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Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection.

Turner KH, Everett J, Trivedi U, Rumbaugh KP, Whiteley M - PLoS Genet. (2014)

Bottom Line: Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes.Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection.Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Institute of Cellular and Molecular Biology, Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

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Reconstruction of in vivo metabolism from RNA-seq data.Metabolic steps as identified by KEGG Orthology are colored according to their relative expression in both burn and chronic wounds as compared to growth in MOPS-succinate (Succ.). Only significant changes (fold change ≥4, P<0.01, negative binomial test) in both burn and chronic wounds are shown. Pathways and metabolic steps of interest are highlighted in yellow, and succinate, the sole carbon and energy source in the control medium, is indicated with an arrow. Generally, biosynthetic pathways were down-regulated and anaplerotic pathways were up-regulated in vivo. (PEPC, PEP carboxylase; THF; tetrahydrofolate).
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pgen-1004518-g003: Reconstruction of in vivo metabolism from RNA-seq data.Metabolic steps as identified by KEGG Orthology are colored according to their relative expression in both burn and chronic wounds as compared to growth in MOPS-succinate (Succ.). Only significant changes (fold change ≥4, P<0.01, negative binomial test) in both burn and chronic wounds are shown. Pathways and metabolic steps of interest are highlighted in yellow, and succinate, the sole carbon and energy source in the control medium, is indicated with an arrow. Generally, biosynthetic pathways were down-regulated and anaplerotic pathways were up-regulated in vivo. (PEPC, PEP carboxylase; THF; tetrahydrofolate).

Mentions: Our analysis of the correlation between differential expression and conditional mutant fitness by COG category (Figure 2B) indicates that expression is a better predictor of fitness contribution for genes involved in primary metabolism. Therefore, to characterize the primary metabolism of P. aeruginosa during wound infection, we projected our transcriptome profiling results onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAYS database (Figure 3). As mentioned previously, our choice of a defined medium (MOPS-succinate) as a control condition provided a reference point from which to understand bacterial physiology and metabolism in the unknown nutritional environment of the infected wound. Our metabolic reconstruction suggests that genes encoding decarboxylating steps of the TCA cycle (isocitrate dehydrogenase and α-ketoglutarate dehydrogenase) are down-regulated, and that the gene encoding the entry point to the glyoxylate shunt (isocitrate lyase) is up-regulated. The glyoxylate shunt is a variation on the TCA cycle not present in mammals that allows bacteria, including P. aeruginosa, to grow on reduced carbon sources such as fatty acids by bypassing TCA cycle reactions that would result in the loss of carbon [33]. This serves to replenish TCA cycle intermediates utilized in biosynthesis, which is known generally as anaplerosis. We also observed up-regulation of ppc, which encodes a second non-mammalian anaplerotic enzyme, PEP carboxylase, in wound infections, though it is much more highly expressed in chronic wounds than in acute wounds (Table S2). Finally, expression of a number of genes associated with oxygen-limited environments is also up-regulated, including those encoding high-affinity terminal oxidases and the denitrification pathway [34], suggesting that at least some bacterial cells in these infections sense decreased oxygen tension. This is consistent with frequent observations of ischemia at wound sites in the clinic, and suggests that oxygen limitation affects the physiology of both the infecting organism and host tissue at wound sites [35]. The transcriptomic results suggest that P. aeruginosa differentially regulates a large portion of its genome in soft tissue infections, and that this reflects a response to differential availability of key metabolic factors such as carbon and energy sources, biosynthetic endproducts, and terminal electron acceptors. In the remainder of this manuscript, we will examine each of these in detail.


Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection.

Turner KH, Everett J, Trivedi U, Rumbaugh KP, Whiteley M - PLoS Genet. (2014)

Reconstruction of in vivo metabolism from RNA-seq data.Metabolic steps as identified by KEGG Orthology are colored according to their relative expression in both burn and chronic wounds as compared to growth in MOPS-succinate (Succ.). Only significant changes (fold change ≥4, P<0.01, negative binomial test) in both burn and chronic wounds are shown. Pathways and metabolic steps of interest are highlighted in yellow, and succinate, the sole carbon and energy source in the control medium, is indicated with an arrow. Generally, biosynthetic pathways were down-regulated and anaplerotic pathways were up-regulated in vivo. (PEPC, PEP carboxylase; THF; tetrahydrofolate).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109851&req=5

pgen-1004518-g003: Reconstruction of in vivo metabolism from RNA-seq data.Metabolic steps as identified by KEGG Orthology are colored according to their relative expression in both burn and chronic wounds as compared to growth in MOPS-succinate (Succ.). Only significant changes (fold change ≥4, P<0.01, negative binomial test) in both burn and chronic wounds are shown. Pathways and metabolic steps of interest are highlighted in yellow, and succinate, the sole carbon and energy source in the control medium, is indicated with an arrow. Generally, biosynthetic pathways were down-regulated and anaplerotic pathways were up-regulated in vivo. (PEPC, PEP carboxylase; THF; tetrahydrofolate).
Mentions: Our analysis of the correlation between differential expression and conditional mutant fitness by COG category (Figure 2B) indicates that expression is a better predictor of fitness contribution for genes involved in primary metabolism. Therefore, to characterize the primary metabolism of P. aeruginosa during wound infection, we projected our transcriptome profiling results onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAYS database (Figure 3). As mentioned previously, our choice of a defined medium (MOPS-succinate) as a control condition provided a reference point from which to understand bacterial physiology and metabolism in the unknown nutritional environment of the infected wound. Our metabolic reconstruction suggests that genes encoding decarboxylating steps of the TCA cycle (isocitrate dehydrogenase and α-ketoglutarate dehydrogenase) are down-regulated, and that the gene encoding the entry point to the glyoxylate shunt (isocitrate lyase) is up-regulated. The glyoxylate shunt is a variation on the TCA cycle not present in mammals that allows bacteria, including P. aeruginosa, to grow on reduced carbon sources such as fatty acids by bypassing TCA cycle reactions that would result in the loss of carbon [33]. This serves to replenish TCA cycle intermediates utilized in biosynthesis, which is known generally as anaplerosis. We also observed up-regulation of ppc, which encodes a second non-mammalian anaplerotic enzyme, PEP carboxylase, in wound infections, though it is much more highly expressed in chronic wounds than in acute wounds (Table S2). Finally, expression of a number of genes associated with oxygen-limited environments is also up-regulated, including those encoding high-affinity terminal oxidases and the denitrification pathway [34], suggesting that at least some bacterial cells in these infections sense decreased oxygen tension. This is consistent with frequent observations of ischemia at wound sites in the clinic, and suggests that oxygen limitation affects the physiology of both the infecting organism and host tissue at wound sites [35]. The transcriptomic results suggest that P. aeruginosa differentially regulates a large portion of its genome in soft tissue infections, and that this reflects a response to differential availability of key metabolic factors such as carbon and energy sources, biosynthetic endproducts, and terminal electron acceptors. In the remainder of this manuscript, we will examine each of these in detail.

Bottom Line: Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes.Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection.Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Institute of Cellular and Molecular Biology, Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

Show MeSH
Related in: MedlinePlus