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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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Biosynthetic requirements of P. aeruginosa during wound infection.(A) P. aeruginosa must biosynthesize several amino acids, cofactors, and metabolic endproducts during growth in vivo. If mutants in >33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ (fold change ≥2, P<0.05, negative binomial test), that metabolite was said to be “Available” to P. aeruginosa in vivo. If 5–33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, or if only one infection matched the criteria for “Available” as described above, that metabolite was said to be “Potentially Available”. If <5% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, that metabolite was said to be “Not Available”. Genes predicted to be involved in synthesis of necessary metabolites were identified from the PseudoCyc database (see Table S7 for details). (B) Kaplan-Meier survival curves of burned mice infected with PAO1 ΔpabC, PAO1 ΔpurF, PAO1 ΔhisE, or PAO1 ΔilvD. The first two strains are auxotrophic for metabolites predicted to be not available, and the last two strains are auxotrophic for metabolites predicted to be available. The experiment was performed with five mice per group, and the percent survival of all mice is shown (*, P<0.05, log-rank (Mantel-Cox) test).
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pgen-1004518-g005: Biosynthetic requirements of P. aeruginosa during wound infection.(A) P. aeruginosa must biosynthesize several amino acids, cofactors, and metabolic endproducts during growth in vivo. If mutants in >33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ (fold change ≥2, P<0.05, negative binomial test), that metabolite was said to be “Available” to P. aeruginosa in vivo. If 5–33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, or if only one infection matched the criteria for “Available” as described above, that metabolite was said to be “Potentially Available”. If <5% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, that metabolite was said to be “Not Available”. Genes predicted to be involved in synthesis of necessary metabolites were identified from the PseudoCyc database (see Table S7 for details). (B) Kaplan-Meier survival curves of burned mice infected with PAO1 ΔpabC, PAO1 ΔpurF, PAO1 ΔhisE, or PAO1 ΔilvD. The first two strains are auxotrophic for metabolites predicted to be not available, and the last two strains are auxotrophic for metabolites predicted to be available. The experiment was performed with five mice per group, and the percent survival of all mice is shown (*, P<0.05, log-rank (Mantel-Cox) test).

Mentions: In addition to identifying primary carbon and energy sources during wound infections, our results also allow identification of biosynthetic end products that are available to P. aeruginosa in wound infections. We reasoned that biosynthetic pathways required in minimal media but dispensable in vivo would likely be responsible for the synthesis of metabolites available to P. aeruginosa in vivo. To identify biosynthetic genes, we used the manually curated PseudoCyc annotation [39]. Metabolites for which 33% or more of the biosynthetic genes contribute more to fitness in MOPS-succinate than in both acute and chronic wounds (P<0.05, negative binomial test, fold change ≥2) were deemed “available”, and include many amino acids, the electron carriers FAD and NAD, and the B vitamin thiamine (Figure 5A and Table S7). Metabolites for which 95% or more of the biosynthetic genes have a similar effect on fitness in MOPS-succinate and in both acute and chronic wounds were deemed “not available”, and include the amino acids glutamate, tyrosine, phenylalanine, aspartate, and asparagine, purines, many other vitamins and cofactors including the folate precursor p-aminobenzoate (PABA) and several other B group vitamins. The remaining metabolites that do not match either of the above sets of criteria were deemed “potentially available”. To confirm the validity of this approach, we constructed two in-frame, unmarked deletion mutants lacking the ability to biosynthesize metabolites predicted to be unavailable in wounds: one lacking pabC (which requires PABA for growth in a minimal medium) and one lacking purF (which requires purines for growth in a minimal medium) (Figure S3B). These two mutants were completely attenuated for virulence in the burn wound (Figure 5B). However, in-frame, unmarked deletion mutants unable to grow without histidine or without isoleucine, leucine, and valine, all of which are predicted to be available in wounds, were significantly more virulent than the pabC or purF mutants. Thus, by comparison with minimal media, we demonstrate that genome-wide bacterial mutant fitness can be used to comprehensively profile bioavailable metabolites in a complex, undefined environment. Bacterial-specific pathways responsible for biosynthesis of any of the unavailable metabolites identified may represent promising targets for therapeutic intervention in wounds.


