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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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Comparison of gene expression fold change in key genes associated with mycolate biosynthesis pathway.Genes associated with mycolate biosynthesis were identified using pathway annotation in Biocyc and by ortholog mapping of genes as defined in mycolate biosynthesis pathway of M. tuberculosis. Gene expression fold change was measured using both microarrays, and qRT-PCR.
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pone-0037884-g008: Comparison of gene expression fold change in key genes associated with mycolate biosynthesis pathway.Genes associated with mycolate biosynthesis were identified using pathway annotation in Biocyc and by ortholog mapping of genes as defined in mycolate biosynthesis pathway of M. tuberculosis. Gene expression fold change was measured using both microarrays, and qRT-PCR.

Mentions: Since leuD gene is part of super pathway of leucine, valine and isoleucine biosynthesis, it is natural to reason that deletion of this gene will have major impact on other cellular pathways of MAP. For analyzing the DEGs in the context of cellular pathways, we used the Biocyc [30] pathway reconstruction of MAP. Pathway tools software [25] was used to visualize 2.0 fold changing DEGs on the cellular pathways (Fig. S1). As expected, fatty acid biosynthesis II, palmitate biosynthesis II, mycolate biosynthesis, fatty acid beta-oxidation, acetate degradation and oleate beta-oxidation pathways were found to be differentially expressed. Other differentially expressed pathways included pathway of assimilatory sulfate reduction, malonate degradation, acetyl-CoA fermentation to butyrate I, glutaryl-CoA degration, arginine biosynthesis II, and pantothenate and coenzyme A biosynthesis I. We used the pathway enrichment tool in Pathway tools to identify statistically significant pathways changes associated with each stress condition. The result of this analysis is given Table 1. As evident in Table 1, there were a total of 53 pathways that were associated with different stress conditions. The following main compounds are either produced or consumed by leucine biosynthesis pathway; (2R, 3S)-3-isopropylmaleate, (2S)-2-isopropyl-3-oxosuccinate, (2S)-2-isopropylmaleate, 2-oxoisovalearate, 4-methyl-2-oxopentanoate, and L-leucine. In addition, the following side compounds in leucine biosynthesis pathway forms links with other pathways; 2-oxoglutarate, acetyl-CoA, coenzyme A, NAD+, NADH and L-glutamate. As shown in Fig.S1, the main effect of leuD deletion is on the pathways dependent on or linked to altered levels of needed primary or side compounds produced or consumed by the leucine biosynthetic pathway. For example, we found that key mycolate biosynthetic pathway genes are modulated in different stress conditions, particularly during growth in minimal medium. Our results measuring mRNA levels mirrored changes in gene activity observed in microarray tests by performing qRT-PCR on the same set of genes in this particular pathway (Fig. 8) showing that both microarray and qRT-PCR test results are in agreement with each other.


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

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

Comparison of gene expression fold change in key genes associated with mycolate biosynthesis pathway.Genes associated with mycolate biosynthesis were identified using pathway annotation in Biocyc and by ortholog mapping of genes as defined in mycolate biosynthesis pathway of M. tuberculosis. Gene expression fold change was measured using both microarrays, and qRT-PCR.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037884-g008: Comparison of gene expression fold change in key genes associated with mycolate biosynthesis pathway.Genes associated with mycolate biosynthesis were identified using pathway annotation in Biocyc and by ortholog mapping of genes as defined in mycolate biosynthesis pathway of M. tuberculosis. Gene expression fold change was measured using both microarrays, and qRT-PCR.
Mentions: Since leuD gene is part of super pathway of leucine, valine and isoleucine biosynthesis, it is natural to reason that deletion of this gene will have major impact on other cellular pathways of MAP. For analyzing the DEGs in the context of cellular pathways, we used the Biocyc [30] pathway reconstruction of MAP. Pathway tools software [25] was used to visualize 2.0 fold changing DEGs on the cellular pathways (Fig. S1). As expected, fatty acid biosynthesis II, palmitate biosynthesis II, mycolate biosynthesis, fatty acid beta-oxidation, acetate degradation and oleate beta-oxidation pathways were found to be differentially expressed. Other differentially expressed pathways included pathway of assimilatory sulfate reduction, malonate degradation, acetyl-CoA fermentation to butyrate I, glutaryl-CoA degration, arginine biosynthesis II, and pantothenate and coenzyme A biosynthesis I. We used the pathway enrichment tool in Pathway tools to identify statistically significant pathways changes associated with each stress condition. The result of this analysis is given Table 1. As evident in Table 1, there were a total of 53 pathways that were associated with different stress conditions. The following main compounds are either produced or consumed by leucine biosynthesis pathway; (2R, 3S)-3-isopropylmaleate, (2S)-2-isopropyl-3-oxosuccinate, (2S)-2-isopropylmaleate, 2-oxoisovalearate, 4-methyl-2-oxopentanoate, and L-leucine. In addition, the following side compounds in leucine biosynthesis pathway forms links with other pathways; 2-oxoglutarate, acetyl-CoA, coenzyme A, NAD+, NADH and L-glutamate. As shown in Fig.S1, the main effect of leuD deletion is on the pathways dependent on or linked to altered levels of needed primary or side compounds produced or consumed by the leucine biosynthetic pathway. For example, we found that key mycolate biosynthetic pathway genes are modulated in different stress conditions, particularly during growth in minimal medium. Our results measuring mRNA levels mirrored changes in gene activity observed in microarray tests by performing qRT-PCR on the same set of genes in this particular pathway (Fig. 8) showing that both microarray and qRT-PCR test results are in agreement with each other.

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