A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes

Abstract

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 bothrelAandspoT, which primarily synthesize and hydrolyze (p)ppGpp inEscherichia coli. We constructedrelAandrelA spoTdeletion mutants to assess the contribution of (p)ppGpp toH. ducreyipathogenesis. Both therelAsingle mutant and therelA spoTdouble mutant failed to synthesize (p)ppGpp, suggesting thatrelAis the primary synthetase of (p)ppGpp inH. 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 intrans. 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 oflspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. InE. coli, overexpression of DksA compensates for the loss of (p)ppGpp; theH. ducreyidouble mutant expressed higher transcript levels ofdksAthan the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.