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

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

Biosynthetic requirements of P. aeruginosa during wound infection.(A) P. aeruginosa must biosynthesize several amino acids, cofactors, and metabolic endproducts during growth in vivo. If mutants in >33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ (fold change ≥2, P<0.05, negative binomial test), that metabolite was said to be “Available” to P. aeruginosa in vivo. If 5–33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, or if only one infection matched the criteria for “Available” as described above, that metabolite was said to be “Potentially Available”. If <5% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, that metabolite was said to be “Not Available”. Genes predicted to be involved in synthesis of necessary metabolites were identified from the PseudoCyc database (see Table S7 for details). (B) Kaplan-Meier survival curves of burned mice infected with PAO1 ΔpabC, PAO1 ΔpurF, PAO1 ΔhisE, or PAO1 ΔilvD. The first two strains are auxotrophic for metabolites predicted to be not available, and the last two strains are auxotrophic for metabolites predicted to be available. The experiment was performed with five mice per group, and the percent survival of all mice is shown (*, P<0.05, log-rank (Mantel-Cox) test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109851&req=5

pgen-1004518-g005: Biosynthetic requirements of P. aeruginosa during wound infection.(A) P. aeruginosa must biosynthesize several amino acids, cofactors, and metabolic endproducts during growth in vivo. If mutants in >33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ (fold change ≥2, P<0.05, negative binomial test), that metabolite was said to be “Available” to P. aeruginosa in vivo. If 5–33% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, or if only one infection matched the criteria for “Available” as described above, that metabolite was said to be “Potentially Available”. If <5% of genes predicted to be in the biosynthetic pathway of a given metabolite were more fit in both burn and chronic wounds than in MOPS-Succ, that metabolite was said to be “Not Available”. Genes predicted to be involved in synthesis of necessary metabolites were identified from the PseudoCyc database (see Table S7 for details). (B) Kaplan-Meier survival curves of burned mice infected with PAO1 ΔpabC, PAO1 ΔpurF, PAO1 ΔhisE, or PAO1 ΔilvD. The first two strains are auxotrophic for metabolites predicted to be not available, and the last two strains are auxotrophic for metabolites predicted to be available. The experiment was performed with five mice per group, and the percent survival of all mice is shown (*, P<0.05, log-rank (Mantel-Cox) test).
Mentions: In addition to identifying primary carbon and energy sources during wound infections, our results also allow identification of biosynthetic end products that are available to P. aeruginosa in wound infections. We reasoned that biosynthetic pathways required in minimal media but dispensable in vivo would likely be responsible for the synthesis of metabolites available to P. aeruginosa in vivo. To identify biosynthetic genes, we used the manually curated PseudoCyc annotation [39]. Metabolites for which 33% or more of the biosynthetic genes contribute more to fitness in MOPS-succinate than in both acute and chronic wounds (P<0.05, negative binomial test, fold change ≥2) were deemed “available”, and include many amino acids, the electron carriers FAD and NAD, and the B vitamin thiamine (Figure 5A and Table S7). Metabolites for which 95% or more of the biosynthetic genes have a similar effect on fitness in MOPS-succinate and in both acute and chronic wounds were deemed “not available”, and include the amino acids glutamate, tyrosine, phenylalanine, aspartate, and asparagine, purines, many other vitamins and cofactors including the folate precursor p-aminobenzoate (PABA) and several other B group vitamins. The remaining metabolites that do not match either of the above sets of criteria were deemed “potentially available”. To confirm the validity of this approach, we constructed two in-frame, unmarked deletion mutants lacking the ability to biosynthesize metabolites predicted to be unavailable in wounds: one lacking pabC (which requires PABA for growth in a minimal medium) and one lacking purF (which requires purines for growth in a minimal medium) (Figure S3B). These two mutants were completely attenuated for virulence in the burn wound (Figure 5B). However, in-frame, unmarked deletion mutants unable to grow without histidine or without isoleucine, leucine, and valine, all of which are predicted to be available in wounds, were significantly more virulent than the pabC or purF mutants. Thus, by comparison with minimal media, we demonstrate that genome-wide bacterial mutant fitness can be used to comprehensively profile bioavailable metabolites in a complex, undefined environment. Bacterial-specific pathways responsible for biosynthesis of any of the unavailable metabolites identified may represent promising targets for therapeutic intervention in wounds.

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