress conditions (Fig. 5). As Fig. 5 shows, leuD deletion influenced the expression profile of most of the virulence associated genes. It is interesting to note that leuD gene itself is annotated as a virulence associated gene in VFDB. Some of the virulence genes in VFDB have also been studied extensively in MAP. For example, the functions of MAP_0216 (fbpA), MAP_0187c (sodA) and MAP_3921(sodC) which were found to be modulated in this study are well defined.

Bottom Line: Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response.The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses.Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

Show MeSH
Related in: MedlinePlus