DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans

Concerta L. Holley, Xinjun Zhang, Kate R. Fortney, Sheila Ellinger, Paula Johnson, Beth Baker, Yunlong Liu, Diane Janowicz, Barry Katz, Robert S. Munson, Stanley Spinola

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.

Original languageEnglish
Pages (from-to)3281-3292
Number of pages12
JournalInfection and Immunity
Volume83
Issue number8
DOIs
StatePublished - 2015

Fingerprint

Haemophilus ducreyi
Genes
Virulence
RNA Sequence Analysis
Foreskin
DNA-Directed RNA Polymerases
Transcriptome
Phagocytosis
Open Reading Frames
Volunteers
Oxidative Stress
Down-Regulation
Fibroblasts
Bacteria
Gene Expression
Food
Survival
Proteins

ASJC Scopus subject areas

  • Immunology
  • Microbiology
  • Parasitology
  • Infectious Diseases

Cite this

DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans. / Holley, Concerta L.; Zhang, Xinjun; Fortney, Kate R.; Ellinger, Sheila; Johnson, Paula; Baker, Beth; Liu, Yunlong; Janowicz, Diane; Katz, Barry; Munson, Robert S.; Spinola, Stanley.

In: Infection and Immunity, Vol. 83, No. 8, 2015, p. 3281-3292.

Research output: Contribution to journalArticle

Holley, Concerta L. ; Zhang, Xinjun ; Fortney, Kate R. ; Ellinger, Sheila ; Johnson, Paula ; Baker, Beth ; Liu, Yunlong ; Janowicz, Diane ; Katz, Barry ; Munson, Robert S. ; Spinola, Stanley. / DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans. In: Infection and Immunity. 2015 ; Vol. 83, No. 8. pp. 3281-3292.
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abstract = "The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28{\%} and 17{\%} of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.",
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AU - Holley, Concerta L.

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AU - Ellinger, Sheila

AU - Johnson, Paula

AU - Baker, Beth

AU - Liu, Yunlong

AU - Janowicz, Diane

AU - Katz, Barry

AU - Munson, Robert S.

AU - Spinola, Stanley

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AB - The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.

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