ABSTRACTTo adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes ofHaemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog ofhfq. Insertional inactivation ofhfqaltered the expression of ~16% of theH. ducreyigenes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in thehfqinactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation ofhfqdownregulated genes in theflp-tadandlspB-lspA2operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarkedhfqdeletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion ofhfqdownregulated Flp1 and impaired the ability ofH. ducreyito form microcolonies, downregulated DsrA and renderedH. ducreyiserum susceptible, and downregulated LspB and LspA2, which allowH. ducreyito resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor ofH. ducreyistationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog.IMPORTANCEPathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, includingHaemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required forH. ducreyito infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.
Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation
