title: DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans.
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.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Concerta L
authors.initials: CL
authors.lastname: Holley
authors.affiliation: School of Informatics and Computing, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Xinjun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Kate R
authors.initials: KR
authors.lastname: Fortney
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Sheila
authors.initials: S
authors.lastname: Ellinger
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Paula
authors.initials: P
authors.lastname: Johnson
authors.affiliation: Center for Microbial Pathogenesis, Nationwide Children's Hospital, Columbus, Ohio, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Baker
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Janowicz
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA Department of Biostatistics, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, Indiana, USA.
authors.forename: Barry P
authors.initials: BP
authors.lastname: Katz
authors.affiliation: Center for Microbial Pathogenesis, Nationwide Children's Hospital, Columbus, Ohio, USA Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Munson
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA Department of Medicine, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA Center for Immunobiology, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana, USA sspinola@iupui.edu.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Spinola
mesh_terms: D000328:Adult; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002602:Chancroid / Q000382:microbiology*; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D006191:Haemophilus ducreyi / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity*; D006801:Humans; D008297:Male; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D006159:Guanosine Tetraphosphate