title: A (p)ppGpp-null mutant of Haemophilus ducreyi is partially attenuated in humans due to multiple conflicting phenotypes.
abstract: (p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi, the causative agent of chancroid, has homologs of both relA and spoT, which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli. We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli, overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.
authors.affiliation: Departments of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Concerta
authors.initials: C
authors.lastname: Holley
authors.affiliation: Departments of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Dharanesh
authors.initials: D
authors.lastname: Gangaiah
authors.affiliation: Departments of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Departments of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Kate R
authors.initials: KR
authors.lastname: Fortney
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Janowicz
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Sheila
authors.initials: S
authors.lastname: Ellinger
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Zwickl
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, 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: Departments of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA Departments of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Center for Immunobiology, Indiana University School of Medicine, Indianapolis, Indiana, USA sspinola@iu.edu.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Spinola
mesh_terms: D000328:Adult; D003872:Dermatitis / Q000382:microbiology / Q000473:pathology; D005260:Female; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006158:Guanosine Pentaphosphate / Q000172:deficiency*; D006191:Haemophilus ducreyi / Q000235:genetics / Q000472:pathogenicity*; D064368:Healthy Volunteers; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*
chemical_list: D006158:Guanosine Pentaphosphate; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases