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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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Long-chain fatty acid oxidation is required for P. aeruginosa virulence and fitness in wounds.(A) Fold change gene expression (above) or mutant abundance (below) in murine wound infections as compared to MOPS-succinate (Succ.) is shown for the long-chain fatty acid oxidation genes faoAB (*, P<0.05, negative binomial test; †, P<0.01, negative binomial test) (B) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, an faoA transposon mutant derivative (faoA::Tn), or an unmarked, in-frame faoA deletion mutant (ΔfaoA). The experiment was performed twice (PAO1, faoA::Tn) or once (ΔfaoA), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.005, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 10 (faoA::Tn), n = 5 (ΔfaoA)). (C) Growth of wild-type PAO1, faoA::Tn, or ΔfaoA in murine chronic wounds four days post infection. Each symbol represents a value obtained from infection of an individual mouse. The central bar indicates the mean, and error bars indicate standard error of the mean (*, P = 0.023, unpaired T-test; †, P = 0.091, unpaired T-test).
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pgen-1004518-g004: Long-chain fatty acid oxidation is required for P. aeruginosa virulence and fitness in wounds.(A) Fold change gene expression (above) or mutant abundance (below) in murine wound infections as compared to MOPS-succinate (Succ.) is shown for the long-chain fatty acid oxidation genes faoAB (*, P<0.05, negative binomial test; †, P<0.01, negative binomial test) (B) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, an faoA transposon mutant derivative (faoA::Tn), or an unmarked, in-frame faoA deletion mutant (ΔfaoA). The experiment was performed twice (PAO1, faoA::Tn) or once (ΔfaoA), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.005, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 10 (faoA::Tn), n = 5 (ΔfaoA)). (C) Growth of wild-type PAO1, faoA::Tn, or ΔfaoA in murine chronic wounds four days post infection. Each symbol represents a value obtained from infection of an individual mouse. The central bar indicates the mean, and error bars indicate standard error of the mean (*, P = 0.023, unpaired T-test; †, P = 0.091, unpaired T-test).

Mentions: Infected host tissue is a complex nutritional environment for a bacterium, with many potential metabolites available for bacterial catabolism. Manipulation of key metabolites during infection has therapeutic potential in much the same way as arginine-auxotrophic cancers can be treated by depletion of available L-arginine [36]; however, the nutrients utilized by bacteria in wound infections are not known. Comparing the expression of primary metabolic genes in vivo to growth in defined minimal media led us to hypothesize that fatty acids are a primary carbon source available to P. aeruginosa in vivo (Figure 3). Examination of our Tn-seq data in detail (Table S4) revealed that the faoAB (or fadBA5) genes, which are required for robust growth on long-chain (C12 or greater) fatty acids [37], contribute to P. aeruginosa fitness in vivo (Figures 4A and S3A). This was confirmed by single mutant infections: both an faoA transposon mutant and an faoA deletion mutant are attenuated in both acute and chronic wounds, indicating that long-chain fatty acids are likely an important energy source in wounds (Figure 4BC). The faoAB genes have also been shown to contribute to resistance to tobramycin, so they could potentially contribute to resistance to an unspecified chemical stress in vivo as well [20]. However, our in vivo gene expression data suggests that growth in wound infections involves pathways active during growth on reduced carbon sources such as long-chain fatty acids (Figure 3). We did not observe an in vivo fitness defect for genes annotated as homologs of the Escherichia coli long-chain fatty acid outer membrane transporter gene fadL (fadL1, fadL2, or fadL3) in P. aeruginosa; however, these genes are not thought to be required for long-chain fatty acid transport in P. aeruginosa[38].


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

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

Long-chain fatty acid oxidation is required for P. aeruginosa virulence and fitness in wounds.(A) Fold change gene expression (above) or mutant abundance (below) in murine wound infections as compared to MOPS-succinate (Succ.) is shown for the long-chain fatty acid oxidation genes faoAB (*, P<0.05, negative binomial test; †, P<0.01, negative binomial test) (B) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, an faoA transposon mutant derivative (faoA::Tn), or an unmarked, in-frame faoA deletion mutant (ΔfaoA). The experiment was performed twice (PAO1, faoA::Tn) or once (ΔfaoA), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.005, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 10 (faoA::Tn), n = 5 (ΔfaoA)). (C) Growth of wild-type PAO1, faoA::Tn, or ΔfaoA in murine chronic wounds four days post infection. Each symbol represents a value obtained from infection of an individual mouse. The central bar indicates the mean, and error bars indicate standard error of the mean (*, P = 0.023, unpaired T-test; †, P = 0.091, unpaired T-test).
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pgen-1004518-g004: Long-chain fatty acid oxidation is required for P. aeruginosa virulence and fitness in wounds.(A) Fold change gene expression (above) or mutant abundance (below) in murine wound infections as compared to MOPS-succinate (Succ.) is shown for the long-chain fatty acid oxidation genes faoAB (*, P<0.05, negative binomial test; †, P<0.01, negative binomial test) (B) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, an faoA transposon mutant derivative (faoA::Tn), or an unmarked, in-frame faoA deletion mutant (ΔfaoA). The experiment was performed twice (PAO1, faoA::Tn) or once (ΔfaoA), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.005, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 10 (faoA::Tn), n = 5 (ΔfaoA)). (C) Growth of wild-type PAO1, faoA::Tn, or ΔfaoA in murine chronic wounds four days post infection. Each symbol represents a value obtained from infection of an individual mouse. The central bar indicates the mean, and error bars indicate standard error of the mean (*, P = 0.023, unpaired T-test; †, P = 0.091, unpaired T-test).
Mentions: Infected host tissue is a complex nutritional environment for a bacterium, with many potential metabolites available for bacterial catabolism. Manipulation of key metabolites during infection has therapeutic potential in much the same way as arginine-auxotrophic cancers can be treated by depletion of available L-arginine [36]; however, the nutrients utilized by bacteria in wound infections are not known. Comparing the expression of primary metabolic genes in vivo to growth in defined minimal media led us to hypothesize that fatty acids are a primary carbon source available to P. aeruginosa in vivo (Figure 3). Examination of our Tn-seq data in detail (Table S4) revealed that the faoAB (or fadBA5) genes, which are required for robust growth on long-chain (C12 or greater) fatty acids [37], contribute to P. aeruginosa fitness in vivo (Figures 4A and S3A). This was confirmed by single mutant infections: both an faoA transposon mutant and an faoA deletion mutant are attenuated in both acute and chronic wounds, indicating that long-chain fatty acids are likely an important energy source in wounds (Figure 4BC). The faoAB genes have also been shown to contribute to resistance to tobramycin, so they could potentially contribute to resistance to an unspecified chemical stress in vivo as well [20]. However, our in vivo gene expression data suggests that growth in wound infections involves pathways active during growth on reduced carbon sources such as long-chain fatty acids (Figure 3). We did not observe an in vivo fitness defect for genes annotated as homologs of the Escherichia coli long-chain fatty acid outer membrane transporter gene fadL (fadL1, fadL2, or fadL3) in P. aeruginosa; however, these genes are not thought to be required for long-chain fatty acid transport in P. aeruginosa[38].

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