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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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Chemotactic flagellar motility is required in burn wounds and not chronic wounds.(A) Fold change mutant abundance in murine wound infections as compared to MOPS-succinate (Succ.) is shown for genes involved in chemotaxis (*, P<0.05, negative binomial test). (B) Log2-transformed fold change gene expression (y axis) and knockout abundance (x axis) of P. aeruginosa in murine chronic wound infections as compared to murine burn wound infections. Significant changes in knockout abundance (fold change ≥4, P<0.05, negative binomial test) are colored purple, and genes annotated as being involved in flagellar assembly or bacterial chemotaxis in the KEGG PATHWAYS database are highlighted green and red, respectively (N.C., no change). (C) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, a cheR1 transposon mutant derivative (cheR1::Tn), or an unmarked, in-frame cheR1 deletion mutant (ΔcheR1). The experiment was performed twice (PAO1, cheR1::Tn) or once (ΔcheR1), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.01, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 9 (cheR1::Tn), n = 5 (ΔcheR1)). (D) Growth of wild-type PAO1 or a cheR1 transposon mutant derivative 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. No significant difference was observed (P = 0.194, unpaired T-test).
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pgen-1004518-g006: Chemotactic flagellar motility is required in burn wounds and not chronic wounds.(A) Fold change mutant abundance in murine wound infections as compared to MOPS-succinate (Succ.) is shown for genes involved in chemotaxis (*, P<0.05, negative binomial test). (B) Log2-transformed fold change gene expression (y axis) and knockout abundance (x axis) of P. aeruginosa in murine chronic wound infections as compared to murine burn wound infections. Significant changes in knockout abundance (fold change ≥4, P<0.05, negative binomial test) are colored purple, and genes annotated as being involved in flagellar assembly or bacterial chemotaxis in the KEGG PATHWAYS database are highlighted green and red, respectively (N.C., no change). (C) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, a cheR1 transposon mutant derivative (cheR1::Tn), or an unmarked, in-frame cheR1 deletion mutant (ΔcheR1). The experiment was performed twice (PAO1, cheR1::Tn) or once (ΔcheR1), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.01, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 9 (cheR1::Tn), n = 5 (ΔcheR1)). (D) Growth of wild-type PAO1 or a cheR1 transposon mutant derivative 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. No significant difference was observed (P = 0.194, unpaired T-test).

Mentions: While our focus on metabolism revealed numerous similarities in chronic and acute infections, the genomic techniques employed here also provided new insight into how these infection types differ (Table S8). As a motile bacterium, P. aeruginosa possesses the ability to detect and move toward nutrients (including long-chain fatty acids [38]), a process referred to as chemotaxis. Examination of our Tn-seq results revealed that several genes with putative roles in chemotaxis, including cheA, cheB, cheR1, and a homolog of cheW (PA3349) are required in burn, but not chronic wounds (Figure 6A). In addition nearly every annotated flagellar gene is required for fitness in burn wounds [29], but is dispensable in chronic wounds (Figure 6B). To further confirm the role of chemotaxis in acute wound infections, single-strain infections with a cheR1 transposon insertion mutant and an in-frame, unmarked cheR1 deletion mutant (Figure S3C) were performed. While both the cheR1 insertion and deletion mutants have virulence defects in burn wounds (Figure 6C), the cheR1 insertion mutant is as fit or more fit than wild-type P. aeruginosa in chronic wounds (Figure 6D). These results suggest that the ability to chemotax along a spatio-chemical gradient by utilizing flagellar motility is a key feature of acute but not chronic wound P. aeruginosa infections.


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

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

Chemotactic flagellar motility is required in burn wounds and not chronic wounds.(A) Fold change mutant abundance in murine wound infections as compared to MOPS-succinate (Succ.) is shown for genes involved in chemotaxis (*, P<0.05, negative binomial test). (B) Log2-transformed fold change gene expression (y axis) and knockout abundance (x axis) of P. aeruginosa in murine chronic wound infections as compared to murine burn wound infections. Significant changes in knockout abundance (fold change ≥4, P<0.05, negative binomial test) are colored purple, and genes annotated as being involved in flagellar assembly or bacterial chemotaxis in the KEGG PATHWAYS database are highlighted green and red, respectively (N.C., no change). (C) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, a cheR1 transposon mutant derivative (cheR1::Tn), or an unmarked, in-frame cheR1 deletion mutant (ΔcheR1). The experiment was performed twice (PAO1, cheR1::Tn) or once (ΔcheR1), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.01, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 9 (cheR1::Tn), n = 5 (ΔcheR1)). (D) Growth of wild-type PAO1 or a cheR1 transposon mutant derivative 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. No significant difference was observed (P = 0.194, unpaired T-test).
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pgen-1004518-g006: Chemotactic flagellar motility is required in burn wounds and not chronic wounds.(A) Fold change mutant abundance in murine wound infections as compared to MOPS-succinate (Succ.) is shown for genes involved in chemotaxis (*, P<0.05, negative binomial test). (B) Log2-transformed fold change gene expression (y axis) and knockout abundance (x axis) of P. aeruginosa in murine chronic wound infections as compared to murine burn wound infections. Significant changes in knockout abundance (fold change ≥4, P<0.05, negative binomial test) are colored purple, and genes annotated as being involved in flagellar assembly or bacterial chemotaxis in the KEGG PATHWAYS database are highlighted green and red, respectively (N.C., no change). (C) Kaplan-Meier survival curves of burned mice infected with wild-type PAO1, a cheR1 transposon mutant derivative (cheR1::Tn), or an unmarked, in-frame cheR1 deletion mutant (ΔcheR1). The experiment was performed twice (PAO1, cheR1::Tn) or once (ΔcheR1), with three to five mice per group, and the percent survival of all mice is shown (*, P<0.01, log-rank (Mantel-Cox) test; n = 8 (PAO1), n = 9 (cheR1::Tn), n = 5 (ΔcheR1)). (D) Growth of wild-type PAO1 or a cheR1 transposon mutant derivative 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. No significant difference was observed (P = 0.194, unpaired T-test).
Mentions: While our focus on metabolism revealed numerous similarities in chronic and acute infections, the genomic techniques employed here also provided new insight into how these infection types differ (Table S8). As a motile bacterium, P. aeruginosa possesses the ability to detect and move toward nutrients (including long-chain fatty acids [38]), a process referred to as chemotaxis. Examination of our Tn-seq results revealed that several genes with putative roles in chemotaxis, including cheA, cheB, cheR1, and a homolog of cheW (PA3349) are required in burn, but not chronic wounds (Figure 6A). In addition nearly every annotated flagellar gene is required for fitness in burn wounds [29], but is dispensable in chronic wounds (Figure 6B). To further confirm the role of chemotaxis in acute wound infections, single-strain infections with a cheR1 transposon insertion mutant and an in-frame, unmarked cheR1 deletion mutant (Figure S3C) were performed. While both the cheR1 insertion and deletion mutants have virulence defects in burn wounds (Figure 6C), the cheR1 insertion mutant is as fit or more fit than wild-type P. aeruginosa in chronic wounds (Figure 6D). These results suggest that the ability to chemotax along a spatio-chemical gradient by utilizing flagellar motility is a key feature of acute but not chronic wound P. aeruginosa infections.

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